United States Patent: 7655774

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United States Patent	7,655,774
Mullins , et al.	February 2, 2010

Ancestral and COT viral sequences, proteins and immunogenic compositions

Abstract

The present invention is directed to ancestral and COT nucleic acid and amino acid sequences, methods for producing such sequences and uses thereof, including prophylactic and diagnostic uses.

Inventors:	Mullins; James I. (Seattle, WA), Rodrigo; Allen G. (Auckland, NZ), Learn; Gerald H. (Kingston, WA), Li; Fusheng (Seattle, WA), Nickle; David C. (Seattle, WA), Jensen; Mark A. (Snohomish, WA)
Assignee:	University of Washington (Seattle, WA)
Appl. No.:	10/780,507
Filed:	February 17, 2004

Related U.S. Patent Documents


Application Number	Filing Date	Patent Number	Issue Date
10204204
PCT/US01/05288	Feb., 2001
60183659	Feb., 2000
60447586	Feb., 2003

Current U.S. Class: 536/23.1 ; 435/252.3; 435/320.1

Current International Class: C07H 21/02 (20060101); C12N 1/20 (20060101); C12N 15/00 (20060101)

References Cited [Referenced By]

U.S. Patent Documents


6958226	October 2005	Gray et al.

Foreign Patent Documents


WO 98/34640	Aug., 1998	WO
WO 00/29561	May., 2000	WO
WO 01/60838	Aug., 2001	WO

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Primary Examiner: Peng; Bo

Government Interests

STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This work was supported by a grant from the US Public Health Service through a grant to the University of Washington Center for AIDS Research (AI-27757) and PHS T32A107509 and T32CA0922925. The Federal Government may have certain rights in this invention.

Parent Case Text

CROSS-REFERENCES TO RELATED APPLICATIONS

This application is a continuation-in-part and claims priority to U.S. patent application Ser. No. 10/204,204, filed Feb. 16, 2001, now abandoned which is a U.S. National Phase application of PCT/US01/05288, filed Feb. 16, 2001, which claims the benefit of U.S. Provisional Patent Application No. 60/183,659, filed Feb. 18, 2000. This application also claims the benefit of U.S. Provisional Application No. 60/447,586, filed Feb. 14, 2003. All of these applications are incorporated by reference herein. All of these patent applications are incorporated herein by reference, in their entirety.

Claims

What is claimed is:

1. An isolated expression construct comprising the following operably linked elements: a transcriptional promoter; a nucleic acid sequence that encodes the polypeptide encoded by SEQ ID NO:25; and a transcriptional terminator.

2. The expression construct of claim 1, wherein the transcriptional promoter is a heterologous promoter.

3. The expression construct of claim 2, wherein the promoter is a cytomegalovirus promoter.

4. An isolated prokaryotic or eukaryotic cell transformed or transfected with the expression construct of claim 1.

5. The eukaryotic cell of claim 4, which is a mammalian cell.

6. The prokaryotic cell of claim 4, which is an E. coli cell.

7. The eukaryotic cell of claim 4, which is an S. cerevisiae cell.

8. The eukaryotic cell of claim 4, which is a human cell.

9. A vector comprising the expression construct of claim 1.

10. The vector of claim 9, wherein the nucleic acid sequence is operably linked to a Semlike Forest Virus replicon, and wherein the resulting recombinant replicon is operably linked to a cytomegalovirus promoter.

11. An isolated host cell comprising the vector of claim 9.

Description

BACKGROUND OF THE INVENTION

HIV-1 has proved to be an extremely difficult target for vaccine development. Immune correlates of protective immunity against HIV-1 infection remain uncertain. The virus persistently replicates in the infected individual, leading inexorably to disease despite the generation of vigorous humoral and cellular immune responses. HIV-1 rapidly mutates during infection, resulting in the generation of viruses that can escape immune recognition. Unlike other highly diverse viruses (e.g., influenza), there does not appear to be a succession of variants where one prototypical strain is replaced by successive uniform strains. Rather, an evolutionary tree of viral sequences sampled from a large number of HIV-infected individuals form a star-burst pattern with most of the variants roughly equidistant from the center of the tree. HIV-1 viruses can also persist indefinitely as latent proviral DNA, capable of replicating in individuals at a later time.

Currently, several HIV-1 vaccine approaches are being developed, each with its own relative strengths and weaknesses. These approaches include the development of live attenuated vaccines, inactivated viruses with adjuvant peptides and subunit vaccines, live vector-based vaccines, and DNA vaccines. Envelope glycoproteins were considered as the prime antigen in the vaccine regimen due to their surface-exposure, until it became evident that they are not ideal immunogens. This is an expected consequence of the immunological selective forces that drive the evolution of these viruses: it appears that the same features of envelope glycoproteins that dictate poor immunogenicity in natural infections have hampered vaccine development. However, modification of the vaccine recipe may overcome these problems. For example, a recent report of successful neutralization (in mice) of primary isolates from infected individuals with a fusion-competent immunogen supports this idea.

Another approach could be to use natural isolates of HIV-1 in a vaccine recipe. Identification of early variants even from stored specimens near the start of the AIDS epidemic is very unlikely, however. Natural isolates are also unlikely to embody features (e.g., epitopes) that are ideal for a vaccine candidate. Furthermore, any given natural virus isolate will have features that reflect adaptations due to specific interactions within that particular human host. These individual-specific features are not expected to be found in all or most strains of the virus, and thus vaccines based on individual isolates are unlikely to be effective against a broad range of circulating virus.

Another approach could be to include as many diverse HIV-1 isolates as possible in the vaccine recipe in an effort to elicit broad protection against HIV-1 challenge. First, one or more strains are chosen from among the many circulating strains of HIV. The advantage of this approach is that such a strain is known to be an infectious form of a viable virus. However, such a strain will be genetically quite dissimilar to other strains in circulation, and thus can fail to elicit broad protection. A related approach is to build a consensus sequence based on circulating strains, or on strains in the database. The consensus sequence is likely to be less distant in a genetic sense from circulating strains, but is not an estimate of any real virus, however, and thus may not provide broad protection.

Accordingly, there is a need in the art for new effective methods of identifying candidate sequences for vaccine development to prevent and treat HIV infection. The present invention fulfills this and other needs.

BRIEF SUMMARY OF THE INVENTION

The present invention provides methods for determining founder sequences from highly diverse virus populations. Also provided are determined founder sequences for highly diverse virus populations.

In one aspect, computational methods are provided for determining an ancestral viral sequence for highly diverse viruses, such as HIV-1, HIV-2 or Hepatitis C. These computational methods use samples of circulating viruses to determine an ancestral viral sequence by maximum likelihood phylogeny analysis. The ancestral viral sequence can be, for example, an HIV-1 ancestral viral gene sequence, an HIV-2 ancestral viral gene sequence, or a Hepatitis C ancestral viral gene sequence. In other embodiments, the ancestral viral gene sequence is of HIV-1 subtype A, B, C, D, E, F, G, H, J, AG, or AGI; HIV-1 Group M, N, or O; or HIV-2 subtype A or B. The ancestral viral gene sequence can be derived from widely dispersed HIV-1 variants, geographically-restricted HIV-1 variants, widely dispersed HIV-2 variants, or geographically-restricted HIV-2 variants. Typically, the ancestor gene is an env gene or a gag gene.

The ancestral viral gene sequence is more closely related, on average, to a gene sequence of any given circulating virus than to any other variant. In some embodiments, the ancestral viral gene sequence has at least 70% identity with the sequence set forth in SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:6, but does not have 100% identity with any circulating viral variant.

In another aspect, ancestral sequences for the env gene of HIV-1 subtype B are provided. HIV-1 subtype B gives rise to most infections in the Western Hemisphere and in Europe. The determined ancestral viral sequence is on average more closely related to any given circulating virus than to any other variant. The env ancestral gene sequence encodes an open reading frame for gp160, the gene product of env. In additional specific embodiments, the ancestral viral sequence has a gene sequence set forth in FIGS. 9 to 17 (designated "mrca").

In another aspect, ancestral sequences for the env gene of HIV-1 subtype C are provided. Subtype C is the most prevalent subtype worldwide. The determined founder sequence is, on average, more closely related to any given circulating virus than to any other variant. This sequence encodes an open reading frame for gp160, the gene product of env. In additional specific embodiments, the ancestral viral sequence has a gene sequence set forth in FIGS. 27 to 35 (designated "mrca").

Isolated HIV ancestor proteins or fragments thereof are also provided. The isolated ancestor protein can be, for example, the contiguous sequence of HIV-1, subtype B, env ancestor protein (SEQ ID NO:2) or HIV-1, subtype C, env ancestor protein (SEQ ID NO:4). The ancestor protein can also be of HIV-1 subtype A, B, C, D, E, F, G, H, J, AG, or AGI; HIV-1 Group M, N, or O; or HIV-2 subtype A or B. In additional specific embodiments, the ancestor protein can have a sequence set forth in FIGS. 18-26 or 36 to 44 (designated "mrca").

In another aspect, computational methods are provided for determining a COT viral sequence for highly diverse viruses, such as HIV-1, HIV-2 or Hepatitis C. These computational methods use samples of circulating viruses to determine a COT viral sequence by Least Squares or Minimum of Means methodologies. The COT viral sequence can be, for example, an HIV-1 COT viral gene sequence, an HIV-2 COT viral gene sequence, or a Hepatitis C COT viral gene sequence. In other embodiments, the COT viral gene sequence is of HIV-1 subtype A, B, C, D, E, F, G, H, J, AG, or AGI; HIV-1 Group M, N, or O; or HIV-2 subtype A or B. The COT viral gene sequence can be derived from widely dispersed HIV-1 variants, geographically-restricted HIV-1 variants, widely dispersed HIV-2 variants, or geographically-restricted HIV-2 variants. Typically, the COT viral gene is an env gene or a gag gene.

The COT viral gene sequence is more closely related, on average, to a gene sequence of any given circulating virus than to any other variant. In certain embodiments, the COT viral gene sequence has at least 70% identity with the LScot and MMcot sequences set forth in FIGS. 9 to 17 or 27 to 35, but does not have 100% identity with any circulating viral variant.

In another aspect, COT sequences for genes of HIV-1 subtype B are provided. HIV-1 subtype B gives rise to most infections in the Western Hemisphere and in Europe. The determined COT viral sequences are, on average, more closely related to any given circulating virus than to any other variant. In specific embodiments, the COT viral gene sequence is an LScot or MMcot sequence set forth in FIGS. 9 to 17, but does not have 100% identity with any circulating viral variant.

In another aspect, COT sequences for genes of HIV-1 subtype C are provided. Subtype C is the most prevalent subtype worldwide. The determined COT sequence is, on average, more closely related to any given circulating virus than to any other variant. In specific embodiments, the COT viral gene sequence is an LScot or MMcot sequence set forth in FIGS. 27 to 35, but does not have 100% identity with any circulating viral variant.

Isolated HIV COT proteins and or fragments thereof are also provided. The isolated ancestor protein can be, for example, an LScot or MMcot amino acid sequence set forth in FIGS. 27 to 25 and 36 to 44.

Also provided are computational methods for determining other ancestral or COT viral sequences. The computational methods can be extended, for example, to determine an ancestral or COT viral sequence for other HIV subtypes, such as, for example, HIV-1 subtype E, which is widely spread in developing countries. The computational methods can also be extended to determine an ancestral or COT viral sequence for all known and newly emerging highly diverse virus, such as, for example, HIV-1 strains, subtypes and groups. For example, ancestral or COT viral sequences can be determined for HIV-1-B in Thailand or Brazil, HIV-1-C in China, India, South Africa or Brazil, and the like. In other embodiments, the ancestral or COT viral sequence is determined for the HIV-1 nef gene or polypeptide, pol gene or polypeptide or other auxiliary genes or polypeptide (see infra).

The present invention also provides an expression construct including a transcriptional promoter; a nucleic acid encoding an ancestor protein; and a transcriptional terminator. The nucleic acid can encode, for example, an HIV-1 ancestor or COT protein (e.g., SEQ ID NO:2 or SEQ ID NO:4). The nucleic acid can be, for example, an HIV-1 subtype B or C env gene sequence (e.g., SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, or SEQ ID NO:6). In one embodiment, the nucleic acid sequence is optimized for expression in a host cell.

The promoter can be a heterologous promoter, such as the cytomegalovirus promoter. The expression construct can be expressed in prokaryotic or eukaryotic cells. Suitable cells include, for example, mammalian cells, human cells, Escherichia coli cells, and Saccharomyces cerevisiae cells. In one embodiment, the expression construct has the nucleic acid sequence operably linked to a Semliki Forest Virus replicon, wherein the resulting recombinant replicon is operably linked to a cytomegalovirus promoter.

In another aspect, compositions are provided for inducing an immune response in a mammal, the compositions include a viral ancestor protein or an immunogenic fragment of an ancestor or COT protein. The ancestor or COT protein can be derived from HIV-1 subtype B or C env ancestor or COT protein, or from other HIV-1, HIV-2 or Hepatitis C ancestor or COT proteins. In other aspects, the composition can be used as a vaccine, such as an AIDS vaccine to protect against infection by the highly diverse human immunodeficiency virus, type 1 (HIV-1), or for protection against HIV-2 or Hepatitis C infections. The ancestral or COT viral sequence can be an HIV-1 group determined founder (e.g., for Group M), for an HIV-1 subtype (e.g., B, C or E), for a widely spread variant, for a geographically-restricted variant, or for a newly emerging variant.

In another aspect, isolated antibodies are provided that bind specifically to a viral ancestor or COT protein and that bind specifically to a plurality of circulating descendant viral ancestor or COT proteins. The ancestor or COT protein can be from, for example, HIV-1, HIV-2, or Hepatitis C. The antibody can be a monoclonal antibody or antigen binding fragment thereof. In one embodiment, the antibody is a humanized monoclonal antibody. Other suitable antibodies or antigen binding fragments thereof can be a single chain antibody, a single heavy chain antibody, an antigen binding F(ab').sub.2 fragment, an antigen binding Fab' fragment, an antigen binding Fab fragment, or an antigen binding Fv fragment.

In addition to determining ancestral and COT viral sequences, the present invention also provides methods for preparing and testing immunogenic compositions based on an ancestral or COT viral sequence. In specific embodiments, immunogenic compositions (based on an ancestral or COT viral sequence) are prepared and administered to a mammal, employing an appropriate model, such as, for example, a mouse model or simian-human immunodeficiency virus (SHIV) macaque model. Immunogenic compositions can be prepared using an isolated ancestral or COT viral gene sequence, or polypeptide sequence, or a portion thereof.

In yet another aspect, diagnostic methods are provided to detect HIV and/or AIDS in a subject, using the nucleic acids, peptides or antibodies based on an ancestral or COT viral sequence.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a phylogenetic classification of HIV-1. The circled nodes approximate the ancestral state of the HIV-1 main group (Group M) and the main group clades A-G, J, AGI and AG.

FIG. 2 shows the phylogenetic relationship of HIV-1 subtype B and the placement of the determined subtype B ancestral node on that tree. The phylogenetic relationship of HIV-1 subtype D is shown as an outgroup.

FIG. 3 shows an ancestral viral sequence reconstruction of the most recent common ancestor using maximum likelihood reconstruction for an SIV inoculum up to three years after infection into macaques. The consensus sequence and the most recent common ancestor sequence were found to differ 1.5% in nucleotide sequence.

FIG. 4 provides an example of the development of a digital vaccine using an ancestral viral sequence.

FIG. 5 shows a comparison of a "most parsimonious reconstruction" methodology and a "maximum likelihood reconstruction methodology."

FIG. 6 shows another comparison of the "most parsimonious reconstruction" methodology and the "maximum likelihood reconstruction methodology." (SEQ ID NOS: 7-23).

FIG. 9 shows a comparison of the Most Recent Common Ancestor ("MRCA"), COT Least Squares ("LScot") and COT Minimum of Means ("MMcot") reconstructions for the Clade B gag gene (SEQ ID NOS: 24-26).

FIG. 10 shows a comparison of the Most Recent Common Ancestor ("MRCA"), COT Least Squares ("LScot") and COT Minimum of Means ("MMcot") reconstructions for the Clade B env (encoding gp160) (SEQ ID NOS: 27-28).

FIG. 11 shows a comparison of the Most Recent Common Ancestor ("MRCA"), COT Least Squares ("LScot") and COT Minimum of Means ("MMcot") reconstructions for the Clade B nef gene (SEQ ID NOS: 29-31).

FIG. 12 shows a comparison of the Most Recent Common Ancestor ("MRCA"), COT Least Squares ("LScot") and COT Minimum of Means ("MMcot") reconstructions for the Clade B pol gene (SEQ ID NOS: 32-34).

FIG. 13 shows a comparison of the Most Recent Common Ancestor ("MRCA"), COT Least Squares ("LScot") and COT Minimum of Means ("MMcot") reconstructions for the Clade B rev gene(SEQ ID NOS: 35-37).

FIG. 14 shows a comparison of the Most Recent Common Ancestor ("MRCA"), COT Least Squares ("LScot") and COT Minimum of Means ("MMcot") reconstructions for the Clade B tat gene (SEQ ID NOS: 38-40).

FIG. 15 shows a comparison of the Most Recent Common Ancestor ("MRCA"), COT Least Squares ("LScot") and COT Minimum of Means ("MMcot") reconstructions for the Clade B vif gene (SEQ ID NOS: 41-42).

FIG. 16 shows a comparison of the Most Recent Common Ancestor ("MRCA"), COT Least Squares ("LScot") and COT Minimum of Means ("MMcot") reconstructions for the Clade B vpr gene (SEQ ID NOS: 43-44).

FIG. 17 shows a comparison of the Most Recent Common Ancestor ("MRCA"), COT Least Squares ("LScot") and COT Minimum of Means ("MMcot") reconstructions for the Clade B vpu gene (SEQ ID NOS: 45-46).

FIG. 18 shows a comparison of the Most Recent Common Ancestor ("MRCA"), COT Least Squares ("LScot") and COT Minimum of Means ("MMcot") reconstructions for the Clade B gag protein (SEQ ID NOS: 47-49).

FIG. 19 shows a comparison of the Most Recent Common Ancestor ("MRCA"), COT Least Squares ("LScot") and COT Minimum of Means ("MMcot") reconstructions for the Clade B gp160 protein (SEQ ID NOS: 50-51).

FIG. 20 shows a comparison of the Most Recent Common Ancestor ("MRCA"), COT Least Squares ("LScot") and COT Minimum of Means ("MMcot") reconstructions for the Clade B nef protein (SEQ ID NOS: 52-54).

FIG. 21 shows a comparison of the Most Recent Common Ancestor ("MRCA"), COT Least Squares ("LScot") and COT Minimum of Means ("MMcot") reconstructions for the Clade B pol protein (SEQ ID NOS: 55-57).

FIG. 22 shows a comparison of the Most Recent Common Ancestor ("MRCA"), COT Least Squares ("LScot") and COT Minimum of Means ("MMcot") reconstructions for the Clade B rev protein (SEQ ID NOS: 58-60).

FIG. 23 shows a comparison of the Most Recent Common Ancestor ("MRCA"), COT Least Squares ("LScot") and COT Minimum of Means ("MMcot") reconstructions for the Clade B tat protein (SEQ ID NOS: 61-62).

FIG. 24 shows a comparison of the Most Recent Common Ancestor ("MRCA"),COT Least Squares ("LScot") and COT Minimum of Means ("MMcot") reconstructions for the Clade B vif protein (SEQ ID NOS: 63-64).

FIG. 25 shows a comparison of the Most Recent Common Ancestor ("MRCA"), COT Least Squares ("LScot") and COT Minimum of Means ("MMcot") reconstructions for the Clade B vpr protein (SEQ ID NOS: 65-67).

FIG. 26 shows a comparison of the Most Recent Common Ancestor ("MRCA"), COT Least Squares ("LScot") and COT Minimum of Means ("MMcot") reconstructions for the Clade B vpu protein (SEQ ID NOS: 68-69).

FIG. 27 shows a comparison of the Most Recent Common Ancestor ("MRCA"), COT Least Squares ("LScot") and COT Minimum of Means ("MMcot") reconstructions for the Clade C gag gene (SEQ ID NOS: 70-72).

FIG. 28 shows a comparison of the Most Recent Common Ancestor ("MRCA"), COT Least Squares ("LScot") and COT Minimum of Means ("MMcot") reconstructions for the Clade C env (encoding gp160) (SEQ ID NOS: 73-75).

FIG. 29 shows a comparison of the Most Recent Common Ancestor ("MRCA"), COT Least Squares ("LScot") and COT Minimum of Means ("MMcot") reconstructions for the Clade C nef gene (SEQ ID NOS: 76-77).

FIG. 30 shows a comparison of the Most Recent Common Ancestor ("MRCA"), COT Least Squares ("LScot") and COT Minimum of Means ("MMcot") reconstructions for the Clade C pol gene (SEQ ID NOS: 78-80).

FIG. 31 shows a comparison of the Most Recent Common Ancestor ("MRCA"), COT Least Squares ("LScot") and COT Minimum of Means ("MMcot") reconstructions for the Clade C rev gene (SEQ ID NOS: 81-83).

FIG. 32 shows a comparison of the Most Recent Common Ancestor ("MRCA"), COT Least Squares ("LScot") and COT Minimum of Means ("MMcot") reconstructions for the Clade C tat gene (SEQ ID NOS: 84-86).

FIG. 33 shows a comparison of the Most Recent Common Ancestor ("MRCA"), COT Least Squares ("LScot") and COT Minimum of Means ("MMcot") reconstructions for the Clade C vif gene (SEQ ID NOS: 87-89).

FIG. 34 shows a comparison of the Most Recent Common Ancestor ("MRCA"), COT Least Squares ("LScot") and COT Minimum of Means ("MMcot") reconstructions for the Clade C vpr gene (SEQ ID NOS: 90-92).

FIG. 35 shows a comparison of the Most Recent Common Ancestor ("MRCA"), COT Least Squares ("LScot") and COT Minimum of Means ("MMcot") reconstructions for the Clade C vpu gene (SEQ ID NOS: 93-95).

FIG. 36 shows a comparison of the Most Recent Common Ancestor ("MRCA"), COT Least Squares ("LScot") and COT Minimum of Means ("MMcot") reconstructions for the Clade C gag protein (SEQ ID NOS: 96-90).

FIG. 37 shows a comparison of the Most Recent Common Ancestor ("MRCA"), COT Least Squares ("LScot") and COT Minimum of Means ("MMcot") reconstructions for the Clade C gp160 protein (SEQ ID NOS: 99-101).

FIG. 38 shows a comparison of the Most Recent Common Ancestor ("MRCA"), COT Least Squares ("LScot") and COT Minimum of Means ("MMcot") reconstructions for the Clade C nef protein (SEQ ID NOS: 102-103).

FIG. 39 shows a comparison of the Most Recent Common Ancestor ("MRCA"), COT Least Squares ("LScot") and COT Minimum of Means ("MMcot") reconstructions for the Clade C pol protein (SEQ ID NOS: 104-106).

FIG. 40 shows a comparison of the Most Recent Common Ancestor ("MRCA"), COT Least Squares ("LScot") and COT Minimum of Means ("MMcot") reconstructions for the Clade C rev protein (SEQ ID NOS: 107-109).

FIG. 41 shows a comparison of the Most Recent Common Ancestor ("MRCA"), COT Least Squares ("LScot") and COT Minimum of Means ("MMcot") reconstructions for the Clade C tat protein (SEQ ID NOS: 110-111).

FIG. 42 shows a comparison of the Most Recent Common Ancestor ("MRCA"), COT Least Squares ("LScot") and COT Minimum of Means ("MMcot") reconstructions for the Clade C vif protein (SEQ ID NOS: 112-114).

FIG. 43 shows a comparison of the Most Recent Common Ancestor ("MRCA"), COT Least Squares ("LScot") and COT Minimum of Means ("MMcot") reconstructions for the Clade C vpr protein (SEQ ID NOS: 115-117).

FIG. 44 shows a comparison of the Most Recent Common Ancestor ("MRCA"), COT Least Squares ("LScot") and COT Minimum of Means ("MMcot") reconstructions for the Clade C vpu protein (SEQ ID NOS: 118-119).

FIG. 45. Phylogenetic relationships of different phylogenetic structures and between HIV-1 group M, Subtype B gp160 sequences. A. thirty-eight Subtype B sequences and three Subtype D (outgroup) sequences used to root the Subtype B sequences (see Table 11). The Subtype B sequences were from nine countries, representing a broad sample of Subtype B diversity: Australia, 8; China, 1; France, 6; Gabon, 1; Germany, 2; Great Britain, 2; The Netherlands, 2; Spain, 1; USA, 15). B. Idealized phylogenetic trees with caterpillar (left) and star (right) shapes.

FIG. 46. Deduced ancestor protein sequences SIVBK28 ancestor (Env segment) and AN1-EnvB (SEQ ID NOS: 120-121).

DESCRIPTION OF THE SPECIFIC EMBODIMENTS

The present invention provides methods for determining founder sequences from highly diverse virus populations. Also provided are determined founder sequences for highly diverse virus populations.

Prior to setting forth the invention in more detail, it may be helpful to a further understanding thereof to set forth definitions of certain terms as used hereinafter.

Definitions

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Although any methods and materials similar to those described herein can be used in the practice or testing of the present invention, only exemplary methods and materials are described. For purposes of the present invention, the following terms are defined below.

An "ancestral sequence" refers to a determined founder sequence, determined through application of maximum likelihood phylogenetic analysis. An ancestral sequence is typically one that is more closely related, on average, to any given variant than to any other variant. An "ancestral viral sequence" refers to a determined founder sequence, typically one that is more closely related, on average, to any given circulating virus than to any other variant. An "ancestral viral sequence" is determined through application of maximum likelihood phylogenetic analysis using the nucleic acid and/or amino acid sequences of circulating viruses. An "ancestor virus" is a virus comprising the "ancestral viral sequence." An "ancestor protein" is a protein, polypeptide or peptide having an amino acid ancestral viral sequence.

A "COT sequence" refers to a determined founder sequence, determined through application of a COT Least Squares Method or a COT Minimum of Means Method. A "COT sequence" is a position at a node or on a branch of a phylogenetic tree having completely specified branch lengths. A "COT viral sequence" refers to a founder nucleic acid sequence determined by COT Least Squares Method or a COT Minimum of Means Method, using the nucleic acid and/or amino acid sequences of circulating viruses. An "COT virus" is a virus comprising the "COT viral sequence." A "COT viral protein" or "COT protein" is a protein, polypeptide or peptide having an amino acid COT viral sequence.

The term "circulating virus" refers to virus found in an infected individual.

The term "variant" refers to a virus, gene or gene product that differs in sequence from other viruses, genes or gene products by one or more nucleotide or amino acids.

The terms "immunological" or "immune response" refer to the development of a beneficial humoral (i.e., antibody mediated) and/or a cellular (i.e., mediated by antigen-specific T-cells or their secretion products) response directed against an HIV peptide in a recipient subject. Such a response can be, in particular, an active response induced by the administration of an immunogen. A cellular immune response is elicited by the presentation of epitopes in association with Class I or Class II MHC molecules to activate antigen-specific CD4.sup.+T helper cells (i.e., Helper T lymphocytes) and/or CD8.sup.+cytotoxic T cells. The presence of a cell-mediated immunological response can be determined by, for example, proliferation assays of CD4.sup.+T cells (i.e., measuring the HTL (Helper T lymphocyte) response) or by CTL (cytotoxic T lymphocyte) assays (see, e.g., Burke et al., J. Inf. Dis. 170:1110-19 (1994); Tigges et al., J. Immunol. 156:3901-10 (1996)). The relative contributions of humoral and cellular responses to the protective or therapeutic effect of an immunogen can be distinguished by separately isolating IgG and T-cells from an immunized syngeneic animal and measuring protective or therapeutic effects in a second subject. For example, the effector cells can be deleted and the resulting response analyzed (see, e.g., Schmitz et al., Science 283:857-60 (1999); Jin et al., J Exp. Med. 189:991-98 (1999)).

"Antibody" refers to a polypeptide substantially encoded by an immunoglobulin gene or immunoglobulin genes, or fragments thereof, that specifically bind and recognize an analyte (antigen). The recognized immunoglobulin genes include the kappa, lambda, alpha, gamma, delta, epsilon and mu constant region genes, as well as the myriad immunoglobulin variable region genes. Light chains are classified as either kappa or lambda. Heavy chains are classified as gamma, mu, alpha, delta, or epsilon, which in turn define the immunoglobulin classes, IgG, IgM, IgA, IgD and IgE, respectively.

An exemplary immunoglobulin (antibody) structural unit comprises a tetramer. Each tetramer is composed of two identical pairs of polypeptide chains, each pair having one "light" (about 25 kD) and one "heavy" chain (about 50-70 kD). The N-terminus of each chain has a variable region of about 100 to 110 or more amino acids primarily responsible for antigen recognition. The terms variable light chain (VL) and variable heavy chain (VH) refer to these light and heavy chains, respectively.

Antibodies exist, for example, as intact immunoglobulins or as a number of well characterized antigen-binding fragments produced by digestion with various peptidases. For example, pepsin digests an antibody below the disulfide linkages in the hinge region to produce an F(ab').sub.2 fragment, a dimer of Fab which itself is a light chain joined to VH-CH1 by a disulfide bond. The F(ab').sub.2 fragment can be reduced under mild conditions to break the disulfide linkage in the hinge region, thereby converting the F(ab').sub.2 dimer into an Fab' monomer. The Fab' monomer is essentially an Fab with part of the hinge region (see, Fundamental Immunology, Third Edition, W. E. Paul (ed.), Raven Press, N.Y. (1993)). While various antibody fragments are defined in terms of the digestion of an intact antibody, one of skill will appreciate that such fragments can be synthesized de novo either chemically or by utilizing recombinant DNA methodology. Thus, the term antibody, as used herein, also includes antibody fragments, such as a single chain antibody, an antigen binding F(ab').sub.2 fragment, an antigen binding Fab' fragment, an antigen binding Fab fragment, an antigen binding Fv fragment, a single heavy chain or a chimeric antibody. Such antibodies can be produced by the modification of whole antibodies or synthesized de novo using recombinant DNA methodologies.

The term "biological sample" refers to any tissue or liquid sample having genomic or viral DNA or other nucleic acids (e.g., mRNA, viral RNA, or the like) or proteins. "Biological sample" further includes fluids, such as serum and plasma, that contain cell-free virus, and also includes both normal healthy cells and cells suspected of HIV infection.

The term "nucleic acid" refers to deoxyribonucleotides or ribonucleotides and polymers thereof in either single or double stranded form. Unless specifically limited, the term encompasses nucleic acids containing known analogues of natural nucleotides that have similar binding properties as the reference nucleic acid. Unless otherwise indicated, a particular nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (e.g., degenerate codon substitutions) and complementary sequences as well as the sequence explicitly indicated. Specifically, degenerate codon substitutions can be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed-base and/or deoxyinosine residues (see, e.g., Batzer et al., Nucleic Acid Res. 19:5081 (1991); Ohtsuka et al., J. Biol. Chem. 260:2605-08 (1985); Rossolini et al., Mol. Cell. Probes 8:91-98 (1994)). Nucleic acids also include fragments of at least 10 contiguous nucleotides (e.g., a hybridizable portion); in other embodiments, the nucleic acids comprise at least 25 nucleotides, 50 nucleotides, 100 nucleotides, 150 nucleotides, 200 nucleotides, or even up to 250 nucleotides or more. The term "nucleic acid" is used interchangeably with gene, cDNA, and mRNA encoded by a gene.

As used herein a "nucleic acid probe" is defined as a nucleic acid capable of binding to a target nucleic acid (e.g., an HIV-1 nucleic acid) of complementary sequence through one or more types of chemical bonds, usually through complementary base pairing, such as by hydrogen bond formation. As used herein, a probe may include natural (e.g., A, G, C, or T) or modified bases (e.g., 7-deazaguanosine, inosine, or the like). In addition, the bases in a probe can be joined by a linkage other than a phosphodiester bond, so long as it does not interfere with hybridization. Thus, for example, probes can be peptide nucleic acids in which the constituent bases are joined by peptide bonds rather than phosphodiester linkages. It will be understood by one of skill in the art that probes can bind target sequences lacking complete complementarity with the probe sequence, at levels that depend upon the stringency of the hybridization conditions.

Nucleic acid probes can be DNA or RNA fragments. DNA fragments can be prepared, for example, by digesting plasmid DNA, by use of PCR, or by chemical synthesis, such as by the phosphoramidite method described by Beaucage and Carruthers (Tetrahedron Lett. 22:1859-62 (1981)), or by the triester method according to Matteucci et al. (J. Am. Chem. Soc. 103:3185 (1981)). A double stranded fragment can then be obtained, if desired, by annealing the chemically synthesized single strands together under appropriate conditions, or by synthesizing the complementary strand using DNA polymerase with an appropriate primer sequence. Where a specific sequence for a nucleic acid probe is given, it is understood that the complementary strand is also identified and included. The complementary strand will work equally well in situations where the target is a double stranded nucleic acid.

A "labeled nucleic acid probe" is a nucleic acid probe that is bound, either covalently, through a linker, or through ionic, van der Waals or hydrogen bonds, to a label such that the presence of the probe can be detected by detecting the presence of the label bound to the probe.

The term "operably linked" refers to functional linkage between a nucleic acid expression control sequence (such as a promoter, signal sequence, or any of an array of transcription factor binding sites) and a second nucleic acid sequence, wherein the expression control sequence affects transcription and/or translation of the nucleic acid corresponding to the second sequence.

"Amplification primers" are nucleic acids, typically oligonucleotides, comprising either natural or analog nucleotides that can serve as the basis for the amplification of a selected nucleic acid sequence. They include, for example, both polymerase chain reaction primers and ligase chain reaction oligonucleotides.

The terms "polypeptide," "peptide" and "protein" are used interchangeably herein to refer to a polymer of amino acid residues. The terms apply to amino acid polymers in which one or more amino acid residue is an artificial chemical mimetic of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers and non-naturally occurring amino acid polymers.

The terms "amino acid" or "amino acid residue", as used herein, refer to naturally occurring L-amino acids or to D-amino acids as described further below. The commonly used one- and three-letter abbreviations for amino acids are used herein (see, e.g., Alberts et al., Molecular Biology of the Cell, Garland Publishing, Inc., New York (3d ed. 1994); Creighton, Proteins, W.H. Freeman and Company (1984)).

A "conservative substitution," when describing a protein, refers to a change in the amino acid composition of the protein that is less likely to substantially alter the protein's activity. Thus, "conservatively modified variations" of a particular amino acid sequence refers to amino acid substitutions of those amino acids that are less likely to be critical for protein activity or substitution of amino acids with other amino acids having similar properties (e.g., acidic, basic, positively or negatively charged, polar or non-polar, or the like) such that the substitutions of even critical amino acids do not substantially alter activity. Conservative substitution tables providing amino acids that are often functionally similar are well known in the art (see, e.g., Creighton, Proteins, W.H. Freeman and Company (1984)). In addition, individual substitutions, deletions or additions which alter, add or delete a single amino acid or a small percentage of amino acids in an encoded sequence are also "conservatively modified variations."

The terms "identical" or "percent identity," in the context of two or more nucleic acids or polypeptide sequences, refer to two or more sequences or subsequences that are the same or have a specified percentage of amino acid residues or nucleotides that are the same (i.e., 60% identity, optionally 65%, 70%, 75%, 80%, 85%, 90%, or 95% identity over a specified region), when compared and aligned for maximum correspondence over a comparison window, or designated region, as measured using one of the following sequence comparison algorithms or by manual alignment and visual inspection. Such sequences are then said to be "substantially identical." This definition also refers to the complement of a test sequence. Optionally, the identity exists over a region that is at least about 30 amino acids or nucleotides in length, typically over a region that is 50, 75 or 150 amino acids or nucleotides. In certain embodiments, the sequences are substantially identical over the entire length of the coding regions.

The terms "similarity," or "percent similarity," in the context of two or more polypeptide sequences, refer to two or more sequences or subsequences that have a specified percentage of amino acid residues that are either the same or similar as defined in the conservative amino acid substitutions defined above (i.e., at least 60%, optionally 65%, 70%, 75%, 80%, 85%, 90%, or 95% similar over a specified region), when compared and aligned for maximum correspondence over a comparison window, or designated region as measured using one of the following sequence comparison algorithms or by manual alignment and visual inspection. Such sequences are then said to be "substantially similar." Optionally, this identity exists over a region that is at least about 25 amino acids in length, or more typically over a region that is at least about 50, 75 or 100 amino acids in length.

For sequence comparison, typically one sequence acts as a reference sequence to which test sequences are compared. When using a sequence comparison algorithm, test and reference sequences are typically input into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. The sequence comparison algorithm then calculates the percent sequence identity for the test sequence(s) relative to the reference sequence, based on the designated program parameters.

Optimal alignment of sequences for comparison can be conducted, for example, by the local homology algorithm of Smith and Waterman (Adv. Appl. Math. 2:482 (1981)), by the homology alignment algorithm of Needleman and Wunsch (J. Mol. Biol. 48:443 (1970)), by the search for identity method of Pearson and Lipman (Proc. Natl. Acad Sci. USA 85:2444 (1988)), by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, Wis.), or by visual inspection (see, generally Ausubel et al., Current Protocols in Molecular Biology, John Wiley and Sons, New York (1996)).

One example of a useful algorithm is PILEUP. PILEUP creates a multiple sequence alignment from a group of related sequences using progressive, pairwise alignments to show relationship and percent sequence identity. It also plots a tree or dendogram showing the clustering relationships used to create the alignment. PILEUP uses a simplification of the progressive alignment method of Feng and Doolittle (J. Mol. Evol. 35:351-60 (1987)). The method used is similar to the CLUSTAL method described by Higgins and Sharp (Gene 73:237-44 (1988); CABIOS 5:151-53 (1989)). The program can align up to 300 sequences, each of a maximum length of 5,000 nucleotides or amino acids. The multiple alignment procedure begins with the pairwise alignment of the two most similar sequences, producing a cluster of two aligned sequences. This cluster is then aligned to the next most related sequence or cluster of aligned sequences. Two clusters of sequences are aligned by a simple extension of the pairwise alignment of two individual sequences. The final alignment is achieved by a series of progressive, pairwise alignments. The program is run by designating specific sequences and their amino acid or nucleotide coordinates for regions of sequence comparison and by designating the program parameters. For example, a reference sequence can be compared to other test sequences to determine the percent sequence identity relationship using the following parameters: default gap weight (3.00), default gap length weight (0.10), and weighted end gaps.

Another example of an algorithm that is suitable for determining percent sequence identity and sequence similarity is the BLAST algorithm, which is described in Altschul et al. (J. Mol. Biol. 215:403-10 (1990)). Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov/). This algorithm involves first identifying high scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence, which either match or satisfy some positive-valued threshold score T when aligned with a word of the same length in a database sequence. T is referred to as the neighborhood word score threshold (Altschul et al., supra). These initial neighborhood word hits act as seeds for initiating searches to find longer HSPs containing them. The word hits are then extended in both directions along each sequence for as far as the cumulative alignment score can be increased. Cumulative scores are calculated using, for nucleotide sequences, the parameters M (reward score for a pair of matching residues; always >0) and N (penalty score for mismatching residues; always <0). For amino acid sequences, a scoring matrix is used to calculate the cumulative score. Extension of the word hits in each direction are halted when: the cumulative alignment score falls off by the quantity X from its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation of one or more negative-scoring residue alignments; or the end of either sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) uses as defaults a wordlength (W) of 11, an expectation (E) of 10, a cutoff of 100, M=5, N=-4, and a comparison of both strands. For amino acid sequences, the BLASTP program uses as defaults a wordlength (W) of 3, an expectation (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff and Henikoff, Proc. Natl. Acad. Sci. USA 89:10915 (1989)).

In addition to calculating percent sequence identity, the BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, e.g., Karlin and Altschul, Proc. Natl. Acad. Sci. USA 90:5873-87 (1993)). One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability by which a match between two nucleotide or amino acid sequences would occur by chance. For example, a nucleic acid is considered similar to a reference sequence if the smallest sum probability in a comparison of the test nucleic acid to the reference nucleic acid is typically between about 0.35 and about 0.1. Another indication that two nucleic acids are substantially identical is that the two molecules hybridize to each other under stringent conditions. The phrase "hybridizing specifically to" refers to the binding, duplexing, or hybridizing of a molecule only to a particular nucleotide sequence under stringent conditions when that sequence is present in a complex mixture (e.g., total cellular) DNA or RNA. "Bind(s) substantially" refers to complementary hybridization between a probe nucleic acid and a target nucleic acid and embraces minor mismatches that can be accommodated by reducing the stringency of the hybridization media to achieve the desired detection of the target polynucleotide sequence.

"Stringent hybridization conditions" and "stringent hybridization wash conditions" in the context of nucleic acid hybridization experiments, such as Southern and northern hybridizations, are sequence-dependent, and are different under different environmental parameters. Longer sequences hybridize specifically at higher temperatures. An extensive guide to the hybridization of nucleic acids is found in Tijssen, Laboratory Techniques in Biochemistry and Molecular Biology--Hybridization with Nucleic Acid Probes, part I, chapter 2 "Overview of principles of hybridization and the strategy of nucleic acid probe assays," Elsevier, N.Y. (1993). Generally, highly stringent hybridization and wash conditions are selected to be about 5.degree. C. lower than the thermal melting point (T.sub.m) for the specific sequence at a defined ionic strength and pH. Typically, under "stringent conditions," a probe will hybridize to its target subsequence, but to no other sequences.

The T.sub.m is the temperature (under defined ionic strength and pH) at which 50% of the target sequence hybridizes to a perfectly matched probe. Very stringent conditions are selected to be equal to the T.sub.m for a particular probe. An example of stringent hybridization conditions for hybridization of complementary nucleic acids which have more than 100 complementary residues on a filter in a Southern or northern blot is 50% formamide in 4-6.times.SSC or SSPE at 42.degree. C., or 65-68.degree. C. in aqueous solution containing 4-6.times.SSC or SSPE. An example of highly stringent wash conditions is 0.15 M NaCl at 72.degree. C. for about 15 minutes. An example of stringent wash conditions is a 0.2.times.SSC wash at 65.degree. C. for 15 minutes. (See generally Sambrook et al., Molecular Cloning, A Laboratory Manual, 2nd ed., Cold Spring Harbor Publish., Cold Spring Harbor, N.Y. (1989)). Often, a high stringency wash is preceded by a low stringency wash to remove background probe signal. An example of medium stringency wash for a duplex of, for example, more than 100 nucleotides, is 1.times.SSC at 45.degree. C. for 15 minutes. An example of low stringency wash for a duplex of, for example, more than 100 nucleotides, is 4-6.times.SSC at 40.degree. C. for 15 minutes. For short probes (e.g., about 10 to 50 nucleotides), stringent conditions typically involve salt concentrations of less than about 1.0 M Na ion, typically about 0.01 to 1.0 M Na ion concentration (or other salts) at pH 7.0 to 8.3, and the temperature is typically at least about 30.degree. C. Stringent conditions can also be achieved with the addition of destabilizing agents such as formamide. In general, a signal to noise ratio of 2.times. (or higher) than that observed for an unrelated probe in the particular hybridization assay indicates detection of a specific hybridization. Nucleic acids that do not hybridize to each other under stringent conditions are still substantially identical if the polypeptides which they encode are substantially identical. This occurs, for example, when a copy of a nucleic acid is created using the maximum codon degeneracy permitted by the genetic code.

A further indication that two nucleic acids or polypeptides are substantially identical is that the polypeptide encoded by the first nucleic acid is immunologically cross reactive with, or specifically binds to, antibodies raised against the polypeptide encoded by the second nucleic acid. Thus, a polypeptide is typically substantially identical to a second polypeptide, for example, where the two peptides differ only by conservative substitutions.

The phrase "specifically (or selectively) binds to an antibody" or "specifically (or selectively) immunoreactive with", when referring to a protein or peptide, refers to a binding reaction which is determinative of the presence of the protein in the presence of a heterogeneous population of proteins and other biologics. Thus, under designated immunoassay conditions, the specified antibodies bind to a particular protein and do not bind in a significant amount to other proteins present in the sample. Specific binding to a protein under such conditions may require an antibody that is selected for its specificity for the particular protein. For example, antibodies raised to the protein with the amino acid sequence encoded by any of the nucleic acids of the invention can be selected to obtain antibodies specifically immunoreactive with that protein and not with other proteins except for polymorphic variants. A variety of immunoassay formats can be used to select antibodies specifically immunoreactive with a particular protein. For example, solid-phase ELISA immunoassays, Western blots, or immunohistochemistry are routinely used to select monoclonal antibodies specifically immunoreactive with a protein (see, e.g., Harlow and Lane, Antibodies, A Laboratory Manual, Cold Spring Harbor Publications, N.Y. (1988), for a description of immunoassay formats and conditions that can be used to determine specific immunoreactivity). Typically, a specific or selective reaction will be at least twice background signal or noise and more typically more than 10 to 100 times background.

The term "immunogenic composition" refers to a composition that elicits an immune response which produces antibodies or cell-mediated immune responses against a specific immunogen. Immunogenic compositions can be prepared as injectables, as liquid solutions, suspensions, emulsions, and the like.

The term "vaccine" refers to an immunogenic composition for in vivo administration to a host, which may be a primate, particularly a human host, to confer protection against disease, particularly a viral disease.

The term "isolated" refers to a virus, nucleic acid or polypeptide that has been removed from its natural cellular environment. An isolated virus, nucleic acid or polypeptide is typically at least partially purified from cellular nucleic acids, polypeptides and other constituents.

In the context of the present invention, a "Coalescent Event" refers to the joining of two lineages on a genealogy at the point of their most recent common ancestor.

A "Coalescent Interval" describes the time between coalescent events. The expected time for each coalescent interval is exponentially distributed with mean E[.sub.t nyn-1]=2N/n(n-1) generations for n<<N.

Determination of Ancestral Sequences

In one aspect, computational methods are provided for determining ancestral sequences. Such methods can be used, for example, to determine ancestral sequences for viruses. These computational methods are typically used to determine an ancestral sequence of a virus that exists as a highly diverse viral population. For example, some highly diverse viruses (including HIV-1, HIV-2, Hepatitis C, and the like) do not appear to evolve through a succession of variants, where one prototypical strain is replaced by successive uniform strains. Instead, an evolutionary tree of viral sequences can form a "star-burst pattern," with most of the variants approximately equidistant from the center of the star-burst. This star-burst pattern indicates that multiple, diverse circulating strains evolve from a common ancestor. The computational methods can be used to determine ancestral sequences for such highly diverse viruses, such as, for example, HIV-1, HIV-2, Hepatitis C, and other viruses.

Methods for determining ancestral sequences are typically based on the nucleic acid sequences of circulating viruses. As a viral nucleic acid sequence is replicated, it acquires base changes due to errors in the replication process. For example, as some nucleic acid sequences are replicated, thymine (T) might bind to a guanine (G) rather than its normal complement, cytosine (C). Most of these base changes (or mutations) are not reproduced in subsequent replication events, but a certain proportion of mutations are passed down to the descendant sequences. With more replication cycles, nucleic acid sequences acquire more mutations. If a nucleic acid sequence bearing one or more mutations gives rise to two separate lineages, then the resulting two lineages will share the same parental nucleic acid sequence, and have the same parental mutation(s). If the "histories" of these lineages are traced backwards, they will have a common branch point, at which the two lineages arose from a common ancestor. Similarly, if the histories of presently circulating viral nucleic acid sequences are traced backwards, the branching points in these histories also correspond to points, designated as nodes, at which a single ancestor gave rise to the descendant lineages.

The present computational methods are based on the principle of maximum likelihood and use samples of nucleic acid sequences of circulating viruses. The sequences of the viruses in the samples typically share a common feature, such as being from the same viral strain, subtype or group. A phylogeny is constructed by using a model of evolution that specifies the probabilities of nucleotide substitutions in the replicating viral nucleic acids. At positions in the sequences where the nucleotides differ (i.e., at the site of a mutation), the methodology assigns one of the nucleotides to the node (i.e., the branch point of the lineages) such that the probability of obtaining the observed viral sequences is maximized. The assignment of nucleotides to the nodes is based on the predicted phylogeny or phylogenies. For each data set, several sequences from a different viral strain, subtype or group are used as an outgroup to root the sequences of interest. A model of sequence substitutions and then a maximum likelihood phylogeny are determined for each data set (e.g., subtype and outgroup). The maximum likelihood phylogeny the one that has the highest probability of giving the observed nucleic acid sequences in the samples. The sequence at the base node of the maximum likelihood phylogeny is referred to as the ancestral sequence (or most recent common ancestor). (See, e.g., FIGS. 1 and 2). This ancestral sequence is thus approximately equidistant from the different sequences within the samples.

Maximum likelihood phylogeny uses samples of the sequences of circulating virus. The sequences of circulating viruses can be determined, for example, by extracting nucleic acids from blood, tissues or other biological samples of virally infected persons and sequencing the viral nucleic acids. (See, e.g., Sambrook et al., Molecular Cloning, A Laboratory Manual, 2nd ed., Cold Spring Harbor Publish., Cold Spring Harbor, N.Y. (1989); Kriegler, Gene Transfer and Expression: A Laboratory Manual, W.H. Freeman, N.Y. (1990); Ausubel et al., supra.) In one embodiment, extracted viral nucleic acids can be amplified by polymerase chain reaction, and then DNA sequenced. Samples of circulating virus can be obtained from stored biological samples and/or prospectively from samples of circulating virus (e.g., sampling HIV-1 subtype C in India versus Ethiopia). Viral sequences can also be identified from databases (e.g., GenBank and Los Alamos sequence databases).

Once samples of circulating viruses are collected (typically about 20 to about 50 samples), the nucleic acid sequences for one or more genes are analyzed using the computational methods according to the present invention. In one method, for any given site in the sequence, the nucleotides at all nodes on a tree are assigned. The configuration of the nucleotides for all nodes that maximizes the probability of obtaining the observed sequences of circulating viruses is determined. With this method, the joint likelihood of the states across all nodes is maximized.

A second method is to choose, for a given nucleotide site and a given node on the tree, the nucleotide that maximizes the probability of obtaining the observed sequences of circulating viruses, allowing for all possible assignments of nucleotides at the other nodes on the tree. This second method maximizes the marginal likelihood of a particular assignment. For these methods, the reconstruction of the ancestral sequence (i.e., ancestral state) need not result in only a single determined sequence, however. It is possible to choose a number of ancestral sequences, ranked in order of their likelihood.

With HIV populations, a second layer of modeling can be added to the maximum likelihood phylogenetic analysis, in particular the layer is added to the model of evolution that is employed in the analysis. This second layer is based on coalescent likelihood analysis. The coalescent is a mathematical description of a genealogy of sequences, taking account of the processes that act on the population. If these processes are known with some certainty, the use of the coalescent can be used to assign prior probabilities to each type of tree. Taken together with the likelihood of the tree, the posterior probability can be determined that a determined phylogenetic tree is correct given the data. Once a tree is chosen, the ancestral states are determined, as described above. Thus, coalescent likelihood analysis can also be applied to determine the sequence of an ancestral viral sequence (e.g., a founder, or Most Recent Common Ancestor (MRCA), sequence).

In a typical embodiment, maximum likelihood phylogeny analysis is applied to determine an ancestor sequence (e.g., an ancestral viral sequence). Typically, between 20 and 50 nucleic acid sequence samples are used that have a common feature, such as a viral strain, subtype or group (e.g., samples encompassing a worldwide diversity of the same subtype). Additional sequences from other viruses (e.g., another strain, subtype, or group) are obtained and used as an outgroup to root the viral sequences being analyzed. The samples of viral sequences are determined from presently circulating viruses, identified from the database (e.g., GenBank and Los Alamos sequence databases), or from similar sources of sequence information. The sequences are aligned using CLUSTALW (Thompson et al., Nucleic Acids Res. 22:4673-80 (1994), the disclosure of which is incorporated by reference herein) and these alignments are refined using GDE (Smith et al., CABIOS 10:671-75 (1994) the disclosure of which is incorporated by reference herein). The amino acid sequences are also translated from the nucleic acid sequences. Gaps are manipulated so that they are inserted between codons. This alignment (alignment I) is modified for phylogenetic analysis so that regions that can not be unambiguously aligned are removed (Learn et al., J. Virol. 70:5720-30 (1996), the disclosure of which is incorporated by reference herein) resulting in alignment II.

An appropriate evolutionary model for phylogeny and ancestral state reconstructions for these sequences (alignment II) is selected using the Akaike Information Criterion (AIC) (Akaike, IEEE Trans. Autom. Contr. 19:716-23 (1974); which is incorporated by reference herein) as implemented in Modeltest 3.0 (Posada and Crandall, Bioinformatics 14:817-8 (1998), which is incorporated by reference herein). For example, for the analysis for the subtype C ancestral sequence the optimal model is equal rates for both classes of transitions and different rates for all four classes of transversions, with invariable sites and a X distribution of site-to-site rate variability of variable sites (referred to as a TVM+I+G model). The parameters of the model in this case can be, for example, equilibrium nucleotide frequencies: f.sub.A=0.3576,f.sub.C=0.1829,f.sub.G=0.2314,f.sub.T=0.2290; proportion of invariable sites=0.2447; shape parameter (.alpha.) of the X distribution=0.7623; rate matrix (R) matrix values: R.sub.A.fwdarw.C=1.7502, R.sub.A.fwdarw.G=R.sub.C.fwdarw.T=4.1332, R.sub.A.fwdarw.T=0.6825, R.sub.C.fwdarw.G=0.6549, R.sub.G.fwdarw.T=1.

Evolutionary trees for the sequences (alignment II) are inferred using maximum likelihood estimation (MLE) methods as implemented in PAUP* version 4.0b (Swofford, PAUP 4.0: Phylogenetic Analysis Using Parsimony (And Other Methods); Sinauer Associates, Inc. (2000) the disclosure of which is incorporated by reference herein). For example, for HIV-1 subtype C sequences, ten different subtree-pruning-regrafting (SPR) heuristic searches can be performed, each using a different random addition order. The ancestral viral nucleotide sequence is determined to be the sequence at the basal node using the phylogeny, the sequences from the databases (alignment II), and the TVM+I+G model above using marginal likelihood estimation (see infra).

In some cases, the determined sequence may not include ancestral sequence for portions of variable regions (e.g., variable regions V1, V2, V4 and V5 for HIV-1-C), and or some short regions may not be unambiguously aligned. The following procedure can optionally be used to predict amino acid sequences for the complete sequence, including the highly variable regions (such as those deleted from alignment I). The determined ancestral sequence is visually aligned to alignment I and translated using GDE (Smith et al., supra). Since the highly variable regions can be deleted as complete codons, the translational reading frame can be preserved and codons can be maintained. The ancestral amino acid sequence for the regions deleted from alignment II can be predicted visually and refined using a parsimony-based sequence reconstruction for these sites using the computer program MacClade, version 3.08a (Maddison and Maddison. MacClade--Analysis of Phylogeny and Character Evolution--Version 3. Sinauer Associates, Inc. (1992)).

The ancestral amino acid sequence is optionally optimized for expression in a particular cell type. Amino acid sequences can converted to a DNA sequence optimized for expression in certain cell types (e.g., human cells) using, for example, the BACKTRANSLATE program of the Wisconsin Sequence Analysis Package (GCG), version 10 and a human gene codon table from the Codon Usage Database (www.kazusa.or.jp/codon/cgi-bin/showcodon.cgi?species=Homo+sapiens+[gbpri- ]), both incorporated by reference herein.

The optimized sequences encode the same amino acid sequence for the gene of interest (e.g., the env gene) as the non-optimized ancestral sequence. A synthetic virus having the optimized sequence may not be fully functional due to the disruption of auxiliary genes in different reading frames the presence of RNA secondary structural feature (e.g., the Rev responsive element (RRE) of HIV-1), and the like. The optimization process may affect the coding region of the auxiliary genes (e.g., vpu, tat and rev genes of HIV-1), and may disrupt RNA secondary structure. Thus, the ancestral sequences can be semi-optimized. A semi-optimized sequence has the optimized sequence for portions of the sequence that do not span other features, where the non-optimized ancestral sequence is used instead. For example, for HIV-1 ancestral sequences, the optimized ancestral sequence is used for portions of the sequence that do not span the vpu, tat, rev and RRE regions, while the "non-optimized" ancestral sequence is used for the portions of the sequence that overlap the vpu, tat, rev and RRE regions.

Determination of HIV Ancestral Viral Sequences

Ancestral viral sequences can be determined for any gene or genes from HIV type 1 (HIV-1), HIV type 2 (HIV-2), or other HIV viruses, including, for example, for an HIV-1 subtype, for an HIV-2 subtype, for other HIV subtypes, for an emerging HIV subtype, and for HIV variants, such as widely dispersed or geographically isolated variants. For example, an ancestral viral gene sequence can be determined for env and gag genes of HIV-1, such as for HIV-1 subtypes A, B, C, D, E, F, G, H, J, AG, AGI, and for groups M, N, 0, or for HIV-2 viruses or HIV-2 subtypes A or B. In specific embodiments, ancestral viral sequences are determined for env genes of HIV-1 subtypes B and/or C, or for gag genes from subtypes B and/or C. In other embodiments, the ancestral viral sequence is determined for other HIV genes or polypeptides, such as nef, pol, or other auxiliary genes or polypeptides.

Nucleic acid sequences of a selected HIV-1 or HIV-2 gene from presently and/or formerly circulating viruses can be identified from existing databases (e.g., from GenBank or Los Alamos sequence databases). The sequence of circulating viruses can also be determined by recombinant DNA methodologies. (See, e.g., Sambrook et al., Molecular Cloning, A Laboratory Manual, 2nd ed., Cold Spring Harbor Publish., Cold Spring Harbor, N.Y. (1989); Kriegler, Gene Transfer and Expression: A Laboratory Manual, W.H. Freeman, N.Y. (1990); Ausubel et al., supra.) For each data set, several sequences from a different viral strain, subtype or group are used as an outgroup to root the sequences of interest. A model of sequence substitutions and then a maximum likelihood phylogeny is determined for each data set (e.g., subtype and outgroup). The ancestral viral sequence is determined as the sequence at the basal node of the variant sequences (see, e.g., FIGS. 1 and 2). This ancestral viral sequence is thus approximately equidistant from the different sequences within the subtype.

In one embodiment, an ancestral HIV-1 group M, subtype B, env sequence was determined using 41 distinct isolates. (The determined nucleic acid and amino acid sequences are depicted in Tables 1 and 2 (SEQ ID NO:1 and SEQ ID NO:2), respectively). Referring to FIG. 2, 38 subtype B sequences and 3 subtype D (outgroup) sequences were used to root the subtype B sequences. The subtype B sequences were from nine countries, representing a broad sample of subtype B diversity: Australia, 8 sequences; China, 1 sequence; France, 5 sequences; Gabon, 1 sequence; Germany, 2 sequences; Great Britain, 2 sequences; the Netherlands, 2 sequences; Spain, 1 sequence; U.S.A., 15 sequences. The determined ancestor protein is 884 amino acids in length. The distances between this ancestral viral sequence and circulating strains used to determine it were on average 12.3% (range: 8.0-21.0%) while the available specimens were 17.3% different from each other (range: 13.3-23.2%). The ancestor sequence is therefore, on average, more closely related to any given circulating virus than to any other variant. When compared with other subtype B strains, the ancestral sequence is most similar to USAD8 (Theodore et al., AIDS Res. Human Retrovir. 12:191-94 (1996)), with an identity of 94.6% at the amino acid level.

Surprisingly, the determined ancestral viral sequence of the HIV-1 subtype B env gene encodes a wide variety of immunologically active peptides when processed for antigen presentation. Nearly all known subtype B CTL epitope consensus amino acids (387/390; 99.23%) are represented in the determined ancestral viral sequence for the subtype B, gp160 sequence. In contrast, most other variants of HIV-1 subtype B have below 95% epitope sequence conservation (although this is a not a necessary feature of ancestral viral sequences, but is a consequence of the rapid expansion of HIV-1). Thus, an immunogenic composition to this subtype B ancestor protein will elicit broad neutralizing antibody against HIV-1 isolates of the same subtype. An immunogenic composition to this subtype B ancestor protein will also elicit a broad cellular response mediated by antigen-specific T-cells.

In another embodiment, similar computational methods were used to determine the ancestral viral sequence of the HIV-1 subtype C env gene sequence. HIV-1 subtype C is widespread in developing countries. Subtype C is the most common subtype worldwide, responsible for an estimated 30% of HIV-1 infections, and a major component of epidemics in Africa, India and China. The ancestral viral sequence for HIV-1 group M, subtype C, env gene was determined using 57 distinct isolates (39 subtype C sequences and 18 outgroup sequences (two from each of the other group M subtypes); FIG. 8). The determined amino acid sequence is depicted in Table 4 (SEQ ID NO:4). The determined nucleic acid sequence, optimized for expression in human cells, is depicted in Table 3 (SEQ ID NO:3).

The subtype C sequences were from twelve African and Asian countries, representing a broad sample of subtype C diversity worldwide: Botswana, 8 sequences; Brazil, 2 sequences; Burundi, 8 sequences; Peoples Republic of China, 1 sequence; Djibouti, 2 sequences; Ethiopia, 1 sequence; India, 8 sequences; Malawi, 3 sequences; Senegal, 1 sequence; Somalia, 1 sequence; Uganda, 1 sequence; and Zambia, 3 sequences. The determined ancestor protein is 853 amino acids in length. The distances between this ancestral viral sequence and circulating strains used to determine it were on average 11.7% (range: 9.3-14.3%) while the available specimens were on average 16.6% different from each other (range: 7.1-21.7%). The ancestor protein sequence is therefore, on average, more closely related to any given circulating virus than to any other variant. When compared with other subtype C strains, the ancestral sequence is most similar to MW965 (Gao et al., J Virol. 70:1651-67 (1996)), with an identity of 89.5% at the amino acid level.

Surprisingly, the determined ancestral viral sequence encodes a wide variety of immunologically active peptides when processed for antigen presentation. Nearly all known subtype C CTL epitope consensus sequences (389/396; 98.23%) are represented in the determined ancestral viral sequence for the subtype C, gp160 sequence. In contrast, typical variants of HIV-1 subtype C (those used to determine the ancestral sequence) have less than 95.19% epitope sequence conservation (average 90.36%, range 64.56-95.19%). Thus, a vaccine to this subtype C ancestral viral sequence will elicit broad neutralizing antibody against HIV-1 isolates of the same subtype. An immunogenic composition to this subtype C ancestor protein will also elicit a broad cellular response mediated by antigen-specific T-cells.

Optimized and semi-optimized sequences for an HIV ancestral sequence are also provided. Ancestral viral sequences can be optimized for expression in particular host cells. While the optimized ancestral sequence encodes the same amino acid sequence for a gene as the non-optimized sequence, the optimized sequence may not be fully functional in a synthetic virus due to the disruption of auxiliary genes in different reading frames, disruption of the RNA secondary structure, and the like. For example, optimization of the HIV-1 env sequence can disrupt the auxiliary genes for vpu, tat and/or rev, and/or the RNA secondary structure Rev responsive element (RRE). Semi-optimized sequences are prepared by using optimized sequences for portions of the sequence that do not span other genes, RNA secondary structure, and the like. For portions of the sequence that overlap such features, the "non-optimized" ancestral sequence is used (e.g., for regions overlapping vpu, tat, rev and/or RRE). In specific embodiments, semi-optimized ancestral viral sequences for HIV-1 subtypes B and C are provided. (See Tables 5 (SEQ ID NO: 5) and 6 (SEQ ID NO:6).)

In other embodiments, ancestral viral sequences are determined for widely circulating variants or geographically-restricted variants. For example, samples can be collected of an HIV-1 subtype which is widely spread (e.g. present in many countries or in regions without obvious geographic boundaries). Similarly, samples can be collected of an HIV-1 subtype which is geographically restricted (e.g., to a country, regions or other physically defined area). The sequences of the genes (e.g., gag or env) in the samples are determined by recombinant DNA methods (see, e.g., Sambrook et al., supra; Kriegler, supra; Ausubel et al., supra), or from information in databases. Typically, the number of samples will range from about 20 to about 50, depending on their current availability and the time the virus has been circulating in the region of interest (e.g., the longer the time the virus has been circulating, the greater the diversity and the greater the information to be gleaned from the samples). The ancestral viral sequence, either nucleic acid or amino acid, is then determined using the computational methods described herein.

Ancestor Proteins

The invention further relates to ancestor proteins based on a determined ancestral viral sequence. Such ancestor proteins include, for example, full-length protein, polypeptides, fragments, derivatives and analogs thereof. In one aspect, the invention provides amino acid sequences of ancestor proteins (see, e.g., Tables 2 and 4; SEQ ID NO:2; SEQ ID NO:4). In certain embodiments, the ancestor protein is functionally active. Ancestor proteins, fragments, derivatives and analogs typically have the desired immunogenicity or antigenicity and can be used, for example, in immunoassays, for immunization, in vaccines, and the like. A specific embodiment relates to an ancestor protein, fragment, derivative or analog that can be bound by an antibody. Such ancestor proteins, fragments, derivatives or analogs can be tested for the desired immunogenicity by procedures known in the art. (See e.g., Harlow and Lane, supra).

In another aspect, a polypeptide is provided which consists of or comprises a fragment that has at least 8-10 contiguous amino acids of the ancestor protein. In other embodiments, the fragment comprises at least 20 or 50 contiguous amino acids of the ancestor protein. In other embodiments, the fragments are not larger than 35, 100 or 200 amino acids.

Ancestor protein derivatives and analogs can be produced by various methods known in the art. The manipulations which result in their production can occur at the gene or protein level. For example, a nucleic acid encoding an ancestor protein can be modified by any of numerous strategies known in the art (see, e.g., Sambrook et al., supra), such as by making conservative substitutions, deletions, insertions, and the like. The nucleic acid sequence can be cleaved at appropriate sites with restriction endonuclease(s), followed by further enzymatic modification, if desired, isolated, and ligated in vitro. In the production of nucleic acids encoding a fragment, derivative or analog of an ancestor protein, the modified nucleic acid typically remains in the proper translational reading frame, so that the reading frame is not interrupted by translational stop signals or other signals that interfere with the synthesis of the fragment, derivative or analog. The ancestral viral sequence nucleic acid can also be mutated in vitro or in vivo to create and/or destroy translation, initiation and/or termination sequences. The ancestral viral sequence-encoding nucleic acid can also be mutated to create variations in coding regions and/or to form new restriction endonuclease sites or destroy preexisting ones and to facilitate further in vitro modification. Any technique for mutagenesis known in the art can be used, including but not limited to chemical mutagenesis, in vitro site-directed mutagenesis, and the like.

Manipulations of the ancestral viral sequence can also be made at the protein level. Included within the scope of the invention are ancestor protein fragments, derivatives or analogs that are differentially modified during or after synthesis (e.g., in vivo or in vitro translation). Such modifications include conservative substitution, glycosylation, acetylation, phosphorylation, phosphorylation, amidation, derivatization by known protecting/locking groups, proteolytic cleavage, linkage to an antibody molecule or other cellular ligand, and the like. Any of numerous chemical modifications can be carried out by known techniques, including, but not limited to, specific chemical cleavage (e.g., by cyanogen bromide); enzymatic cleavage (e.g., by trypsin, chymotrypsin, papain, V8 protease, and the like); modification by, for example, NaBH.sub.4 acetylation, formylation, oxidation and reduction; metabolic synthesis in the presence of tunicamycin; and the like.

In addition, fragments, derivatives and analogs of ancestor proteins can be chemically synthesized. For example, a peptide corresponding to a portion, or fragment, of an ancestor protein, which comprises a desired domain, can be synthesized by use of chemical synthetic methods using, for example, an automated peptide synthesizer. (See also Hunkapiller et al., Nature 310:105-11 (1984); Stewart and Young, Solid Phase Peptide Synthesis, 2nd ed., Pierce Chemical Co., Rockford, Ill., (1984).) Furthermore, if desired, nonclassical amino acids or chemical amino acid analogs can be introduced as a substitution or addition into the polypeptide sequence. Non-classical amino acids include, but are not limited to, the D-isomers of the common amino acids, .alpha.-amino isobutyric acid, 4-aminobutyric acid, 2-amino butyric acid, 6-amino hexanoic acid, 2-amino isobutyric acid, 3-amino propionic acid, ornithine, norleucine, norvaline, hydroxyproline, sarcosine, citrulline, cysteic acid, t-butylglycine, t-butylalanine, phenylglycine, cyclohexylalanine, .beta.-alanine, selenocysteine, fluoro-amino acids, designer amino acids such as .beta.-methyl amino acids, C .alpha.-methyl amino acids, N .alpha.-methyl amino acids, and other amino acid analogs. Furthermore, the amino acid can be D (dextrorotary) or L (levorotary).

The ancestor protein, fragment, derivative or analog can also be a chimeric, or fusion, protein comprising an ancestor protein, fragment, derivative or analog thereof (typically consisting of at least a domain or motif of the ancestor protein, or at least 10 contiguous amino acids of the ancestor protein) joined at its amino- or carboxy-terminus via a peptide bond to an amino acid sequence of a different protein. In one embodiment, such a chimeric protein is produced by recombinant expression of nucleic acid encoding the chimeric protein. The chimeric nucleic acid can be made by ligating the appropriate nucleic acid sequences to each other in the proper reading frame and expressing the chimeric product by methods commonly known in the art. Alternatively, the chimeric protein can be made by protein synthetic techniques (e.g., by use of an automated peptide synthesizer).

Ancestor protein can be isolated and purified by standard methods including chromatography (e.g., ion exchange, affinity, sizing column chromatography, high pressure liquid chromatography), centrifugation, differential solubility, or by any other standard technique for the purification of proteins.

Determination of COT Sequences

The present invention also provides methods for determining a nucleic acid and protein sequences using Center of Tree (or COT) analysis.

COT provides a method for identifying a position at a node or on a branch of a phylogenetic tree having completely specified branch lengths. This position, called the "center of tree" or COT, is the point at which a specified function F of the lengths from any point to all tips of the tree is minimized. By way of explanation, suppose a tree T has n tips or leaves, labelled a.sub.1, a.sub.2, . . . , a.sub.n, and p is a point on a branch or is a node of the tree. Let l.sub.i be the distance along the tree branches from p to a.sub.i. Then a COT of T for the function F, is a point {circumflex over (p)} satisfying the following relationship: F({circumflex over (p)}:{circumflex over (l)}.sub.1,{circumflex over (l)}.sub.2, . . . , {circumflex over (l)}.sub.n).ltoreq.F(p:l.sub.1,l.sub.2,. . . ,l.sub.n), for all points p, where the notation F(p:l.sub.1,l.sub.2, . . . ,l.sub.n) highlights the fact that the distances l.sub.1 depend on the point p.

In this description, the form of F is general; specific choices for F can be made, based on the intended application. A general algorithm that is applicable for most useful continuous Fs is described (infra). For a given choice of F, an algorithm based on COT analysis can be selected. A COT-based algorithm can be selected, for example, to be more efficient. Such an algorithm is described to find COT when F is the mean of squares (MS) of the ls. Depending upon F, one or more COTs may exist for a given tree, but for many reasonable choices of F, the COT will be unique.

General Algorithm

First, for a certain large class of functions F:R.sup.n.fwdarw.R, namely those which are continuous and have finitely many extreme points (which includes those functions described infra), that there is a finite number of points along the tree which are possible COTs, that can be enumerated constructively, and determine which points are in fact COTs.

For an unrooted tree T of n tips, there are u.ltoreq.2n-2 nodes, counting tips and internal branches, and w.ltoreq.2n-3 branches, including internal and external branches. (u and w are less than their maxima when polytomies exist in the tree.) For each node q.sub.j, 1.ltoreq.j.ltoreq.u, C.sub.j=F(q.sub.j:l.sub.1,l.sub.2, . . . ,l.sub.n) is calculated. Each q.sub.j is a candidate COT.

The branches are enumerated b.sub.k, 1.ltoreq.k.ltoreq.w. Candidate COTs are determined for each branch. Note each branch, say branch k, is flanked by two nodes, call them R.sub.k and L.sub.k (suggesting right and left nodes). Let the branch length of b.sub.k be l. Now the tree is divided into two parts, call them right and left, so that if the tree had a root within branch k, the tips a.sub.1, a.sub.2, . . . , a.sub.n would be divided into two groups, those descended from R.sub.k and those descended from L.sub.k. Suppose there are s right tips and t left tips. Let the distances from the right tips to R.sub.k be written .rho..sub.1, . . . .rho..sub.s, and the distances from the left tips to L.sub.k be written .lamda..sub.1, . . . .lamda..sub.t. Now, let a point p lying on branch b.sub.k be a distance x from the right node R.sub.k. Then the distance from p to L.sub.k is l-x.

Then for branch b.sub.k and p defined along it as described above, F(p:l.sub.1, . . . ,l.sub.n)=F(p:.rho..sub.1+x, . . . ,.rho..sub.s+x,.lamda..sub.1+l-x, . . . ,.lamda..sub.t+l-x)={tilde over (F)}.sub.b.sub.k(x), 021 x<l. In other words, on any branch k of T, the function F of n distances can be expressed for every point p along that branch as a function of a single variable x. By this assumption that F has a finite number of extreme points, the functions {tilde over (F)}.sub.b.sub.k (x) have a finite number of minima for x between 0 and l. Because F is continuous, those minima can be found by standard numerical methods, and each minimum {circumflex over (x)} is associated with a point p.sub.{circumflex over (x)}as described above. Suppose there are v such points over all w branches (v may be greater than, equal to, or less than w). Then the following equation can be written: d.sub.i=F(p.sub.i: l.sub.1, . . . ,l.sub.n)={tilde over (F)}.sub.b(p.sub.i.sub.)({circumflex over (x)}.sub.i), for 1.ltoreq.i.ltoreq.v, where b(p.sub.i) is the branch associated with point p.sub.i (not necessarily the ith branch). Then each p.sub.i is a candidate COT, since if p.sub.i is to minimize F among all points on the tree, it must at least minimize F on those points comprising the branch on which p resides. Since the nodes and branches contain all points on the tree, all possible COTs in the q.sub.j and the p.sub.i have been enumerated.

Therefore, according to the definition of a COT in the first paragraph, any point p.epsilon.{q.sub.1, . . . ,q.sub.u,p.sub.1, . . . ,p.sub.v} is a COT that satisfies F(p:l.sub.1, . . . ,l.sub.n)=min{c.sub.1, . . . ,c.sub.u,d.sub.1, . . . ,d.sub.v} and all such points p are the only COTs for tree T given function F.

This decomposition of possible COTs into separate consideration of nodes and branches allows phylogenetic trees to be expressed in computer programs as data structures that can be efficiently traversed by recursive routines which isolate each node and branch individually and systematically. The above decomposition formally describes the tasks to be performed upon consideration of each node and branch. During the algorithm, the points and function values are stored, and the final determination of COTs is easily accomplished by identifying the minima of the list of values and their associated points after the tree data structure has been completely traversed.

Algorithm to Find Points Minimizing the Mean Squared Distance from the Points to Tips (Least-Squares or LS-COT)

In this special case, let

.function..times..times..times..times..times..times..times. ##EQU00001## the mean of the squared distances from the tips to point p. The COT obtained by minimizing this function essentially balances the average length of the branches on either side of point p, and as such provides a point which will yield a single reconstructed sequence that has the greatest amount of sequence similarity to all the tips as possible, given the evolutionary constraints of nucleotide change along the tree branches. As in the general algorithm, the tree is decomposed into nodes and branches, all possible COTs are enumerated, and F is calculated for each possibility. The point with the minimum F is the COT. The function F can be expressed in terms of quantities that can be efficiently calculated as the tree is traversed recursively; this allows the algorithm to accumulate the quantities c.sub.i and d.sub.i. First, the method of identifying possible COTs and calculating c.sub.i and d.sub.i based on these quantities is described; then the recursion equations for these quantities is described that can be using in the tree-traversal algorithm.

Nodes: Consider each node q.sub.i as a temporary root of the tree, and suppose q.sub.i has k descendant branches, each of which defines a subtree with t.sub.m tips, 1.ltoreq.m.ltoreq.k. Then F can be written

.times..times..times..gamma..function..times..times..times..gamma..functi- on..times..times..times..gamma..times..times..times..gamma..times..times..- times. ##EQU00002## where

.gamma. ##EQU00003## and (l.sub.j.sup.(m)), 1.ltoreq.j.ltoreq.t.sub.m are the distances from q.sub.i to each of the t.sub.m tips of the mth subtree.

Each MS.sub.m is therefore the mean of squared distances of the branches of subtree m, considering node q.sub.i as the root, and each .gamma..sub.m is the proportion of the n tips of the entire tree associated with subtree m.

Branches: With this function F, there exists at most one possible COT on any branch. Consider a branch of length l with left and right nodes as described in the general algorithm above, and consider a point p within the branch. Let M.sub.L be the simple average of distances from point p to the left tips, and M.sub.R be the average of distances from p to the right tips. Suppose there are t left tips and s right tips, and let

.gamma. ##EQU00004## Now, define .alpha. as follows:

.alpha..gamma..times..gamma..times..times..gamma. ##EQU00005##

Then there is a possible COT within the branch if 0<.alpha.<1, and it is the distance .alpha.l from the left node along the branch. If there is such a point, then the value of F at that point, d.sub.i, can be written as d.sub.i=.gamma.(1-.gamma.)(M.sub.L+M.sub.R+l)-.gamma.(M.sub.L.sup.2-MS.su- b.L)-(1-.gamma.)(M.sub.R.sup.2-MS.sub.R) where MS is the mean of summed squared distances from the left or right nodes to their descendant tips as indicated.

Finally, the COT is the point which is associated with the smallest value among the c.sub.i and d.sub.i.

Algorithm to Find Points Minimizing the Mean Distance from the Points to Tips ("Minimum of Means " or MM-COT)

Another useful and simpler function is

.function..times..times..times..times..times..times..times. ##EQU00006## for points within branches, and M.sub.q for nodes. For points within branches, the condition that must be met such that p is a possible COT is the inequality involving .alpha. above, with .gamma. set equal to 0.5. The "minimum of means" COT is then min{c.sub.1, . . . ,c.sub.u,d.sub.1, . . . ,d.sub.v}, where now c.sub.i=M.sub.q(i) for each node i, and

.function..function. ##EQU00007## for points on branches.

Recursions to Calculate M and MS for the Above Algorithms

Suppose node q has k descendant nodes. Each of the k nodes is connected to q by a branch of length l.sub.i, and each is the root of a subtree having s.sub.i tips, 1.ltoreq.i.ltoreq.k. Suppose for each subtree, the mean distance M.sub.i and the mean squared distances MS.sub.i from node to tips have been calculated. Then the mean distance M.sub.q and mean squared distance MS.sub.q from q to all s=s.sub.1+ . . . +S.sub.k descendant tips are given by: M.sub.q=.gamma..sub.1(M.sub.1+l.sub.1)+ . . . +.gamma..sub.k(M.sub.k+l.sub.k), and MS.sub.q=.gamma..sub.1(MS.sub.1+2l.sub.1M.sub.1+l.sub.1.sup.2)+ . . . +.gamma..sub.k(MS.sub.k+2l.sub.kM.sub.k+l.sub.k.sup.2), where

.gamma. ##EQU00008## for 1.ltoreq.i.ltoreq.k.

These quantities can thus be built up as a tree is recursively traversed, and can be used in the calculations described above.

As will be appreciated by the skilled, the methodology for COT analysis can be applied to determine the nucleic acid sequences for a highly disperse viruses. In addition to the embodiments of the determination of HIV COT nucleic acid and protein sequences (infra), COT analysis can be used to determine nucleic acid and protein sequences for other highly disperse viruses.

HIV COT Sequences

COT viral sequences can be determined for any gene or genes from HIV type 1 (HIV-1), HIV type 2 (HIV-2), or other HIV viruses, including, for example, for an HIV-1 subtype, for an HIV-2 subtype, for other HIV subtypes, for an emerging HIV subtype, and for HIV variants, such as widely dispersed or geographically isolated variants. For example, an COT viral gene sequence can be determined for env and gag genes of HIV-1, such as for HIV-1 subtypes A, B, C, D, E, F, G, H, J, AG, AGI, and for groups M, N, 0, or for HIV-2 viruses or HIV-2 subtypes A or B. In specific embodiments, COT viral sequences are determined for env genes of HIV-1 subtypes B and/or C, or for gag genes from subtypes B and/or C. In other embodiments, the COT viral sequence is determined for other HIV genes or polypeptides, such as nef, pol, or other auxiliary genes or polypeptides.

Nucleic acid sequences of a selected HIV-1 or HIV-2 gene from presently and/or formerly circulating viruses can be identified from existing databases (e.g., from GenBank or Los Alamos sequence databases). The sequence of circulating viruses can also be determined by recombinant DNA methodologies. (See, e.g., Sambrook et al., Molecular Cloning, A Laboratory Manual, 2nd ed., Cold Spring Harbor Publish., Cold Spring Harbor, N.Y. (1989); Kriegler, Gene Transfer and Expression: A Laboratory Manual, W.H. Freeman, N.Y. (1990); Ausubel et al., supra.) For each data set, several sequences from a different viral strain, subtype or group are used as an outgroup to root the sequences of interest.

The determined COT viral gene sequence is more closely related, on average, to a gene sequence of any given circulating virus than to any other variant. In certain embodiments, the COT viral gene sequence has at least 70% identity with the LScot and MMcot sequences set forth in FIGS. 9 to 17 or 27-35, but does not have 100% identity with any circulating viral variant. In specific embodiments, the COT viral gene sequence is an LScot or MMcot sequence set forth in FIGS. 9 to 17, but does not have 100% identity with any circulating viral variant. In additional specific embodiments, the COT viral gene sequence is an LScot or MMcot sequence set forth in FIGS. 27 to 35, but does not have 100% identity with any circulating viral variant.

Optimized and semi-optimized sequences for an HIV COT sequence are also provided. COT viral sequences can be optimized for expression in particular host cells. While the optimized COT sequence encodes the same amino acid sequence for a gene as the non-optimized sequence, the optimized sequence may not be fully functional in a synthetic virus due to the disruption of auxiliary genes in different reading frames, disruption of the RNA secondary structure, and the like. For example, optimization of the HIV-1 env sequence can disrupt the auxiliary genes for vpu, tat and/or rev, and/or the RNA secondary structure Rev responsive element (RRE). Semi-optimized sequences are prepared by using optimized sequences for portions of the sequence that do not span other genes, RNA secondary structure, and the like. For portions of the sequence that overlap such features, the "non-optimized" COT sequence is used (e.g., for regions overlapping vpu, tat, rev and/or RRE). In specific embodiments, semi-optimized COT viral sequences for HIV-1 subtypes B and C are provided.

In other embodiments, COT viral sequences are determined for widely circulating variants or geographically-restricted variants. For example, samples can be collected of an HIV-1 subtype which is widely spread (e.g. present in many countries or in regions without obvious geographic boundaries). Similarly, samples can be collected of an HIV-1 subtype which is geographically restricted (e.g., to a country, regions or other physically defined area). The sequences of the genes (e.g., gag or env) in the samples are determined by recombinant DNA methods (see, e.g., Sambrook et al., supra; Kriegler, supra; Ausubel et al., supra), or from information in databases. Typically, the number of samples will range from about 20 to about 50, depending on their current availability and the time the virus has been circulating in the region of interest (e.g., the longer the time the virus has been circulating, the greater the diversity and the greater the information to be gleaned from the samples). The COT viral sequence, either nucleic acid or amino acid, is then determined using the computational methods described herein.

COT Proteins

The invention further relates to COT proteins based on a determined COT viral sequence. Such COT proteins include, for example, full-length protein, polypeptides, fragments, derivatives and analogs thereof. In one aspect, amino acid sequences of COT proteins are provided. In certain embodiments, the COT protein is functionally active. COT proteins, fragments, derivatives and analogs typically are immunogenic or antigenic and can be used, for example, in immunoassays, for immunization, in vaccines, and the like. A specific embodiment relates to an COT protein, fragment, derivative or analog that can be bound by an antibody. Such COT proteins, fragments, derivatives or analogs can be tested for the desired immunogenicity by procedures known in the art. (See e.g., Harlow and Lane, supra). In specific embodiments, an isolated COT protein has the sequence of an LScot or MMcot amino acid sequence set forth in FIGS. 27 to 25 and 36 to 44.

In another aspect, a polypeptide is provided which consists of or comprises a fragment that has at least 8-10 contiguous amino acids of the COT protein. In other embodiments, the fragment comprises at least 20 or 50 contiguous amino acids of the COT protein. In other embodiments, the fragments are not larger than 35, 100 or 200 amino acids. In specific embodiments, an isolated antigenic COT protein fragment is an antigenic fragment of an LScot or MMcot amino acid sequence set forth in FIGS. 27 to 25 and 36 to 44.

COT protein derivatives and analogs can be produced by various methods known in the art. The manipulations which result in their production can occur at the gene or protein level. For example, a nucleic acid encoding a COT protein can be modified by any of numerous strategies known in the art (see, e.g., Sambrook et al., supra), such as by making conservative substitutions, deletions, insertions, and the like. The nucleic acid sequence can be cleaved at appropriate sites with restriction endonuclease(s), followed by further enzymatic modification, if desired, isolated, and ligated in vitro. In the production of nucleic acids encoding a fragment, derivative or analog of a COT protein, the modified nucleic acid typically remains in the proper translational reading frame, so that the reading frame is not interrupted by translational stop signals or other signals that interfere with the synthesis of the fragment, derivative or analog. The COT viral sequence nucleic acid can also be mutated in vitro or in vivo to create and/or destroy translation, initiation and/or termination sequences. The COT viral sequence-encoding nucleic acid can also be mutated to create variations in coding regions and/or to form new restriction endonuclease sites or destroy preexisting ones and to facilitate further in vitro modification. Any technique for mutagenesis known in the art can be used, including but not limited to chemical mutagenesis, in vitro site-directed mutagenesis, and the like.

Manipulations of the COT viral sequence can also be made at the protein level. Included within the scope of the invention are COT protein fragments, derivatives or analogs that are differentially modified during or after synthesis (e.g., in vivo or in vitro translation). Such modifications include conservative substitution, glycosylation, acetylation, phosphorylation, amidation, derivatization by known protecting/blocking groups, proteolytic cleavage, linkage to an antibody molecule or other cellular ligand, and the like. Any of numerous chemical modifications can be carried out by known techniques, including, but not limited to, specific chemical cleavage (e.g., by cyanogen bromide); enzymatic cleavage (e.g., by trypsin, chymotrypsin, papain, V8 protease, and the like); modification by, for example, NaBH.sub.4 acetylation, formylation, oxidation and reduction; metabolic synthesis in the presence of tunicamycin; and the like.

In addition, fragments, derivatives and analogs of COT proteins can be chemically synthesized. For example, a peptide corresponding to a portion, or fragment, of an COT protein, which comprises a desired domain, can be synthesized by use of chemical synthetic methods using, for example, an automated peptide synthesizer. (See also Hunkapiller et al., Nature 310:105-11 (1984); Stewart and Young, Solid Phase Peptide Synthesis, 2nd ed., Pierce Chemical Co., Rockford, Ill., (1984).) Furthermore, if desired, nonclassical amino acids or chemical amino acid analogs can be introduced as a substitution or addition into the polypeptide sequence. Non-classical amino acids include, but are not limited to, the D-isomers of the common amino acids, .alpha.-amino isobutyric acid, 4-aminobutyric acid, 2-amino butyric acid, 6-amino hexanoic acid, 2-amino isobutyric acid, 3-amino propionic acid, ornithine, norleucine, norvaline, hydroxyproline, sarcosine, citrulline, cysteic acid, t-butylglycine, t-butylalanine, phenylglycine, cyclohexylalanine, .beta.-alanine, selenocysteine, fluoro-amino acids, designer amino acids such as .beta.-methyl amino acids, C .alpha.-methyl amino acids, N .alpha.-methyl amino acids, and other amino acid analogs. Furthermore, the amino acid can be D (dextrorotary) or L (levorotary).

The COT protein, fragment, derivative or analog can also be a chimeric, or fusion, protein comprising an COT protein, fragment, derivative or analog thereof (typically consisting of at least a domain or motif of the COT protein, or at least 10 contiguous amino acids of the COT protein) joined at its amino- or carboxy-terminus via a peptide bond to an amino acid sequence of a different protein. In one embodiment, such a chimeric protein is produced by recombinant expression of nucleic acid encoding the chimeric protein. The chimeric nucleic acid can be made by ligating the appropriate nucleic acid sequences to each other in the proper reading frame and expressing the chimeric product by methods commonly known in the art. Alternatively, the chimeric protein can be made by protein synthetic techniques (e.g., by use of an automated peptide synthesizer).

COT protein can be isolated and purified by standard methods including chromatography (e.g., ion exchange, affinity, sizing column chromatography, high pressure liquid chromatography), centrifugation, differential solubility, or by any other standard technique for the purification of proteins.

Nucleic Acids Encoding Ancestral or COT Viral Sequences

Once an ancestral or COT viral sequence is determined by the methods described herein, recombinant DNA methods can be used to prepare nucleic acids encoding the ancestral or COT viral sequence of interest. Suitable methods include, but are not limited to: (1) modifying an existing viral strain most similar to the ancestor viral sequence; (2) synthesizing a nucleic acid encoding the ancestral or COT viral sequence by joining shorter oligonucleotides (e.g., 160-200 nucleotides in length); or (3) a combination of these methods (e.g., by modifying an existing sequence using fragments with very high similarity to the ancestral or COT viral sequence, while synthesizing de novo more divergent sequences).

The nucleic acid sequences can be produced and manipulated using routine techniques. (See, e.g., Sambrook et al., supra; Kriegler, supra; Ausubel et al., supra.)

TABLE-US-00001 TABLE 1 1 ATGCGCGTGA AGGGCATCCG CAAGAACTAC CAGCACCTGT GGCGCTGGGG (SEQ ID NO: 1) 51 CACCATGCTG CTGGGGATGC TGATGATCTG CTCCGCGGCC GAGAAGCTGT 101 GGGTGACCGT GTACTACGGC GTGCCCGTGT GGAAGGAGGC CACCACCACC 151 CTGTTCTGCG CCAGCGACGC CAAGGCTTAC GACACCGAGG TCCACAACGT 201 GTGGGCCACC CACGCCTGCG TGCCCACCGA CCCCAACCCC CAGGAGGTGG 251 TGCTGGAGAA CGTGACCGAG AACTTCAACA TGTGGAAGAA CAACATGGTG 301 GAGCAGATGC ACGAGGACAT CATCAGCCTG TGGGACCAGA GCCTGAAGCC 351 CTGCGTGAAG TTAACCCCCC TGTGCGTGAC CCTGAACTGC ACCGACGACC 401 TGCGCACCAA CGCCACCAAC ACCACCAACA GCAGCGCCAC CACCAACACC 451 ACCAGCAGCG GCGGCGGCAC GATGGAGGGC GAGAAGGGCG AGATCAAGAA 501 CTGCAGCTTC AACGTGACCA CCAGCATCCG CGACAAGATG CAGAAGGAGT 551 ACGCCCTGTT CTACAAGCTG GACGTGGTGC CCATCGACAA CGACAACAAC 601 AACACCAACA ACAACACCAG CTACCGCCTC ATCAACTGCA ACACCAGCGT 651 GATCACCCAG GCCTGCCCCA AGGTGAGCTT CGAGCCCATC CCCATCCACT 701 ACTGCACCCC CGCCGGCTTC GCCATCCTGA AGTGCAACGA CAAGAAGTTC 751 AACGGCACCG GCCCCTGCAC CAACGTGAGC ACCGTGCAGT GCACCCACGG 801 CATCCGCCCC GTGGTGAGCA CCCAGCTGCT GCTGAACGGC AGCCTGGCCG 851 AGGAGGAGGT GGTGATCCGC AGCGAGAACT TCACCGACAA CGCCAAGACC 901 ATCATCGTGC AGCTGAACGA GAGCGTGGAG ATCAACTGCA CGCGTCCCAA 951 CAACAACACC CGCAAGAGCA TCCCCATCGG CCCTGGCCGC GCCCTGTACG 1001 CCACCGGCAA GATCATCGGC GACATCCGCC AGGCCCACTG CAACCTGTCG 1051 CGAGCCAAGT GGAACAACAC CCTGAAGCAG ATCGTGACCA AGCTGCGCGA 1101 GCAGTTCGGC AACAACAAGA CCACCATCGT GTTCAACCAG AGCAGCGGCG 1151 GCGACCCCGA GATCGTGATG CACAGCTTCA ACTGCGGCGG CGAATTCTTC 1201 TACTGCAACA GCACCCAGCT GTTCAACAGC ACCTGGCACT TCAACGGCAC 1251 CTGGGGCAAC AACAACACCG AGCGCAGCAA CAACGCCGCC GACGACAACG 1301 ACACCATCAC CCTGCCCTGC CGCATCAAGC AGATCATCAA CATGTGGCAG 1351 GAGGTGGGCA AGGCCATGTA CGCCCCCCCC ATCAGCGGCC AGATCCGCTG 1401 CAGCAGCAAC ATCACCGGCC TGCTGCTGAC TCGAGACGGC GGCAACAACG 1451 AGAACACCAA CAACACCGAC ACCGAGATCT TCCGCCCCGG GGGCGGCGAC 1501 ATGCGCGACA ACTGGCGCAG CGAGCTGTAC AAGTACAAGG TGGTGAAGAT 1551 CGAGCCCCTG GGCGTGGCCC CCACCAAGGC CAAGCGCCGC GTGGTGCAGC 1601 GCGAGAAGCG CGCCGTGGGC ATGCTGGGCG CCATGTTCCT GGGCTTCCTG 1651 GGCGCCGCCG GCAGCACCAT GGGCGCCGCC AGCATGACCC TGACCGTGCA 1701 GGCGCGCCAG CTGCTGAGCG GCATCGTGCA GCAGCAGAAC AACCTGCTGC 1751 GCGCCATCGA GGCCCAGCAG CACCTGCTGC AGCTGACCGT GTGGGGCATC 1801 AAGCAGCTGC AGGCCCGCGT GCTGGCCGTG GAGCGGTACC TGAAGGACGA 1851 GCAGCTGCTG GGCATCTGGG GCTGCAGCGG CAAGCTGATC TGCACCACCG 1901 CGGTGCCCTG GAACGCCAGC TGGAGCAACA AGAGCCTGGA CAAGATCTGG 1951 AACAACATGA CCTGGATGGA GTGGGAGCGC GAGATCGACA ACTACACCGG 2001 CCTGATCTAC ACCCTGATCG AGGAGAGCCA GAACCAGCAG GAGAAGAACG 2051 AGCAGGAGCT GCTGGAGCTG GACAAGTGGG CCAGGCTGTG GAACTGGTTC 2101 GATATCACCA ACTGGCTGTG GTACATCAAG ATCTTCATCA TGATCGTGGG 2151 CGGCCTGGTG GGCCTGCGCA TCGTGTTCGC CGTGCTGAGC ATCGTGAACC 2201 GCGTGCGCCA GGGCTACAGC CCCCTGAGCT TCCAGACCCG CCTGCCCGCC 2251 CCCCGCGGCC CCGACCGCCC CGAGGGCATC GAGGAGGAGG GCGGCGAGCG 2301 CGACCGCGAC CGCAGCGGGC GCCTGGTGAA CGGCTTCCTG GCCCTGATCT 2351 GGGACGACCT GCGCAGCCTG TGCCTGTTCA GCTACCACCG CCTGCGCGAC 2401 CTGCTGCTGA TCGTGGCCCG CATCGTGGAG CTGCTGGGCC GGCGCGGCTG 2451 GGAGGCCCTG AAGTATTGGT GGAACCTGCT GCAGTACTGG AGCCAGGAGC 2501 TGAAGAACAG CGCCGTGAGC CTGCTGAACG CCACCGCCAT CGCCGTGGCC 2551 GAGGGCACCG ACCGCGTGAT CGAGGTGGTG CAGCGCGCCT GCCGCGCCAT 2601 CCTGCACATC CCCCGCCGCA TCCGCCAGGG CCTGGAGCGC GCCCTGCTGT 2651 GA

TABLE-US-00002 TABLE 2 MRVKGIRKNY QHLWRWGTML LGMLMICSAA EKLWVTVYYG VPVWKEATTT LFCASDAKAY (SEQ ID NO: 2) DTEVHNVWAT HACVPTDPNP QEVVLENVTE NFNMWKNNMV EQMHEDIISL WDQSLKPCVK LTPLCVTLNC TDDLRTNATN TTNSSATTNT TSSGGGTMEG EKGEIKNCSF NVTTSIRDKM QKEYALFYKL DVVPIDNDNN NTNNNTSYRL INCNTSVITQ ACPKVSFEPI PIHYCTPAGF AILKCNDKKF NGTGPCTNVS TVQCTHGIRP VVSTQLLLNG SLAEEEVVIR SENFTDNAKT IIVQLNESVE INCTRPNNNT RKSIPIGPGR ALYATGKIIG DIRQAHCNLS RAKWNNTLKQ IVTKLREQFG NNKTTIVFNQ SSGGDPEIVM HSFNCGGEFF YCNSTQLFNS TWHFNGTWGN NNTERSNNAA DDNDTITLPC RIKQIINMWQ EVGKAMYAPP ISGQIRCSSN ITGLLLTRDG GNNENTNNTD TEIFRPGGGD MRDNWRSELY KYKVVKIEPL GVAPTKAKRR VVQREKRAVG MLGAMFLGFL GAAGSTMGAA SMTLTVQARQ LLSGIVQQQN NLLRAIEAQQ HLLQLTVWGI KQLQARVLAV ERYLKDQQLL GIWGCSGKLI CTTAVPWNAS WSNKSLDKIW NNMTWMEWER EIDNYTGLIY TLIEESQNQQ EKNEQELLEL DKWASLWNWF DITNWLWYIK IFIMIVGGLV GLRIVFAVLS IVNRVRQGYS PLSFQTRLPA PRGPDRPEGI EEEGGERDRD RSGRLVNGFL ALIWDDLRSL CLFSYHRLRD LLLIVARIVE LLGRRGWEAL KYWWNLLQYW SQELKNSAVS LLNATAIAVA EGTDRVIEVV QRACRAILHI PRRIRQGLER ALL

TABLE-US-00003 TABLE 3 ATGCGGGTGATGGGCATCCTGCGGAACTGCCAGCAGTGCTGGATCTGGGGCATCCTGGGC (SEQ ID NO: 3) TTCTGGATGCTGATGATCTGCAGCGTGATGGGCAACCTGTGGGTGACCGTGTACTACGGC GTGCCCGTGTGGAAGGAGGCCAAGACCACCCTGTTCTGCGCCAGCGACGCCAAGGCCTAC GAGCGGGAGGTGCACAACGTGTGGGCCACCCACGCCTGCGTGCCCACCGACCCCAACCCC CAGGAGATGGTGCTGGAGAACGTGACCGAGAACTTCAACATGTGGAAGAACGACATGGTG GACCAGATGCACGAGGACATCATCAGCCTGTGGGACCAGAGCCTGAAGCCCTGCGTGAAG CTGACCCCCCTGTGCGTGACCCTGAACTGCACCAAGGTGACCAACAGCAACAACAACAAC AACACCAGCATGGGCGGGGAGATCAAGAACTGCAGCTTGAACATCACCAGCGAGCTGCGG GACAAGAAGCAGAAGGTGTACGCCCTGTTCTACCGGCTGGACATCGTGCCCCTGAACGAG AACAGCAACAGCAACAGCAGCGAGTACCGGCTGATCAACTGCAACACCAGCGCCATCACC CAGGCCTGCCCCAAGGTGAGCTTCGACCCCATCCCCATCCACTACTGCGCCCCCGCCGGC TACGCCATCCTGAAGTGCAACAACAAGACCTTCAACGGCACCGGCCCCTGCAACAACGTG AGCACCGTGCAGTGCACCCACGGCATCAAGCCCGTGGTGAGCACCCAGCTGCTGCTGAAC GGCAGCCTGGCCGAGGAGGAGATCATCATCCGGAGCGAGAACCTGACCAACAACGCCAAG ACCATCATCGTGCACCTGAACGAGAGCGTGGAGATCGTGTGCACCCGGCCCAACAACAAC ACCCGGAAGAGCATCCGGATCGGCCCCGGCCAGACCTTCTACGCGACCGGCGACATCATC GGCGACATCCGGCAGGCCCACTGCAACATCAGCGAGAAGGAGTGGAACAAGACCCTGCAG CGGGTGGGCAAGAAGCTGAAGGAGCACTTCCCCAACAAGACCATCAAGTTCGAGCCCAGC AGCGGCGGCGACCTGGAGATCACCACCCACAGCTTCAACTGCCGGGGCGAGTTCTTCTAC TGCAACACCAGCAAGCTGTTCAACAGCACCTACAACAGCACCAACAACGGCACCACCAGC AACAGCACCATCACCCTGCCCTGCCGGATCAAGCAGATCATCAACATGTGGCAGGGCGTG GGCCGGGCCATCTACGCCCCCCCCATCGCCGGCAACATCACCTGCAAGAGCAACATCACC GGCCTGCTGCTGACCCGGGACGGCGGCAACACCAACAACACCACCGAGACCTTCCGGCCC GGCGGCGGCGACATGCGGGACAACTGGCGGAGCGAGCTGTACAAGTACAAGGTGGTGGAG ATCAAGCCCCTGGGCGTGGCCCCCACCGAGGCCAAGCGGCGGGTGGTGGAGCGGGAGAAG CGGGCCGTGGGCATCGGCGCCGTGTTCCTGGGCTTCCTGGGCGCCGCCGGCAGCACCATG GGCGCCGCCAGCATCACCCTGACCGTGCAGGCCCGGCAGCTGCTGAGCGGCATCGTGCAG CAGCAGAGCAACCTGCTGCGGGCCATCGAGGCCCAGCAGCACATGCTGCAGCTGACCGTG TGGGGCATCAAGCAGCTGCAGACCCGGGTGCTGGCCATCGAGCGGTACCTGAAGGACCAG CAGCTGCTGGGCATCTGGGGCTGCAGCGGCAAGCTGATCTGCACCACCGCCGTGCCCTGG AACAGCAGCTGGAGCAACAAGAGCCAGGACGACATCTGGGACAACATGACCTGGATGCAG TGGGACCGGGAGATCAGCAACTACACCGACACCATCTACCGGCTGCTGGAGGACAGCCAG AACCAGCAGGAGAAGAACGAGAAGGACCTGCTGGCCCTGGACAGCTGGAAGAACCTGTGG AACTGGTTCGACATCACCAACTGGCTGTGGTACATCAAGATCTTCATCATGATCGTGGGC GGCCTGATCGGCCTGCGGATCATCTTCGCCGTGCTGAGCATCGTGAACCGGGTGCGGCAG GGCTACAGCCCCCTGAGCTTCCAGACCCTGACCCCCAACCCGCGGGGCCCCGACCGGCTG GGCGGCATCGAGGAGGAGGGCGGCGAGCAGGACCGGGACCGGAGCATCCGGCTGGTGAGC GGCTTCCTGGCCCTGGCCTGGGACGACCTGCGGAGCCTGTGCCTGTTCAGCTACCACCGG CTGCGGGACTTCATCCTGATCGCCGCCCGGGGCGTGAACCTGCTGGGCCGGAGCAGCCTG CGGGGCCTGCAGCGGGGGTGGGAGGCCCTGAAGTACCTGGGCAGGCTGGTGCAGTACTGG GGCCTGGAGCTGAAGAAGAGCGCCATCAGCCTGGTGGACACCATCGCGATCGCCGTGGCC GAGGGCACCGACCGGATCATCGAGCTGGTGCAGCGGATCTGCCGGGCCATCCGGAACATC CCCCGGCGGATCCGGCAGGGCTTCGAGGCCGCCCTGCAGTGA

TABLE-US-00004 TABLE 4 (SEQ ID NO: 4) MRVMGILRNCQQWWIWGILGFWMLMICSVMGNLWVTVYYGVPVWKEAKTT LFCASDAKAYEREVHNVWATHACVPTDPNPQEMVLENVTENFNMWKNDMV DQMHEDIISLWDQSLKPCVKLTPLCVTLNCTNVTNTNNNNNTSMGGEIKN CSFNITTELRDKKQKVYALFYRLDIVPLNENSNSNSSEYRLINCNTSAIT QACPKVSFDPIPIHYCAPAGYAILKCNNKTFNGTGPCNNVSTVQCTHGIK PVVSTQLLLNGSLAEEEIIIRSENLTNNAKTIIVHLNESVEIVCTRPNNN start* TRKSIRIGPGQTFYATGDIIGDIRQAHCNISEKEWNKTLQRVGKKLKEHF PNKTIKFEPSSGGDLEITTHSFNCRGEFFYCNTSKLFNSTYNSTNNGTTS NSTITLPCRIKQIINMWQGVGRAMYAPPIAGNITCKSNITGLLLTRDGGN TNNTTETFRPGGGDMRDNWRSELYKYKVVEIKPLGVAPTEAKRRVVEREK RAVGIGAVFLGFLGAAGSTMGAASITLTVQARQLLSGIVQQQSNLLRAIE AQQHMLQLTVWGIKQLQTRVLAIERYLKDQQLLGIWGCSGKLICTTAVPW NSSWSNKSQDDIWDNMTWMQWDREISNYTDTIYRLLEDSQNQQEKNEKDL LALDSWKNLWNWFDITNWLWYIKIFIMIVGGLIGLRIIFAVLSIVNRVRQ GYSPLSFQTLTPNPRGPDRLGGIEEEGGEQDRDRSIRLVSGFLALAWDDL RSLCLFSYHRLRDFILIAARGVNLLGRSSLRGLQRGWEALKYLGSLVQYW GLELKKSAISLLDTIAIAVAEGTDRIIELVQRICRAIRNIPRRIRQGFEA ALQ

TABLE-US-00005 TABLE 5 (SEQ ID NO: 5) ATGAGAGTGAAGGGGATCAGGAAGAACTATCAGCACTTGTGGAGATGGGG CACCATGCTCCTTGGGATGTTGATGATCTGTAGCGCCGCCGAGAAGCTGT GGGTGACCGTGTACTACGGCGTGCCCGTGTGGAAGGAGGCCACCACCACC CTGTTCTGCGCCAGCGACGCCAAGGCTTACGACACCGAGGTCCACAACGT GTGGGCCACCCACGCCTGCGTGCCCACCGACCCCAACCCCCAGGAGGTGG TGCTGGAGAACGTGACCGAGAACTTCAACATGTGGAAGAACAACATGGTG GAGCAGATGCACGAGGACATCATCAGCCTGTGGGACCAGAGCCTGAAGCC CTGCGTGAAGTTAACCCCCCTGTGCGTGACCCTGAACTGCACCGACGACC TGCGCACCAACGCCACCAACACCACCAACAGCAGCGCCACCACCAACACC ACCAGCAGCGGCGGCGGCACGATGGAGGGCGAGAAGGGCGAGATCAAGAA CTGCAGCTTCAACGTGACCACCAGCATCCGCGACAAGATGCAGAAGGAGT ACGCCCTGTTCTACAAGCTGGACGTGGTGCCCATCGACAACGACAACAAC AACACCAACAACAACACCAGCTACCGCCTCATCAACTGCAACACCAGCGT GATCACCCAGGCCTGCCCCAAGGTGAGCTTCGAGCCCATCCCCATCCACT ACTGCACCCCCGCCGGCTTCGCCATCCTGAAGTGCAACGACAAGAAGTTC AACGGCACCGGCCCCTGCACCAACGTGAGCACCGTGCAGTGCACCCACGG CATCCGCCCCGTGGTGAGCACCCAGCTGCTGCTGAACGGCAGCCTGGCCG AGGAGGAGGTGGTGATCCGCAGCGAGAACTTCACCGACAACGCCAAGACC ATCATCGTGCAGCTGAACGAGAGCGTGGAGATCAACTGCACGCGTCCCAA CAACAACACCCGCAAGAGCATCCCCATCGGCCCTGGCCGCGCCCTGTACG CCACCGGCAAGATCATCGGCGACATCCGCCAGGCCCACTGCAACCTGTCG CGAGCCAAGTGGAACAACACCCTGAAGCAGATCGTGACCAAGCTGCGCGA GCAGTTCGGCAACAACAAGACCACCATCGTGTTCAACCAGAGCAGCGGCG GCGACCCCGAGATCGTGATGCACAGCTTCAACTGCGGCGGCGAATTCTTC TACTGCAACAGCACCCAGCTGTTCAACAGCACCTGGCACTTCAACGGCAC CTGGGGCAACAACAACACCGAGCGCAGCAACAACGCCGCCGACGACAACG ACACCATCACCCTGCCCTGCCGCATCAAGCAGATCATCAACATGTGGCAG GAGGTGGGCAAGGCCATGTACGCCCCCCCCATCAGCGGCCAGATCCGCTG CAGCAGCAACATCACCGGCCTGCTGCTGACTCGAGACGGCGGCAACAACG AGAACACCAACAACACCGACACCGAGATCTTCCGCCCCGGGGGCGGCGAC ATGCGCGACAACTGGCGCAGCGAGCTGTACAAGTACAAGGTGGTGAAGAT CGAGCCCCTGGGCGTAGCACCCACCAAGGCAAAGAGAAGAGTGGTGCAGA GAGAAAAAAGCGCAGTGGGAATGCTAGGAGCTATGTTCCTTGGGTTCTTG GGAGCAGCAGGAAGCACTATGGGCGCAGGGTCAATGACGCTGACCGTACA GGCCAGACAATTATTGTCTGGTATAGTGCAGCAGCAGAACAATCTGCTGA GGGCTATTGAGGCGCAACAGCATCTGTTGCAACTCACAGTCTGGGGCATC AAGCAGCTCCAGGCAAGAGTCCTGGCTGTGGAAAGATACCTAAAGGATCA GCAGCTCCTGGGGATTTGGGGTTGCTCTGGAAAACTCATCTGCACCACTG CTGTGCCTTGGAATGCTAGCTGGAGCAACAAGAGCCTGGACAAGATCTGG AACAACATGACCTGGATGGAGTGGGAGCGCGAGATCGACAACTACACCGG CCTGATCTACACCCTGATCGAGGAGAGCCAGAACCAGCAGGAGAAGAACG AGCAGGAGCTGCTGGAGCTGGACAAGTGGGCCAGCCTGTGGAACTGGTTC GATATCACCAACTGGCTGTGGTACATCAAGATCTTCATCATGATCGTGGG CGGCCTGGTGGGCCTGCGCATCGTGTTCGCCGTGCTGAGCATCGTGAACC GCGTGCGCCAGGGCTACAGCCCCCTGAGCTTCCAGACCCACCTGCCAGCC CCGAGGGGACCCGACAGGCCCGAAGGAATCGAAGAAGAAGGTGGAGAGAG AGACAGAGACAGATCCGGTCGATTAGTGAATGGATTCTTAGCACTTATCT GGGACGACGTGCGGAGCCTGTGCCTCTTCAGCTACCACCGCTTGAGCGAC TTACTCTTGATTGTAGCGAGGATTGTGGAACTTCTGGGACGCAGGGGGTG GGAGGCCCTCAAATATTGGTGGAATCTCCTGCAGTACTGGAGTCAGGAAC TAAAGAATAGCGCCGTGAGCCTGCTGAACGCGACCGCCATCGCCGTGGCC GAGGGCACCGACCGCGTGATCGAGGTGGTGCAGCGCGCCTGCCGCGCCAT CCTGCACATCCCCCGCCGCATCCGCCAGGGCCTGGAGCGCGCCCTGCTGT GA

TABLE-US-00006 TABLE 6 (SEQ ID NO: 6) ATGAGAGTGATGGGGATACTGAGGAATTGTCAACAATGGTGGATATGGGG CATCCTAGGCTTTTGGATGCTAATGATTTGTGACGTGATGGGCAACCTGT GGGTGACCGTGTACTACGGCGTGCCCGTGTGGAAGGAGGCCAAGACCACC CTGTTCTGCGCCAGCGACGCCAAGGCCTACGAGCGGGAGGTGCACAACGT GTGGGCCACCCACGCCTGCGTGCCCACCGACCCCAACCCCCAGGAGATGG TGCTGGAGAACGTGACCGAGAACTTCAACATGTGGAAGAACGACATGGTG GACCAGATGCACGAGGACATCATCAGCCTGTGGGACCAGAGCCTGAAGCC CTGCGTGAAGCTGACCCCCCTGTGCGTGACCCTGAACTGCACCAACGTGA CCAACACCAACAACAACAACAACACCAGCATGGGCGGCGAGATCAAGAAC TGCAGCTTCAACATCACCACCGAGCTGCGGGACAAGAAGCAGAAGGTGTA CGCCCTGTTCTACCGGCTGGACATCGTGCCCCTGAACGAGAACAGCAACA GCAACAGCAGCGAGTACCGGCTGATCAAGTGCAACACCAGCGCCATCACC CAGGCCTGCCCCAAGGTGAGCTTCGACCCCATCCCCATCCACTACTGCGC CCCCGCCGGCTACGCCATCCTGAAGTGCAACAACAAGACCTTCAACGGCA CCGGCCCCTGCAACAACGTGAGCACCGTGCAGTGCACCCACGGCATCAAG CCCGTGGTGAGCACCCAGCTGCTGCTGAACGGCAGCCTGGCCGAGGAGGA GATCATCATCCGGAGCGAGAACCTGACCAACAACGCCAAGACCATCATCG TGCACCTGAACGAGAGCGTGGAGATCGTGTGCACCCGGCCCAACAACAAC ACCCGGAAGAGCATCCGGATCGGCCCCGGCCAGACCTTCTACGCCACCGG CGACATCATCGGCGACATCCGGCAGGCCCACTGCAACATCAGCGAGAAGG AGTGGAACAAGACCCTGCAGCGGGTGGGCAAGAAGCTGAAGGAGCACTTC CCCAACAAGACCATCAAGTTCGAGCCCAGCAGCGGCGGCGACCTGGAGAT CACCACCCACAGCTTCAACTGCCGGGGCGAGTTCTTCTACTGCAACACCA GCAAGCTGTTCAACAGCACCTACAACAGCACCAACAACGGCACCACCAGC AACAGCACCATCACCCTGCCCTGCCGGATCAAGCAGATCATCAACATGTG GCAGGGCGTGGGCCGGGCCATGTACGCCCCCCCCATCGCCGGCAACATCA CCTGCAAGAGCAACATCACCGGCCTGCTGCTGACCCGGGACGGCGGCAAC ACCAACAACACCACCGAGACCTTCCGGCCCGGCGGCGGCGACATGCGGGA CAACTGGCGGAGCGAGCTGTACAAGTACAAGGTGGTGGAGATCAAGCCCC TGGGCGTAGCACCCACTGAGGCAAAAAGGAGAGTGGTGGAGAGAGAAAAA AGAGCAGTGGGAATAGGAGCTGTGTTCCTTGGGTTCTTGGGAGCAGCAGG AAGCACTATGGGCGCGGCGTCAATAACGCTGACGGTACAGGCCAGACAAT TATTGTCTGGTATAGTGCAACAGCAAAGCAATTTGCTGAGGGCTATAGAG GCGCAACAGCATATGTTGCAACTCACGGTCTGGGGCATTAAGCAGCTCCA GACAAGAGTCCTGGCTATAGAAAGATACCTAAAGGATCAGCAGCTCCTGG GCATTTGGGGCTGCTCTGGAAAACTCATCTGCACCACTGCTGTGCCTTGG AACTCTAGCTGGAGCAACAAGAGCCAGGACGACATCTGGGACAACATGAC CTGGATGCAGTGGGACCGGGAGATCAGCAACTACACCGACACCATCTACC GGCTGCTGGAGGACAGCCAGAACCAGCAGGAGAAGAACGAGAAGGACCTG CTGGCCCTGGACAGCTGGAAGAACCTGTGGAACTGGTTCGACATCACCAA CTGGCTGTGGTACATCAAGATCTTCATCATGATCGTGGGCGGCCTGATCG GCCTGCGGATCATCTTCGCCGTGCTGAGCATCGTGAACCGGGTGCGGCAG GGCTACAGCCCCCTGAGCTTGCAGACCCTTACCCCAAACCCGAGGGGACC CGACAGGCTCGGAGGAATCGAAGAAGAAGGTGGAGAGCAAGACAGAGACA GATCCATTCGATTAGTGAGCGGATTCTTAGCACTGGCCTGGGACGACCTG CGGAGCCTGTGCCTCTTCAGCTACCACCGATTGAGAGACTTCATATTGAT TGCAGCCAGAGGGTGGGAACTTCTGGGACGCAGCAGTCTCAGGGGACTGC AGAGGGGGTGGGAAGCCCTTAAGTATCTGGGAAGTCTTGTGCAGTATTGG GGTCTGGAGCTAAAAAAGAGTGCTATTAGCCTGGTGGACACCATCGCCAT CGCCGTGGCCGAGGGCACGGAGCGGATCATCGAGCTGGTGCAGCGGATCT GCCGGGCCATCCGGAACATCCCCCGGCGGATCCGGCAGGGGTTCGAGGCC GCCCTGCAGTGA

Unless otherwise stated, all enzymes are used in accordance with the manufacturer's instructions.

In a typical embodiment, a nucleic acid encoding the ancestral or COT viral sequence is synthesized by joining long oligonucleotides. By synthesizing a nucleic acid de novo, desired features are easily incorporated into the gene. Such features include, but are not limited to, the incorporation of convenient restriction sites to enable further manipulation of the nucleic acid sequence, optimization of the codon frequencies (e.g., human codon frequencies) to greatly enhance in vivo expression levels, which can favor the immunogenicity of the polypeptide sequence, and the like. Long oligonucleotides can be synthesized with a very low error rate using the solid-phase method. Long oligonucleotides designed with a 20-25 nucleotide complementary sequence at both 5' and 3' ends can be joined using DNA polymerase, DNA ligase, and the like. If necessary, the sequence of the synthesized nucleic acid can be verified by DNA sequence analysis.

Oligonucleotides that are not commercially available can be chemically synthesized. Suitable methods include, for example, the solid phase phosphoramidite triester method first described by Beaucage and Caruthers (Tetrahedron Letts 22(20):1859-62 (1981)), and the use of an automated synthesizer (see, e.g., Needham Van Devanter et al., Nucleic Acids Res. 12:6159-68 (1984)). Purification of oligonucleotides is, for example, by native acrylamide gel electrophoresis or by anion-exchange HPLC, as described in Pearson and Reanier (J. Chrom. 255:137-49 (1983)).

The sequence of the nucleic acids can be verified, for example, using the chemical degradation method of Maxam et al. (Methods in Enzymology 65:499-560 (1980)), or the chain termination method for sequencing double stranded templates (see, e.g., Wallace et al., Gene 16:21-26 (1981)). Southern blot hybridization techniques can be carried out according to Southern et al. (J. Mol. Biol. 98:503 (1975)), Sambrook et al. (supra), or Ausubel et al. (supra).

Expression of Ancestral or COT Viral Sequences

The nucleic acids encoding ancestral or COT viral sequences can be inserted into an appropriate expression vector (i.e., a vector which contains the necessary elements for the transcription and translation of the inserted polypeptide-coding sequence). A variety of host-vector systems can be utilized to express the polypeptide-coding sequence(s). These include, for example, mammalian cell systems infected with virus (e.g., vaccinia virus, adenovirus, sindbis virus, Venezuelan equine encephalitis (VEE) virus, and the like), insect cell systems infected with virus (e.g., baculovirus), microorganisms such as yeast containing yeast vectors, or bacteria transformed with bacteriophage DNA, plasmid DNA, or cosmid DNA. The expression elements of vectors vary in their strengths and specificities. Depending on the host-vector system utilized, any one of a number of suitable transcription and translation elements can be used. In specific embodiments, the ancestral or COT viral sequence is expressed in human cells, other mammalian cells, yeast or bacteria. In yet another embodiment, a fragment of an ancestral or COT viral sequence comprising an immunologically active region of the sequence is expressed.

Any suitable method can be used for insertion of nucleic acids encoding ancestral or COT viral sequences into an expression vector. Suitable expression vectors typically include appropriate transcriptional and translational control signals. Suitable methods include in vitro recombinant DNA and synthetic techniques and in vivo recombination techniques (genetic recombination). Expression of nucleic acid sequences can be regulated by a second nucleic acid sequence so that the encoded nucleic acid is expressed in a host transformed with the recombinant DNA molecule. For example, expression of an ancestral or COT viral sequence can be controlled by any suitable promoter/enhancer element known in the art. Suitable promoters include, for example, the SV40 early promoter region (Benoist and Chambon, Nature 290:304-10 (1981)), the promoter contained in the 3' long terminal repeat of Rous sarcoma virus (Yamamoto et al., Cell 22:787-97 (1980)), the herpes thymidine kinase promoter (Wagner et al., Proc. Natl. Acad Sci. USA 78:1441-45 (1981)), the Cytomegalovirus promoter, the translational elongation factor EF-1.alpha. promoter, the regulatory sequences of the metallothionein gene (Brinster et al., Nature 296:39-42 (1982)), prokaryotic promoters such as, for example, the .beta.-lactamase promoter (Villa-Komaroff et al., Proc. Natl. Acad Sci. USA 75:3727-31 (1978)) or the tac promoter (deBoer et al., Proc. Natl. Acad. Sci. USA 80:21-25 (1983)), plant expression vectors including the cauliflower mosaic virus 35S RNA promoter (Gardner et al., Nucl. Acids Res. 9:2871-88 (1981)), and the promoter of the photosynthetic enzyme ribulose biphosphate carboxylase (Herrera-Estrella et al., Nature 310:115-20 (1984)), promoter elements from yeast or other fungi such as the GAL7 and GAL4 promoters, the ADH (alcohol dehydrogenase) promoter, the PGK (phosphoglycerol kinase) promoter, the alkaline phosphatase promoter, and the like.

Other exemplary mammalian promoters include, for example, the following animal transcriptional control regions, which exhibit tissue specificity: the elastase I gene control region which is active in pancreatic acinar cells (Swift et al., Cell 38:639-46 (1984); Ornitz et al., Cold Spring Harbor Symp. Quant. Biol. 50:399-409 (1986); MacDonald, Hepatology 7(1 Suppl.):42S-51S (1987); the insulin gene control region which is active in pancreatic beta cells (Hanahan, Nature 315:115-22 (1985)), the immunoglobulin gene control region which is active in lymphoid cells (Grosschedl et al., Cell 38:647-58 (1984); Adams et al., Nature 318:533-38 (1985); Alexander et al., Mol. Cell. Biol. 7:1436-44 (1987)), the mouse mammary tumor virus control region which is active in testicular, breast, lymphoid and mast cells (Leder et al., Cell 45:485-95 (1986)), the albumin gene control region which is active in liver (Pinkert et al., Genes Dev. 1:268-76 (1987)), the alpha-fetoprotein gene control region which is active in liver (Krumlauf et al., Mol. Cell. Biol. 5:1639-48 (1985); Hammer et al., Science 235:53-58 (1987); the alpha 1-antitrypsin gene control region which is active in the liver (Kelsey et al., Genes and Devel. 1:161-71 (1987)); the beta-globin gene control region which is active in myeloid cells (Magram et al., Nature 315:338-40 (1985); Kollias et al., Cell 46:89-94 (1986); the myelin basic protein gene control region which is active in oligodendrocyte cells in the brain (Readhead et al., Cell 48:703-12 (1987)); the myosin light chain-2 gene control region which is active in skeletal muscle (Shani, Nature 314:283-86 (1985)); and the gonadotropic releasing hormone gene control region which is active in the hypothalamus (Mason et al., Science 234:1372-78 (1986)).

In a specific embodiment, a vector is used that comprises a promoter operably linked to the ancestral or COT viral sequence encoding nucleic acid, one or more origins of replication, and, optionally, one or more selectable markers (e.g., an antibiotic resistance gene). Suitable selectable markers include, for example, those conferring resistance to ampicillin, tetracycline, neomycin, G418, and the like. An expression construct can be made, for example, by subcloning a nucleic acid encoding an ancestral or COT viral sequence into a restriction site of the pRSECT expression vector. Such a construct allows for the expression of the ancestral or COT viral sequence under the control of the T7 promoter with a histidine amino terminal flag sequence for affinity purification of the expressed polypeptide.

In an exemplary embodiment, a high efficiency expression system can be used which employs a high-efficiency DNA transfer vector (the pJW4304 SV40/EBV vector) with a very high efficiency RNA/protein expression component (e.g., from the Semliki Forest Virus) to achieve maximal protein expression, as further discussed infra. pJW4304 SV40/EBV was prepared from pJW4303, which is described by Robinson et al. (Ann. New York Acad. Sci. 27:209-11 (1995)) and Yasutomi et al. (J. Virol. 70:678-81 (1996)).

Expression vector/host systems expressing an ancestral or COT viral sequences can be identified by general approaches well known to the skilled artisan, including: (a) nucleic acid hybridization, (b) the presence or absence of "marker" gene function, (c) expression of inserted sequences; or (d) screening transformed cells by standard recombinant DNA methods. In the first approach, the presence of an ancestral or COT viral sequence nucleic acid inserted in host cells can be detected by nucleic acid hybridization using probes comprising sequences that are homologous to an inserted nucleic acid. In the second approach, the expression vector/host system can be identified and selected based upon the presence or absence of certain "marker" gene functions (e.g., thymidine kinase activity, resistance to antibiotics, transformation phenotype, occlusion body formation in baculovirus, and the like) caused by the insertion of a vector containing the desired nucleic acids. For example, if the nucleic acid is inserted within the marker gene sequence of the vector, recombinants containing the ancestral or COT viral sequence can be identified by the absence of the marker gene function.

In the third approach, expression vector/host systems can be identified by assaying for the ancestral or COT viral sequence polypeptide expressed by the recombinant host organism. Such assays can be based, for example, on the physical or functional properties of the ancestral or COT viral sequence polypeptide in in vitro assay systems (e.g., binding by antibody). In the fourth approach, expression vector/host cells can be identified by screening transformed host cells by known recombinant DNA methods.

Once a suitable expression vector host system and growth conditions are established, methods that are known in the art can be used to propagate it. In addition, host cells can be chosen that modulate the expression of the inserted nucleic acid sequences, or that modify or process the gene product in the specific fashion desired. Expression from certain promoters can be elevated in the presence of certain inducers; thus, expression of the ancestral or COT viral sequence can be controlled. Furthermore, different host cells having characteristic and specific mechanisms for the translational and post-translational processing and modification (e.g., glycosylation or phosphorylation) of polypeptides can be used. Appropriate cell lines or host systems can be chosen to ensure the desired modification and processing of the expressed polypeptide. For example, expression in a bacterial system can be used to produce an unglycosylated polypeptide.

Antibodies to Ancestor or COT Proteins, Fragments, Derivatives and Analogs:

Ancestor or COT proteins (including fragments, derivatives, and analogs thereof), can be used as an immunogen to generate antibodies which immunospecifically bind such ancestor or COT proteins and to circulating variants. Such antibodies include but are not limited to polyclonal antibodies, monoclonal antibodies, chimeric antibodies, single chain antibodies, antigen binding antibody fragments (e.g., Fab, Fab', F(ab').sub.2, Fv, or hypervariable regions), and an Fab expression library. In some embodiments, polyclonal and/or monoclonal antibodies to an ancestor or COT protein are produced. In other embodiments, antibodies to a domain of an ancestor or COT protein are produced. In yet other embodiments, fragments of an ancestor or COT protein that are identified as immunogenic (e.g., hydrophilic) are used as immunogens for antibody production.

Various procedures known in the art can be used for the production of polyclonal antibodies. For the production of such antibodies, various host animals (including, but not limited to, rabbits, mice, rats, sheep, goats, camels, and the like) can be immunized by injection with the ancestor or COT protein, fragment, derivative or analog. Various adjuvants can be used to increase the immunological response, depending on the host species including, but not limited to, Freund's adjuvant (complete and incomplete), mineral gels such as aluminum hydroxide, surface active substances such as lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanins, dinitrophenol, and potentially useful human adjuvants such as BCG (bacille Calmette-Guerin) and Corynebacterium parvum.

For preparation of monoclonal antibodies directed toward an ancestor or COT protein, fragment, derivative, or analog thereof, any technique that provides for the production of antibody molecules by continuous cell lines in culture can be used. Such techniques include, for example, the hybridoma technique originally developed by Kohler and Milstein (see, e.g., Nature 256:495-97 (1975)), the trioma technique (see, e.g., Hagiwara and Yuasa, Hum. Antibodies Hybridomas. 4:15-19 (1993); Hering et al., Biomed. Biochim. Acta 47:211-16 (1988)), the human B-cell hybridoma technique (see, e.g., Kozbor et al., Immunology Today 4:72 (1983)), and the EBV-hybridoma technique to produce human monoclonal antibodies (see, e.g., Cole et al., In: Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp. 77-96 (1985)). Human antibodies can be used and can be obtained by using human hybridomas (see, e.g., Cote et al., Proc. Natl. Acad. Sci. USA 80:2026-30 (1983)) or by transforming human B cells with EBV virus in vitro (see, e.g., Cole et al., supra).

Further to the invention, "chimeric" or "humanized" antibodies (see, e.g., Morrison et al., Proc. Natl. Acad Sci. USA 81:6851-55 (1984); Neuberger et al., Nature 312:604-08 (1984); Takeda et al., Nature 314:452-54 (1985)) can be prepared. Such chimeric antibodies are typically prepared by splicing the non-human genes for an antibody molecule specific for ancestor or COT protein together with genes from a human antibody molecule of appropriate biological activity. It can be desirable to transfer the antigen binding regions (e.g., Fab', F(ab').sub.2, Fab, Fv, or hypervariable regions) of non-human antibodies into the framework of a human antibody by recombinant DNA techniques to produce a substantially human molecule. Methods for producing such "chimeric" molecules are generally well known and described in, for example, U.S. Pat. Nos. 4,816,567; 4,816,397; 5,693,762; and 5,712,120; International Patent Publications WO 87/02671 and WO 90/00616; and European Patent Publication EP 239 400 (the disclosures of which are incorporated by reference herein). Alternatively, a human monoclonal antibody or portions thereof can be identified by first screening a human B-cell cDNA library for DNA molecules that encode antibodies that specifically bind to an ancestor or COT protein according to the method generally set forth by Huse et al. (Science 246:1275-81 (1989)). The DNA molecule can then be cloned and amplified to obtain sequences that encode the antibody (or binding domain) of the desired specificity. Phage display technology offers another technique for selecting antibodies that bind to ancestor or COT proteins, fragments, derivatives or analogs thereof. (See, e.g., International Patent Publications WO 91/17271 and WO 92/01047; Huse et al., supra.)

According to another aspect of the invention, techniques described for the production of single chain antibodies (see, e.g., U.S. Pat. Nos. 4,946,778 and 5,969,108) can be adapted to produce single chain antibodies. An additional aspect of the invention utilizes the techniques described for the construction of a Fab expression library (see, e.g., Huse et al., supra) to allow rapid and easy identification of monoclonal Fab fragments with the desired specificity for ancestor or COT proteins, fragments, derivatives, or analogs thereof.

Antibody that contains the idiotype of the molecule can be generated by known techniques. For example, such fragments include but are not limited to, the F(ab').sub.2 fragment which can be produced by pepsin digestion of the antibody molecule, the Fab' fragments which can be generated by reducing the disulfide bridges of the F(ab').sub.2 fragment, the Fab fragments which can be generated by treating the antibody molecule with papain and a reducing agent, and Fv fragments. Recombinant Fv fragments can also be produced in eukaryotic cells using, for example, the methods described in U.S. Pat. No. 5,965,405.

In the production of antibodies, screening for the desired antibody can be accomplished by techniques known in the art (e.g., ELISA (enzyme-linked immunosorbent assay)). In one example, antibodies that recognize a specific domain of an ancestor or COT protein can be used to assay generated hybridomas for a product which binds to polypeptide containing that domain. Antibodies specific to a domain of an ancestor or COT protein are also provided.

Antibodies against ancestor or COT proteins (including fragments, derivatives and analogs) can be used for passive antibody treatment, according to methods known in the art. Antibodies can be introduced into an individual to prevent or treat viral infection. Typically, such antibody therapy is practiced as an adjuvant to the vaccination protocols. The antibodies can be produced as described supra and can be polyclonal or monoclonal antibodies and administered intravenously, enterally (e.g., as an enteric coated tablet form), by aerosol, orally, transdermally, transmucosally, intrapleurally, intrathecally, or by other suitable routes.

Immunogenic Compositions and Vaccines

The present invention also provides immunogenic compositions, such as vaccines. An example of the development of a vaccine ("digital vaccine") using the sequences of the invention is illustrated in FIG. 4. The present invention also provides a new way to produce vaccines, using HIV ancestral or COT viral sequences (e.g., HIV env or gag genes or polypeptides). Such ancestral or COT viral sequences typically correspond to the structure of a real biological entity--the founding virus (i.e., "the viral Eve").

Formulations

Immunogenic compositions and vaccines that contain an immunogenically effective amount of one or more ancestor or COT viral protein sequences, or fragments, derivatives, or analogs thereof, are provided. Immunogenic epitopes in an ancestral or COT protein sequence can be identified according to methods known in the art, and proteins, fragments, derivatives, or analogs containing those epitopes can be delivered by various means, in a vaccine composition. Suitable compositions can include, for example, lipopeptides (e.g., Vitiello et al., J. Clin. Invest. 95:341 (1995)), peptide compositions encapsulated in poly(DL-lactide-co-glycolide) ("PLG") microspheres (see, e.g., Eldridge et al., Molec. Immunol. 28:287-94 (1991); Alonso et al., Vaccine 12:299-306 (1994); Jones et al., Vaccine 13:675-81 (1995)), peptide compositions contained in immune stimulating complexes (ISCOMS) (see, e.g., Takahashi et al., Nature 344:873-75 (1990); Hu et al., Clin. Exp. Immunol. 113:235-43 (1998)), multiple antigen peptide systems (MAPs) (see, e.g., Tam, Proc. Natl. Acad. Sci. U.S.A. 85:5409-13 (1988); Tam, J. Immunol. Methods 196:17-32 (1996)), viral delivery vectors (see, e.g., Perkus et al., In: Concepts in vaccine development, Kaufmann (ed.), p. 379 (1996)), particles of viral or synthetic origin (see, e.g., Kofler et al., J. Immunol. Methods. 192:25-35 (1996); Eldridge et al., Sem. Hematol. 30:16 (1993); Falo et al., Nature Med. 7:649 (1995)), adjuvants (see, e.g., Warren et al., Annu. Rev. Immunol. 4:369 (1986); Gupta et al., Vaccine 11:293 (1993)), liposomes (see, e.g., Reddy et al., J. Immunol. 148:1585 (1992); Rock, Immunol. Today 17:131 (1996)), or naked or particle absorbed cDNA (see, e.g., Shiver et al., In: Concepts in vaccine development, Kaufmann (ed.), p. 423 (1996)). Toxin-targeted delivery technologies, also known as receptor-mediated targeting, such as those of Avant Immunotherapeutics, Inc. (Needham, Mass.) can also be used.

Furthermore, useful carriers that can be used with immunogenic compositions and vaccines of the invention are well known in the art, and include, for example, thyroglobulin, albumins such as human serum albumin, tetanus toxoid, polyamino acids such as poly L-lysine, poly L-glutamic acid, influenza, hepatitis B virus core protein, and the like. The compositions and vaccines can contain a physiologically tolerable (i.e., acceptable) diluent such as water, or saline, typically phosphate buffered saline. The compositions and vaccines also typically include an adjuvant. Adjuvants such as incomplete Freund's adjuvant, aluminum phosphate, aluminum hydroxide, or alum are examples of materials well known in the art. Additionally, as disclosed herein, CTL responses can be primed by conjugating ancestor or COT proteins (or fragments, derivative or analogs thereof) to lipids, such as tripalmitoyl-S-glycerylcysteinyl-seryl-serine (P.sub.3CSS).

As disclosed in greater detail herein, upon immunization with a composition or vaccine containing an ancestor or COT viral sequence protein composition in accordance with the invention, via injection, aerosol, oral, transdermal, transmucosal, intrapleural, intrathecal, or other suitable routes, the immune system of the host responds to the composition or vaccine by producing large amounts of CTL's, HTL's and/or antibodies specific for the desired antigen. Consequently, the host typically becomes at least partially immune to later infection, or at least partially resistant to developing an ongoing chronic infection, or derives at least some therapeutic benefit.

For therapeutic or prophylactic immunization, ancestor or COT proteins (including fragments, derivatives and analogs) can also be expressed by viral or bacterial vectors. Examples of expression vectors include attenuated viral hosts, such as vaccinia or fowlpox. In one embodiment, this approach involves the use of vaccinia virus, for example, as a vector to express nucleotide sequences that encode the polypeptide. Upon introduction into an acutely or chronically infected host, or into a non-infected host, the recombinant vaccinia virus expresses the immunogenic protein, and thereby elicits a host CTL, HTL and/or antibody response. Vaccinia vectors and methods useful in immunization protocols are described in, for example, U.S. Pat. No. 4,722,848, the disclosure of which is incorporated by reference herein. A wide variety of other vectors useful for therapeutic administration or immunization of the peptides of the invention, for example, adeno and adeno-associated virus vectors, retroviral vectors, Salmonella typhimurium vectors, detoxified anthrax toxin vectors, Alphavirus, and the like, can also be used, as will be apparent to those skilled in the art from the description herein. Alphavirus vectors that can be used include, for example, Sindbis and Venezuelan equine encephalitis (VEE) virus. (See, e.g., Coppola et al., J. Gen. Virol. 76:635-41 (1995); Caley et al., Vaccine 17:3124-35 (1999); Loktev et al., J. Biotechnol. 44:129-37 (1996).)

Polynucleotides (e.g, DNA or RNA) encoding one or more ancestral or COT proteins (including fragments, derivative or analogs) can also be administered to a patient. This approach is described in, for example, Wolff et al., (Science 247:1465 (1990)), in U.S. Pat. Nos. 5,580,859; 5,589,466; 5,804,566; 5,739,118; 5,736,524; 5,679,647; and WO 98/04720; and in more detail below. Examples of DNA-based delivery technologies include "naked DNA", facilitated (bupivicaine, polymer, or peptide-mediated) delivery, cationic lipid complexes, particle-mediated ("gene gun"), or pressure-mediated delivery (see, e.g., U.S. Pat. No. 5,922,687).

The direct injection of naked plasmid DNA encoding a protein antigen as a means of vaccination is, among several HIV delivery and expression systems that have been developed in the last decade, one that has attracted much attention. In mouse models, as well as in large animal models, both humoral and cellular immune responses are readily induced, resulting in protective immunity against challenge infections in some instances. A Semliki Forest Virus (SFV) replicon can also be used, for example, in the context of naked DNA immunization. SFV belongs to the Alphavirus family wherein the genome consists of a single stranded RNA of positive polarity encoding its own replicase. By replacing the SFV structural genes with the gene of interest, expression levels as high as 25% of the total cell protein are obtained. Another advantage of this alphavirus over plasmid vectors is its non-persistence: the antigen of interest is expressed at high levels but for a short period (typically <72 hours). In contrast, plasmid vectors generally induce synthesis of the antigen of interest over extended time periods, risking chromosomal integration of foreign DNA and cell transformation. Furthermore, antigen persistence or repeated inoculations of small amounts of antigen has been shown experimentally to induce tolerance. Prolonged antigen synthesis, therefore, can theoretically result in unresponsiveness rather than immunity.

Ancestor or COT proteins, fragments, derivative, and analogs can also be introduced into a subject in vivo or ex vivo. For example, ancestral or COT viral sequences can be transferred into defined cell populations. Suitable methods for gene transfer include, for example:

1) Direct gene transfer. (See, e.g., Wolff et al., Science 247:1465-68 (1990)).

2) Liposome-mediated DNA transfer. (See, e.g, Caplen et al., Nature Med. 3:39-46 (1995); Crystal, Nature Med. 1:15-17 (1995); Gao and Huang, Biochem. Biophys. Res. Comm. 179:280-85 (1991).)

3) Retrovirus-mediated DNA transfer. (See, e.g., Kay et al., Science 262:117-19 (1993); Anderson, Science 256:808-13 (1992).) Retroviruses from which the retroviral plasmid vectors can be derived include lentiviruses. They further include, but are not limited to, Moloney Murine Leukemia Virus, spleen necrosis virus, retroviruses such as Rous Sarcoma Virus, Harvey Sarcoma Virus, avian leukosis virus, gibbon ape leukemia virus, human immunodeficiency virus, Myeloproliferative Sarcoma Virus, and mammary tumor virus. In one embodiment, the retroviral plasmid vector is derived from Moloney Murine Leukemia Virus. Examples illustrating the use of retroviral vectors in gene therapy further include the following: Clowes et al. (J. Clin. Invest. 93:644-51 (1994)); Kiem et al. (Blood 83:1467-73 (1994)); Salmons and Gunzberg (Human Gene Therapy 4:129-41 (1993)); and Grossman and Wilson (Curr. Opin. in Genetics and Devel. 3:110-14 (1993)).

4) DNA Virus-mediated DNA transfer. Such DNA viruses include adenoviruses (e.g., Ad-2 or Ad-5 based vectors), herpes viruses (typically herpes simplex virus based vectors), and parvoviruses (e.g., "defective" or non-autonomous parvovirus based vectors, or adeno-associated virus based vectors, such as AAV-2 based vectors). (See, e.g., Ali et al., Gene Therapy 1:367-84 (1994); U.S. Pat. Nos. 4,797,368 and 5,139,941, the disclosures of which are incorporated herein by reference.) Adenoviruses have the advantage that they have a broad host range, can infect quiescent or terminally differentiated cells, such as neurons or hepatocytes, and appear essentially non-oncogenic. Adenoviruses do not appear to integrate into the host genome. Because they exist extrachromosomally, the risk of insertional mutagenesis is greatly reduced. Adeno-associated viruses exhibit similar advantages as adenoviral-based vectors. However, AAVs exhibit site-specific integration on human chromosome 19.

Kozarsky and Wilson (Current Opinion in Genetics and Development 3:499-503 (1993)) present a review of adenovirus-based gene therapy. Bout et al. (Human Gene Therapy 5:3-10 (1994)) demonstrated the use of adenovirus vectors to transfer genes to the respiratory epithelia of rhesus monkeys. Herman et al. (Human Gene Therapy 10:1239-49 (1999)) describe the intraprostatic injection of a replication-deficient adenovirus containing the herpes simplex thymidine kinase gene into human prostate, followed by intravenous administration of the prodrug ganciclovir in a phase I clinical trial. Other instances of the use of adenoviruses in gene therapy can be found in Rosenfeld et al. (Science 252:431-34 (1991)); Rosenfeld et al. (Cell 68:143-55 (1992)); Mastrangeli et al. (J. Clin. Invest. 91:225-34 (1993)); Thompson (Oncol. Res. 11:1-8 (1999)).

The choice of a particular vector system for transferring the ancestral or COT viral sequence of interest will depend on a variety of factors. One important factor is the nature of the target cell population. Although retroviral vectors have been extensively studied and used in a number of gene therapy applications, these vectors are generally unsuited for infecting non-dividing cells. In addition, retroviruses have the potential for oncogenicity. However, recent developments in the field of lentiviral vectors may circumvent some of these limitations. (See Naldini et al., Science 272:263-67 (1996).)

The skilled artisan will appreciate that any suitable expression vector containing nucleic acid encoding an ancestor or COT protein, or fragment, derivative or analog thereof can be used in accordance with the present invention. Techniques for constructing such a vector are known. (See, e.g., Anderson, Nature 392:25-30 (1998); Verma, Nature 389:239-42 (1998).) Introduction of the vector to the target site can be accomplished using known techniques.

In another one embodiment, a novel expression system employing a high-efficiency DNA transfer vector (the pJW4304 SV40/EBV vector (pJW4304 SV40/EBV was prepared from pJW4303, which is described by Robinson et al., Ann. New York Acad. Sci. 27:209-11 (1995) and Yasutomi et al., J. Virol. 70:678-81 (1996)) with a very high efficiency RNA/protein expression system (the Semliki Forest Virus) is used to achieve maximal protein expression in vaccinated hosts with a safe and inexpensive vaccine. SFV cDNA is placed, for example, under the control of a cytomegalovirus (CMV) promoter (see FIG. 7). Unlike conventional DNA vectors, the CMV promoter does not directly drive the expression of the antigen encoding nucleic acids. Instead, it directs the synthesis of recombinant SFV replicon RNA transcript. Translation of this RNA molecule produces the SFV replicase complex, which catalyzes cytoplasmic self-amplification of the recombinant RNA, and eventual high-level production of the actual antigen-encoding mRNA. Following vector delivery, the transfected host cell dies within a few days. In the context of the present invention, env and/or gag genes are typically cloned into this vector. In vitro experiments using Northern blot, Western blot, SDS-PAGE, immunoprecipitation assay, and CD4 binding assays can be performed, as described infra, to determine the efficiency of this system by assessing protein expression level, protein characteristics, duration of expression, and cytopathic effects of the vector.

In some embodiments, ancestor or COT protein (or a fragment, derivative or analog thereof) is administered to a subject in need thereof. The dosage for an initial therapeutic immunization generally occurs in a unit dosage range where the lower value is about 1, 5, 50, 500, or 1,000 .mu.g and the higher value is about 10,000; 20,000; 30,000; or 50,000 .mu.g. Dosage values for a human typically range from about 500 .mu.g to about 50,000 .mu.g per 70 kilogram patient. Boosting dosages of between about 1.0 .mu.g to about 50,000 .mu.g of polypeptide pursuant to a boosting regimen over weeks to months can be administered depending upon the patient's response and condition as determined by measuring the antibody levels or specific activity of CTL and HTL obtained from the patient's blood.

A human unit dose form of the protein or nucleic acid composition is typically included in a pharmaceutical composition that comprises a human unit dose of an acceptable carrier, typically an aqueous carrier, and is administered in a volume of fluid that is known by those of skill in the art to be used for administration of such compositions to humans (see, e.g., Remington "Pharmaceutical Sciences", 17 Ed., Gennaro (ed.), Mack Publishing Co., Easton, Pa. (1985)).

The ancestor or COT proteins and nucleic acids can also be administered via liposomes, which serve to target the peptides to a particular tissue, such as lymphoid tissue, or to target selectively to infected cells, as well as to increase the half-life of the composition. Liposomes include emulsions, foams, micelles, insoluble monolayers, liquid crystals, phospholipid dispersions, lamellar layers and the like. In these preparations, the protein or nucleic acid to be delivered is incorporated as part of a liposome, alone or in conjunction with a molecule that binds to a receptor prevalent among lymphoid cells, such as monoclonal antibodies that bind to the CD45 antigen, or with other therapeutic or immunogenic compositions. Thus, liposomes either filled or decorated with a desired protein or nucleic acid can be directed to the site of lymphoid cells, where the liposomes then deliver the protein compositions to the cells. Liposomes for use in accordance with the invention are formed from standard vesicle-forming lipids, which generally include neutral and negatively charged phospholipids and a sterol, such as cholesterol. The selection of lipids is generally guided by consideration of, for example, liposome size, acid lability and stability of the liposomes in the blood stream. A variety of methods are available for preparing liposomes, as described in, for example, Szoka et al., Ann. Rev. Biophys. Bioeng. 9:467 (1980), and U.S. Pat. Nos. 4,235,871; 4,501,728; 4,837,028; and 5,019,369.

For targeting cells of the immune system, a ligand to be incorporated into the liposome can include, for example, antibodies or fragments thereof specific for cell surface determinants of the desired immune system cells. A liposome suspension containing a protein or nucleic acid can be administered, for example, intravenously, locally, topically, etc., in a dose which varies according to, inter alia, the manner of administration, the protein or nucleic acid being delivered, and the like.

For solid compositions, conventional nontoxic solid carriers can be used which include, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin, talcum, cellulose, glucose, sucrose, magnesium carbonate, and the like. For oral administration, a pharmaceutically acceptable nontoxic composition is formed by incorporating any of the normally employed excipients, such as those carriers previously listed, and generally 10-95% of active ingredient, that is, the ancestor or COT proteins or nucleic acids, and typically at a concentration of 25%-75%.

For aerosol administration, the immunogenic proteins or nucleic acids are typically in finely divided form along with a surfactant and propellant. Suitable percentages of peptides are about 0.01% to about 20% by weight, typically about 1% to about 10%. The surfactant is, of course, nontoxic, and typically soluble in the propellant. Representative of such agents are the esters or partial esters of fatty acids containing from 6 to 22 carbon atoms, such as caproic, octanoic, lauric, palmitic, stearic, linoleic, linolenic, stearic and oleic acids with an aliphatic polyhydric alcohol or its cyclic anhydride. Mixed esters, such as mixed or natural glycerides can be employed. The surfactant can constitute about 0.1% to about 20% by weight of the composition, typically 0.25-5%. The balance of the composition is ordinarily propellant. A carrier can also be included, as desired, as with, for example, lecithin for intranasal delivery.

Immune Responses Elicited by the Ancestral or COT Viral Sequences

Ancestor or COT proteins (including fragments, derivative and analogs) can be used as a vaccine, as described supra. Such vaccines, referred to as a "digital vaccine", are typically screened for those that elicit neutralizing antibody and/or viral (e.g., HIV) specific CTLs against a larger fraction of circulating strains than a vaccine comprising a protein antigen encoded by any sequences of existing viruses or by consensus sequences. Such a digital vaccine will typically provide protection when challenged by the same subtype of virus (e.g., HIV-1 virus) as the subtype from which the ancestral or COT viral sequence was derived.

The invention also provides methods to analyze the function of ancestral or COT viral gene sequences. For example, in one embodiment, the gp160 ancestor or COT viral gene sequence is analyzed by assays for functions, such as, for example, CD4 binding, co-receptor binding, receptor specificity (e.g., binding to the CCR5 receptor), protein structure, and the ability to cause cell fusion. Although the ancestor or COT sequences can result in a viable virus, such a viable virus is not necessary for obtaining a successful vaccine. For example, a gp160 ancestor or COT not correctly folded can be more immunogenic by exposing epitopes that are normally buried to the immune system. Further, although the ancestor or COT viral sequence can be successfully used as a vaccine, such a sequence need not include alternate open reading frames that encode proteins such a tat or rev, when used as an immunogen (e.g., a vaccine).

Accordingly, in one aspect, mice are immunized with an ancestor or COT protein and tested for humoral and cellular immune responses. Typically, 5-10 mice are intradermally or intramuscularly injected with a plasmid containing a gag and/or env gene encoding an ancestral or COT viral sequence in, for example, 50 .mu.l volume. Two control groups are typically used to interpret the results. One control group is injected with the same vector containing the gag or env gene from a standard laboratory strain (e.g., HIV-1-IIIB). A second control group is injected with same vector without any insert. Antibody titration against gag or env protein is performed using standard immunoassays (e.g., ELISA), as described infra. The neutralizing antibody is analyzed by subtype-specific laboratory HIV-1 strains, such as for example pNL4-3 (HIV-1-IIIB), as well as primary isolates from HIV-1 infected individuals. The ability of an ancestor or COT viral sequence protein-elicited neutralizing antibody to neutralize a broad primary isolates is one factor indicative of an immunogenic or vaccine composition. Similar studies can be performed in large animals, such as non-human animals (e.g, macques) or in humans.

Immunoassays for Titrating the Ancestor or COT Protein-Elicited Antibodies

There are a variety of assays known to those of ordinary skill in the art for detecting antibodies in a sample (see, e.g., Harlow and Lane, supra). In general, the presence or absence of antibodies in a subject immunized with an ancestor or COT protein vaccine can be determined by (a) contacting a biological sample obtained from the immunized subject with one or more ancestor or COT proteins (including fragments, derivatives or analogs thereof); (b) detecting in the sample a level of antibody that binds to the ancestor or COT protein(s); and (c) comparing the level of antibody with a predetermined cut-off value.

In a typical embodiment, the assay involves the use of an ancestor or COT protein (including fragment, derivative or analog) immobilized on a solid support to bind to and remove the antibody from the sample. The bound antibody can then be detected using a detection reagent that contains a reporter group. Suitable detection reagents include antibodies that bind to the antibody/ancestor or COT protein complex and free protein labeled with a reporter group (e.g., in a semi-competitive assay). Alternatively, a competitive assay can be utilized, in which an antibody that binds to the ancestor or COT protein of interest is labeled with a reporter group and allowed to bind to the immobilized antigen after incubation of the antigen with the sample. The extent to which components of the sample inhibit the binding of the labeled antibody to the ancestor or COT protein of interest is indicative of the reactivity of the sample with the immobilized ancestor or COT protein.

The solid support can be any solid material known to those of ordinary skill in the art to which the antigen may be attached. For example, the solid support can be a test well in a microtiter plate or a nitrocellulose or other suitable membrane. Alternatively, the support can be a bead or disc, such as glass, fiberglass, latex or a plastic material such as polystyrene or polyvinylchloride. The support may also be a magnetic particle or a fiber optic sensor, such as those disclosed, for example, in U.S. Pat. No. 5,359,681, the disclosure of which is incorporated by reference herein.

The ancestor or COT proteins can be bound to the solid support using a variety of techniques known to those of ordinary skill in the art, which are amply described in the patent and scientific literature. In the context of the present invention, the term "bound" refers to both non-covalent association, such as adsorption, and covalent attachment (see, e.g., Pierce Immunotechnology Catalog and Handbook, at A12-A13 (1991)).

In certain embodiments, the assay is an enzyme-linked immunosorbent assay (ELISA). This assay can be performed by first contacting an ancestor or COT protein that has been immobilized on a solid support, commonly the well of a microtiter plate, with the sample, such that antibodies present within the sample that recognize the ancestor or COT protein of interest are allowed to bind to the immobilized protein. Unbound sample is then removed from the immobilized ancestor or COT protein and a detection reagent capable of binding to the immobilized antibody-protein complex is added. The amount of detection reagent that remains bound to the solid support is then determined using a method appropriate for the specific detection reagent.

More specifically, once the ancestor or COT protein is immobilized on the support as described above, the remaining protein binding sites on the support are typically blocked. Any suitable blocking agent known to those of ordinary skill in the art, such as bovine serum albumin or Tween 2O (Sigma Chemical Co., St. Louis, Mo.), can be employed. The immobilized ancestor or COT protein is then incubated with the sample, and the antibody is allowed to bind to the protein. The sample can be diluted with a suitable diluent, such as phosphate-buffered saline (PBS) prior to incubation. In general, an appropriate contact time (i.e., incubation time) is a period of time that is sufficient to detect the presence of antibody within a biological sample of an immunized subject. Those of ordinary skill in the art will recognize that the time necessary to achieve equilibrium can be readily determined by assaying the level of binding that occurs over a period of time. At room temperature, an incubation time of about 30 minutes is generally sufficient.

Unbound sample can then be removed by washing the solid support with an appropriate buffer, such as PBS containing 0. 1% Tween 20. Detection reagent can then be added to the solid support. An appropriate detection reagent is any compound that binds to the immobilized antibody-protein complex and that can be detected by any of a variety of means known to those in the art. Typically, the detection reagent contains a binding agent (such as, for example, Protein A, Protein G, immunoglobulin, lectin or free antigen) conjugated to a reporter group. Suitable reporter groups include enzymes (such as horseradish peroxidase or alkaline phosphatase), substrates, cofactors, inhibitors, dyes, radionuclides, luminescent groups, fluorescent groups, and biotin. The conjugation of a binding agent to the reporter group can be achieved using standard methods known to those of ordinary skill in the art. Common binding agents, pre-conjugated to a variety of reporter groups, can be purchased from many commercial sources (e.g., Zymed Laboratories, San Francisco, Calif., and Pierce, Rockford, Ill.).

The detection reagent is then incubated with the immobilized antibody-protein complex for an amount of time sufficient to detect the bound antibody. An appropriate amount of time can generally be determined from the manufacturer's instructions or by assaying the level of binding that occurs over a period of time. Unbound detection reagent is then removed and bound detection reagent is detected using the reporter group. The method employed for detecting the reporter group depends upon the nature of the reporter group. For radioactive groups, scintillation counting or autoradiographic methods are generally appropriate. Spectroscopic methods can be used to detect dyes, luminescent groups and fluorescent groups. Biotin can be detected using avidin, coupled to a different reporter group (commonly a radioactive or fluorescent group or an enzyme). Enzyme reporter groups can generally be detected by the addition of substrate (generally for a specific period of time), followed by spectroscopic or other analysis of the reaction products.

To determine the presence or absence of anti-ancestor or COT protein antibodies in the sample, the signal detected from the reporter group that remains bound to the solid support is generally compared to a signal that corresponds to a predetermined cut-off value. In one embodiment, the cut-off value is the average mean signal obtained when the immobilized ancestor or COT protein is incubated with samples from non-immunized subject.

In a related embodiment, the assay is performed in a rapid flow-through or strip test format, wherein the ancestor or COT protein is immobilized on a membrane, such as, for example, nitrocellulose, nylon, PVDF, and the like. In the flow-through test, antibodies within the sample bind to the immobilized polypeptide as the sample passes through the membrane. A detection reagent (e.g., protein A-colloidal gold) then binds to the antibody-protein complex as the solution containing the detection reagent flows through the membrane. The detection of bound detection reagent can then be performed as described above. In the strip test format, one end of the membrane to which the ancestor or COT protein is bound is immersed in a solution containing the sample. The sample migrates along the membrane through a region containing the detection reagent and to the area of immobilized ancestor or COT protein. The concentration of the detection reagent at the protein indicates the presence of anti-ancestor or COT protein antibodies in the sample. Typically, the concentration of detection reagent at that site generates a pattern, such as a line, that can be read visually. The absence of such a pattern indicates a negative result. In general, the amount of protein immobilized on the membrane is selected to generate a visually discernible pattern when the biological sample contains a level of antibodies that would be sufficient to generate a positive signal (e.g., in an ELISA) as discussed supra. Typically, the amount of protein immobilized on the membrane ranges from about 25 ng to about 1 .mu.g, and more typically from about 50 ng to about 500 ng. Such tests can typically be performed with a very small amount (e.g., one drop) of subject serum or blood.

Cytotoxic T-Lymphocyte Assay

Another factor in treating HIV-1 infection is the cellular immune response, in particular the cellular immune response involving the CD8.sup.+ cytotoxic T lymphocytes (CTL's). A cytotoxic T lymphocyte assay can be used to monitor the cellular immune response following sub-genomic immunization with an ancestral or COT viral sequence against homologous and heterologous HIV strains, as above using standard methods (see, e.g., Burke et al., supra; Tigges et al., supra).

Conventional assays utilized to detect T cell responses include, for example, proliferation assays, lymphokine secretion assays, direct cytotoxicity assays, limiting dilution assays, and the like. For example, antigen-presenting cells that have been incubated with an ancestor or COT protein can be assayed for the ability to induce CTL responses in responder cell populations. Antigen-presenting cells can be cells such as peripheral blood mononuclear cells or dendritic cells. Alternatively, mutant non-human mammalian cell lines that are deficient in their ability to load class I molecules with internally processed peptides and that have been transfected with the appropriate human class I gene, can be used to test the capacity of an ancestor or COT peptide of interest to induce in vitro primary CTL responses.

Peripheral blood mononuclear cells (PBMCs) can be used as the responder cell source of CTL precursors. The appropriate antigen-presenting cells are incubated with the ancestor or COT protein, after which the protein-loaded antigen-presenting cells are incubated with the responder cell population under optimized culture conditions. Positive CTL activation can be determined by assaying the culture for the presence of CTLs that kill radio-labeled target cells, both specific peptide-pulsed targets as well as target cells expressing endogenously processed forms of the antigen from which the peptide sequence was derived.

Another suitable method allows direct quantification of antigen-specific T cells by staining with Fluorescein-labeled HLA tetrameric complexes (Altman et al., Proc. Natl. Acad. Sci. USA 90:10330 (1993); Altman et al., Science 274:94 (1996)). Other relatively recent technical developments include staining for intracellular lymphokines, and interferon release assays or ELISPOT assays. Tetramer staining, intracellular lymphokine staining and ELISPOT assays are typically at least 10-fold more sensitive than more conventional assays (Lalvani et al., J. Exp. Med. 186:859 (1997); Dunbar et al., Curr. Biol. 8:413 (1998); Murali-Krishna et al., Immunity 8:177 (1998)).

Diagnosis

The present invention also provides methods for diagnosing viral (e.g., HIV) infection and/or AIDS, using the ancestor or COT viral sequences described herein. Diagnosing viral (e.g., HIV) infection and/or AIDS can be carried out using a variety of standard methods well known to those of skill in the art. Such methods include, but are not limited to, immunoassays, as described supra, and recombinant DNA methods to detect the presence of nucleic acid sequences. The presence of a viral gene sequence can be detected, for example, by Polymerase Chain Reaction (PCR) using specific primers designed using the sequence, or a portion thereof, set forth in Tables 1 or 3, using standard techniques (see, e.g. Innis et al., PCR Protocols A Guide to Methods and Application (1990); U.S. Pat. Nos. 4,683,202; 4,683,195; and 4,889,818; Gyllensten et al., Proc. Natl. Acad. Sci. USA 85:7652-56 (1988); Ochman et al., Genetics 120:621-23 (1988); Loh et al., Science 243:217-20 (1989)). Alternatively, a viral gene sequence can be detected in a biological sample using hybridization methods with a nucleic acid probe having at least 70% identity to the sequence set forth in Tables 1 or 3, according to methods well known to those of skill in the art (see, e.g., Sambrook et al., supra).

EXAMPLES

Example 1

Determination of Ancestral Viral Sequences

Sequences representing genes of a HIV-1 subtype C were selected from the GenBank and Los Alamos sequence databases. 39 subtype C sequences were used. 18 outgroup sequences (two from each of the other group M subtypes (FIG. 8) were used as an outgroup to root the subtype C sequences. The sequences were aligned using CLUSTALW (Thompson et al., Nucleic Acids Res. 22:4673-80 (1994)), the alignments were refined using GDE (Smith et al., CABIOS 10:671-5 (1994)), and amino acid sequences translated from them. Gaps were manipulated so that they were inserted between codons. This alignment (alignment I) was modified for phylogenetic analysis so that regions that could not be unambiguously aligned were removed (Learn et al., J. Virol. 70:5720-30 (1996)) resulting in alignment II.

An appropriate evolutionary model for phylogeny and ancestral state reconstructions for these sequences (alignment II) was selected using the Akaike Information Criterion (AIC) (Akaike, IEEE Trans. Autom. Contr. 19:716-23 (1974)) as implemented in Modeltest 3.0 (Posada and Crandall, Bioinformatics 14: 817-8 (1998)). For the analysis for the subtype C ancestral sequence the optimal model is equal rates for both classes of transitions and different rates for all four classes of transversions, with invariable sites and a X distribution of site-to-site rate variability of variable sites (referred to as a TVM+I+G model). The parameters of the model in this case were: equilibrium nucleotide frequencies: f.sub.A=0.3576,f.sub.C=0.1829,f.sub.G=0.2314,f.sub.T=0.2290; proportion of invariable sites=0.2447; shape parameter (.alpha.) of the X distribution=0.7623; rate matrix (R) matrix values: R.sub.A.fwdarw.C=1.7502, R.sub.A.fwdarw.G=R.sub.C.fwdarw.T=4.1332, R.sub.A.fwdarw.T=0.6825, R.sub.C.fwdarw.G=0.6549, R.sub.G.fwdarw.T=1.

Evolutionary trees for the sequences (alignment II) were inferred using maximum likelihood estimation (MLE) methods as implemented in PAUP* version 4.0b (Swofford, PAUP 4.0: Phylogenetic Analysis Using Parsimony (And Other Methods). Sinauer Associates, Inc. (2000)). Specifically for the subtype C sequences, ten different subtree-pruning-regrafting (SPR) heuristic searches were performed each using a different random addition order. All ten searches found the same MLE phylogeny (LnL=-33585.74). The ancestral nucleotide sequence for subtype C was inferred to be the sequence at the basal node of this subtype using this phylogeny, the sequences from the databases (alignment II), and the TVM+I+G model above using marginal likelihood estimation (see below).

This inferred sequence does not include predicted ancestral sequence for portions of several variable regions (V1, V2, V4 and V5) and four additional short regions that could not be unambiguously aligned (these eight regions were removed from alignment I to produce alignment II). The following procedure was used to predict amino acid sequences for the complete gp160 including the highly variable regions. The inferred ancestral sequence was visually aligned to alignment I and translated using GDE (Smith et al., supra). Since the highly variable regions were deleted as complete codons, the translation was in the correct reading frame and codons were properly maintained. The ancestral amino acid sequence for the regions deleted from alignment II were predicted visually and refined using a parsimony-based sequence reconstruction for these sites using the computer program MacClade, version 3.08a (Maddison and Maddison. MacClade--Analysis of Phylogeny and Character Evolution--Version 3. Sinauer Associates, Inc. (1992)). This amino acid sequences was converted to DNA sequence optimized for expression in human cells using the BACKTRANSLATE program of the Wisconsin Sequence Analysis Package (GCG), version 10 and a human gene codon table from the Codon Usage Database (www.kazusa.or.jp/codon/cgi-bin/showcodon.cgi?species=Homo+sapiens+[gbpri- ]).

Example 2

Different methods are available to determine the maximum likelihood phylogeny for a given subtype. One such method is based on the coalescent theory, which is a mathematical description of the genealogy of a sample of gene sequences drawn from a large evolving population. Coalescence analysis takes into account the HIV population in vivo and in the larger epidemic and offers a way of understanding how sampled genealogies behave when different processes operate on the HIV population. This theory can be used to determine the sequence of the ancestral viral sequence, such as a founder, or MRCA. Exponentially growing populations have decreasing coalescent intervals going back in time, while the converse is true for a declining population.

Epidemics in the USA and Thailand are growing exponentially. The coalescent dates for subtype B epidemics in the USA and Thailand are in accordance with the epidemiologic data. The coalescent date for subtype E epidemic in Thailand is earlier than predicted from the epidemiologic data. Potential reasons that can account for this discrepancy include, for example, the existence of multiple introductions of HIV-1 (there is no evidence from phylogenetics on this point), the absence of HIV-1 detection in Thailand for about 7 years, and the difference in the mutation rates for env gene in the HIV-1 subtypes E and B.

The Unit of Reconstruction

This unit of reconstruction relates to the ancestral viral sequence (i.e., state) state that is reconstructed. There are three possible units of reconstruction: nucleotides, amino acids or codons. In one embodiment, the states of the individual nucleotides are reconstructed and the amino acid sequences are then determined on the basis of this reconstruction. In another embodiment, the amino acid ancestral states are directly reconstructed. In a typical embodiment, the codons are reconstructed using a likelihood-based procedure that uses a codon model of evolution. A codon model of evolution takes into account the frequencies of the codons and implicitly the probability of substituting one nucleotide for another--in other words, it incorporates both nucleotide and amino acid substitutions in a single model. Computer programs capable of doing this are available or can readily be developed, as will be appreciated by the skilled artisan.

Use of Marginal or Joint Likelihoods for Estimating the Ancestral States

The ancestral state can be estimated using either a marginal or a joint likelihood. The marginal and joint likelihoods differ on the basis of how ancestral states at other nodes in the phylogenetic tree estimated. For any particular tree, the probability that the ancestral state of a given site on a sequence alignment at the root is, for example, an A can be determined in different ways.

The likelihood that the nucleotide is an adenine (A) can be determined regardless of whether higher nodes (i.e., those nodes closer to the ancestral viral sequence, founder or MRCA) have an adenine, cytosine (C), guanine(G), or thymine (T). This is the marginal likelihood of the ancestral state being A.

Alternatively, the likelihood that the nucleotide is an A can be determined depending on whether the nodes above are A, C, G, or T. This estimation is the joint likelihood of A with all the other ancestral reconstructions for that site.

The joint likelihood is a preferred method when all the ancestral states along the entire tree need to be determined. To establish the most likely states at one given node, the marginal likelihood is preferably used. In case of uncertainty at a particular site, a likelihood estimate of the ancestral state allows testing whether one state is statistically better than another. If two possible ancestral states do not have statistically different likelihoods, or if one ends up with multiple states over a number of sites building all possible sequences is not desirable. The likelihoods of all combinations can however be computed and ranked, and only those above a certain critical value are used. For example, when two sites on a sequence, each with different likelihoods for A, C, G, T, are considered: L(A) L(C) L(G) L(T)* * L represents the -lnL (the negative log-likelihood); therefore, the smaller the more likely. Site 1 3 2 1.5 1 Site 2 10 7 5 1 there are 16 possible sequence configurations, each with its own log-likelihood, that is simply the sum of the log-likelihoods for each base, which are:

TABLE-US-00007 AA 13 CA 12 GA 11.5 TA 11 AC 10 CC 9 GC 8.5 TC 8 AG 8 CG 7 GG 6.5 TG 6 AT 4 CT 3 GT 2.5 TT 2

In order of likelihood the ranking is:

TABLE-US-00008 TT, GT, CT, AT, TG, GG, CG, AG, TC, GC, CC, AC, TA, GA, CA, AA

The first four sequences have T at the second site. This results from the likelihood at that site being spread over a large range, resulting into a very low probability of having any nucleotide other than T at this site. At Site 1, however, any nucleotide tends to give quite similar likelihoods. This kind of ranking is one way of whittling down the number of possible sequences to look at if variation is to be taken into account.

The above variation in reconstructed ancestral states deals with variation that comes about because of the stochastic nature of the evolutionary process, and because of the probabilistic models of that process that are typically used. Another source of variation results from the sampling of sequences. One way of testing how sampling affects ancestral state reconstruction is to perform jackknife re-sampling on an existing data set. This involves deleting randomly without replacement of some portion (e.g., half) of the sequences, and reconstructing the ancestral state. Alternatively, the ancestral state can be estimated for each of a set of bootstrap trees, and the number of times a particular nucleotide was estimated can be reported as the ancestral state for a given site. The bootstrap trees are generated using bootstrapped data, but the ancestral state reconstructions use the bootstrap trees on the original data.

Different models of evolution can be used to reconstruct the ancestral states for the root node. Examples of models are known and can be chosen on a multitude of levels. For example, a model of evolution can be chosen by some heuristic means or by picking one that gives the highest likelihood for the ancestral sequence (obtained by summing the likelihoods over all sites). Alternatively the ancestral states are reconstructed at each site over all models of evolution, all of the likelihoods obtained summed, and the ancestral state chosen that has the maximum likelihood.

Example 3

The conservation of HIV-1 subtype C CTL amino acid consensus epitopes was analyzed. The total number of epitopes was 395. The table below summarize the results of the similarly of each circulating viral sequence to the C subtype CTL consensus sequence. The determined ancestor viral sequence for the HIV-1 subtype C env protein (SEQ ID NO:4) has the highest score (98.48%). Note that the scores for several strains are below 65%, because truncated sequences were used.

TABLE-US-00009 Sequence Name Total AA number Percentage CTL to Consensus cCanc95-mod1 389 98.48% cBR.92BR025 376 95.19% cBI.BU910717 363 91.90% cIN.21068 368 93.16% cIN.301905 370 93.67% cMW959.U08453 358 90.63% cBW.96BW1210 365 92.41% cBI.BU910316 367 92.91% cZAM176.U86778 352 89.11% cMW965.U08455 364 92.15% cZAM174.16.U86768 351 88.86% c84ZR085.U88822 322 81.52% cSN.SE364A 370 93.67% cMW960.U08454 365 92.41% cBI.BU910812 368 93.16% cET.ETH2220 358 90.63% cBI.BU910518 361 91.39% cIN.94IN11246 361 91.39% cBW.96BW15B03 359 90.89% cDJ.DJ259A 355 89.87% cBI.BU910213 365 92.41% cBW.96BW01B03 362 91.65% cIND760.L07655 255 64.56% cIN.301904 372 94.18% cSO.SM145A 354 89.62% cCHN19.AF268277 356 90.13% cIND747.L07653 255 64.56% cBW.96BW0402 364 92.15% cBI.BU910611 367 92.91% cBI.BU910423 359 90.89% cBW.96BW17B05 355 89.87% cBW.96BW0502 367 92.91% cUG.UG268A2 372 94.18% cZAM18.L22954 365 92.41% cIN.301999 368 93.16% c91BR15.U39238 371 93.92% cDJ.DJ373A 361 91.39% cBI.BU910112 369 93.42% c93IN101.AB023804 365 92.41% cBW.96BW16B01 361 91.39% cBW.96BW11B01 361 91.39% cINdiananc66 363 91.90%

Example 4

Ancestor sequence reconstruction was performed on simian immunodeficiency viruses grown in macaques. Macaques were infected and challenged with a relatively homogeneous SIV inoculum. Viral sequences were obtained up to three years following infection and were used to deduce an MRCA using maximum likelihood phylogeny analysis. The resulting sequence was compared to the consensus sequence of the inoculum. The MRCA sequence was found to be 97.4% identical to the virus inoculum. This figure improved to 98.2% when convergence at 5 glycosylation sites was removed--this convergence was due to readaptation of the virus from tissue culture to growth in the animal (Edmonson et al., J. Virol. 72:405-14 (1998)). The MRCA sequence and the consensus sequence were found to differ at 1.5% at the nucleotide level. FIG. 3 illustrates the determination of simian immunodeficiency virus MRCA phylogeny.

Example 5

An experiment to test the biological activity of the HIV-1 subtype B ancestral viral env gene sequence was performed. A nucleic acid sequence encoding the HIV-1 subtype B ancestral viral env gene sequence was assembled from long (160-200 base) oligonucleotides; the assembled gene was designated ANC1. The biological activity of ANC1 HIV-1-B Env was evaluated in co-receptor binding and syncytium formation assays. The plasmid pANC1, harboring the determined and chemically synthesized HIV-1 subtype B Ancestor gp160 Env sequence, or a positive control plasmid containing the HIV-1 subtype B 89.6 gp160 Env, was transfected into COS7 cells. These cells are capable of taking up and expressing foreign DNA at high efficiencies and thus are routinely used to produce viral proteins for presentation to other cells. The transfected COS7 cells were then mixed with GHOST cells expressing either one of the two major HIV-1 co-receptor proteins, CCR5 or CXCR4. CCR5 is the predominant receptor used by HIV early in infection. CXCR4 is used later in infection, and use of the latter receptor is temporally associated with the development of disease. The COS7-GHOST-co-receptor+ cells were then monitored for giant cell formation by light microscopy and for expression of viral Env protein by HIV-Env-specific antibody staining and fluorescence detection.

Cells expressing the ANC1 Env were shown to be expressed by virtue of binding to HIV-specific antibody and fluorescent detection, and to cause the formation of giant multinucleated cells in the presence of the CCR5 co-receptor, but not the CXCR4 co-receptor. The positive control 89.6 Env uses both CCR5 and CXCR4 and formed syncytia with cells expressing either co-receptor. Thus, the ANC1 Env protein was shown to be biologically active by co-receptor binding and syncytium formation.

Example 6

Maximum likelihood phylogeny reconstruction differs from traditional consensus sequence determinations because a consensus sequence represents a sequence of the most common nucleotide or amino acid residue at each site in the sequence. Thus, a consensus sequence is subject to biased sampling. In particular, the determination of a consensus sequence can be biased if many samples have the same sequence. In addition, the consensus sequence is a real viral sequence.

In contrast, maximum likelihood phylogeny analysis is less likely to be affected by biased sample because it does not determine the sequence of a most recent common ancestor based solely on the frequencies of the each nucleotide at each position. The determined ancestral viral sequence is an estimate of a real virus, the virus that is the common ancestor of the sampled circulating viruses.

In the simplest of methods for determining an ancestral sequence, for a single site on a sequence alignment nucleotides are assigned to ancestral nodes such that the total number of changes between nodes is minimized; this approach is called a "most parsimonious reconstruction." An alternative methodology, based on the principle of maximum likelihood, assigns nucleotides at the nodes such that the probability of obtaining the observed sequences, given a phylogeny, is maximized. The phylogeny is constructed by using a model of evolution that specifies the probabilities of nucleotide substitutions. The maximum likelihood phylogeny is the one that has the highest probability of giving the observed data.

Referring to FIG. 5, a comparison is presented of parsimony methodology and maximum likelihood methodology of determining an ancestral viral sequence (e.g., a founder sequence or a most recent common ancestor sequence (MRCA)). The most parsimonious reconstruction ("MP") can have the undesirable problem of creating an ambiguous state at the ancestral branch point (i.e., node). In this example, the two descendant sequences from this node have an adenine (A) or guanine (G) at a particular position in the sequence. The most parsimonious reconstruction ("MP Reconstruction") for the ancestral sequence at this site is ambiguous, because there can be either an A or G (symbolized by "R") at this position. In contrast, a maximum likelihood phylogeny analysis applies knowledge about sequence evolution. For example, likelihood analysis relies, in part, on the identity of nucleotides at the same position in other variants. Thus, in this example, a G to A mutation is more likely than an A to G change because variant at the adjacent node also has a G at the same position.

Referring to FIG. 6, another example illustrates the differences in these methodologies to determine a most recent common ancestor. In this example, twelve sequences of seven nucleotides are presented. These sequences share the illustrated evolutionary history. A consensus sequence calculated from these sequences is CATACTG (SEQ ID NO: 19). In panel A, the maximum likelihood reconstruction of the determined ancestral node is shown as GATCCTG (SEQ ID NO: 7). Other determined sequences are presented adjacent the other internal nodes. In panel B, the most parsimonious reconstruction at the same nodes is presented. As shown, the most parsimonious reconstruction predicts the consensus sequence GAWCCTG (SEQ ID NO: 8), where "W" symbolizes that either an A or T is equally possible to be at the third position. Similarly other most parsimonious reconstructions are shown at the various internal nodes. At the seventh internal node, the last nucleotide is indicated with the symbol "V" representing that an A, C or G might be present. Also note in this example, the consensus sequence differs in at least two sites (the 1.sup.st and 4.sup.th positions) from either the maximum likelihood- or parsimony-determined sequence for the MRCA.

Example 7

MRCA and COT state reconstructions were performed. Complete HIV-1 genome sequences were obtained from the Los Alamos National Laboratory HIV Sequence database or from research done in support of the HIV Vaccine Trials Network (HVTN) in the Mullins laboratory (HVTN1925c1.US98, HVTN3605c9.US98, HVTN8229c6.US98, and HVTN941c16.US98). Each sequence set, subtype B (Table 7) and subtype C (Table 9), was aligned using CLUSTALW (Thompson, 1994 #6159). Resulting alignments were examined and adjusted using amino acid alignments as guides with MacClade version 4 (Maddison, 2001 #13641). Alignment gaps were inserted between adjacent codons. Regions that could not be unambiguously aligned were omitted from the following phylogenetic analyses but included in the ancestral state reconstructions (see below). The complete genome alignments were partitioned into 9 gene coding regions (gag, env (encoding gp160), nef, pol, rev, tat, vif, vpr, and vpu); tat and rev exon sequences were spliced together after the intervening intron sequence was removed to produce a single continuous protein coding region.

Parameter values of optimal evolutionary models for each of these nucleotide alignments, except for subtype C env and gag, were estimated using MODELTEST (Posada, 1998 #10104) in conjunction with PAUP* (Swofford, 1999 #9501) based on the Akaike Information Criterion (Akaike, 1974 #10404). For subtype C env and gag, PAUP* was used to estimate HKY parameter values (Hasegawa, 1985 #9867) based on a neighbor-joining (Saitou, 1987 #5332) phylogenetic tree. Evolutionary model parameter values are presented for the subtype B genes (Table 8) and the subtype C genes (Table 10). These values were used for maximum-likelihood phylogenetic analyses using PAUP* (Swofford, 1999 #9501). For all genes except subtype C env and gag, 10 random-addition subtree-pruning-regrafting iterations were done. For subtype C env and gag, a single iteration based on a neighbor-joining starting trees were used.

After phylogenetic trees were obtained, the regions of ambiguous alignment that were removed (see above) were included in their original positions in the alignments. For the MRCA estimation, the sequence states at the ancestral node (the point at which the subtype D sequences attach to the portion of the tree that solely included subtype B sequences) were derived using marginal maximum-likelihood estimation with PAUP* {Swofford, 1999 #9501}. The MRCA protein sequence is the translation of the MRCA nucleotide sequence. For the COT state reconstructions, the outgroup sequences (subtype D sequences in Table 7; subtype other than C in Table 9) were pruned from the phylogenetic tree for each gene using PAUP* (Swofford, 1999 #9501). The remaining gene trees were analyzed using perl computer programs to determine the point at the center of the phylogenetic tree via the Minimum of Means, and Least-Squares Method. Evolutionary states at this point for each method for each gene tree were then estimated using marginal state estimation using PAUP* (Swofford, 1999 #9501).

TABLE-US-00010 TABLE 7 Sequence Names, GenBank Accession Numbers and Country of Isolation for the sequences used in the complete genome MRCA and COT estimation of subtype B. Accession Sequence number Country Subtype.sup..sctn. 1WK.KR97 AF224507 Korea B 3202A21.NL86 U34604 Netherlands B 89SP061.ES89 AJ006287 Spain B AD8.US86 AF004394 U.S.A. B ARCH054.AR98 AY037268 Argentina B ARMA173.AR99 AY037274 Argentina B ARMS008.AR00 AY037269 Argentina B BK132.TH90 AY173951 Thailand B BOL122.BO99 AY037270 Bolivia B BZ167.BR89 AY173956 Brazil B CAM1.GB83 D10112 United Kingdom B D31.DE86 U43096 Germany B DH123.US91 AF069140 U.S.A. B HAN.DE86 U43141 Germany B HVTN1925c1.US98 * U.S.A. B HVTN3605c9.US98 * U.S.A. B HVTN8229c6.US98 * U.S.A. B HVTN941c16.US98 * U.S.A. B HXB2.FR83 K03455 France B JRCSF.US86 M38429 U.S.A. B MBC200.AU86 AF042100 Australia B MBC925.AU87 AF042101 Australia B MBCC18B.AU93 AF042102 Australia B MBCC98.AU96 AF042104 Australia B MBCD36.AU96 AF042105 Australia B MN.US84 M17449 U.S.A. B NY5.US84 M38431 U.S.A. B OYI.GA88 M26727 Gabon B P896.US89 U39362 U.S.A. B RF.US83 M17451 U.S.A. B RL42.CN U71182 China B SF2.US83 K02007 U.S.A. B TWCYS.TW94 AF086817 Taiwan B WCIPR9018.US90 U69591 U.S.A. B WEAU160.US90 U21135 U.S.A. B WR27.US88 U26546 U.S.A. B YU2.US86 M93258 U.S.A. B D.ELI.CD83 K03454 D. R. Congo D D.UG1141.UG94 U88824 Uganda D D.ZR085.CD84 U88822 D. R. Congo D .sup..sctn.subtype D sequences included as an outgroup to root the clade B phylogeny; *unpublished complete genome sequences from HVTN project.

TABLE-US-00011 TABLE 8 Evolutionary model parameters for phylogenetic analysis and ancestral and COT state reconstruction for subtype B gene sequences. Assumed proportion of Shape invariable gene R.sub.A<->C R.sub.A<->G R.sub.A<->T R.sub.C<->- ;G R.sub.C<->T R.sub.G<->T parameter sites freq(A) freq(C) fre- q(G) freq(T) gag 1.787 4.4321 0.8198 0.9897 5.2536 1 0.5253 0.3028 0.3832 0.1751 0.2268- 0.2149 gp160 2.469 5.6546 1.1659 0.9752 5.6546 1 0.5833 0.2469 0.3575 0.1858 0.22- 36 0.2331 nef 1.2074 3.3345 1.193 0.5574 3.3345 1 0.5329 none 0.346 0.2094 0.2613 0.- 1833 pol 2.6829 11.9199 1.1681 1.342 11.9199 1 0.6476 0.369 0.3937 0.1717 0.213- 8 0.2208 rev 1 2.1615 0.3786 0.3786 2.1615 1 0.4075 none 0.3224 0.2751 0.2411 0.161- 4 tat 1.9279 3.0288 0.3625 1.2287 3.0288 1 0.5314 0.1979 0.3543 0.2489 0.219- 1 0.1777 vif 1.7739 3.7387 0.3906 0.5473 3.7387 1 0.5248 0.2459 0.3796 0.1809 0.231- 6 0.2079 vpr 3.657 7.8789 1.0026 1.3814 15.7753 1 0.6295 0.3193 0.3439 0.1894 0.238- 6 0.2281 vpu 2.3344 3.1513 1.4396 2.2202 8.3028 1 0.5218 none 0.368 0.1192 0.26 0.2- 528

TABLE-US-00012 TABLE 10 Evolutionary model parameters for phylogenetic analysis and ancestral and COT state reconstruction for subtype C gene sequences. Assumed proportion of Shape invariable gene R.sub.A<->C R.sub.A<->G R.sub.A<->T R.sub.C<->- ;G R.sub.C<->T R.sub.G<->T parameter sites freq(A) freq(C) fre- q(G) freq(T) Ti/Tv gag -- -- -- -- -- -- 0.3348 none 0.36904 0.18619 0.24234 0.20244 3.09542 gp160 -- -- -- -- -- -- 0.405447 none 0.34797 0.17109 0.23644 0.24449 2.39- 53 nef 2.0998 4.0826 1.4418 1.3501 5.7703 1 0.6877 0.1885 0.3426 0.2063 0.257- 9 0.1932 -- pol 1.9799 8.7785 1.078 0.7924 11.9393 1 0.7828 0.3661 0.3961 0.1705 0.223- 1 0.2103 -- rev 2.1477 5.7666 0.7273 0.3826 5.7666 1 0.71 0.1773 0.3116 0.2212 0.2668 - 0.2004 -- tat 4.1494 7.2595 1.082 1.0846 9.5448 1 0.6771 0.3018 0.3463 0.2367 0.2339- 0.1831 -- vif 1.6981 4.1865 0.8836 0.7624 5.8585 1 0.525 0.2597 0.3842 0.1794 0.2363- 0.2001 -- vpr 2.571 6.3751 0.9793 0.8462 9.8567 1 0.829 0.3024 0.3448 0.1814 0.2495 - 0.2243 -- vpu 1.5454 2.1322 0.5312 0.2609 5.7821 1 0.635 0.1585 0.4191 0.1036 0.2453- 0.232 --

TABLE-US-00013 TABLE 9 Sequence Names, GenBank Accession Numbers and Country of Isolation for the sequences used in the complete genome MRCA and COT estimation of subtype C. Accession Sequence number Country SUBTYPE.sup..sctn. 86ETH2220 U46016 Ethiopia C 92BR025 U52953 Brazil C 93IN904 AF067157 India C 94IN476 AF286223 India C 95IN21068 AF067155 India C 96BW0402 AF110962 Botswana C 96BW0408 AF110964 Botswana C 96BW0502 AF110967 Botswana C 96BW06J4 AF290028 Botswana C 96BW1106 AF110970 Botswana C 96BW1210 AF110972 Botswana C 96BW15B03 AF110973 Botswana C 96BW16B01 AF110976 Botswana C 96BW17A09 AF110979 Botswana C 96BW96BW01B03 AF110959 Botswana C 96BWM032 AF443075 Botswana C 96ZM651 AF286224 Zambia C 96ZM751 AF286225 Zambia C 97TZ04 AF361874 Tanzania C 97TZ05 AF361875 Tanzania C 97ZA012 AF286227 South Africa C 98BR004 AF286228 Brazil C 98BWMO1410 AF443079 Botswana C 98BWMO18D5 AF443080 Botswana C 98BWMO36A5 AF443081 Botswana C 98BWMO37D5 AF443082 Botswana C 98IN012 AF286231 India C 98IS002 AF286233 Israel C 98TZ013 AF286234 Tanzania C 98TZ017 AF286235 Tanzania C 99BW393212 AF443083 Botswana C 99BW46424 AF443084 Botswana C 99BW47458 AF443085 Botswana C 99BW47547 AF443086 Botswana C 00BW076820 AF443089 Botswana C 00BW087421 AF443090 Botswana C 00BW147127 AF443091 Botswana C 00BW16162 AF443092 Botswana C 00BW1686. AF443093 Botswana C 00BW17593 AF443094 Botswana C 00BW17835 AF443096 Botswana C 00BW17956 AF443097 Botswana C 00BW18113 AF443098 Botswana C 00BW18802 AF443100 Botswana C 00BW22767 AF443107 Botswana C 00BW38713 AF443110 Botswana C 00BW38769 AF443111 Botswana C 00BW38868 AF443112 Botswana C 00BW50311 AF443115 Botswana C ZA. .CTSC2 AY043176 South Africa C ZA. .DU151 AY043173 South Africa C ZA. .DU179 AY043174 South Africa C A KE.Q2317 AF004885 Kenya A B US.JRFL U63632 U.S.A. B D UG.94UG1 U88824 Uganda D F BE.VI850 AF077336 Belgium F G SE.SE616 AF061642 Sweden G J SE.SE928 AF082394 Sweden J H CF.90CF0 AF005496 C.A.R. H .sup..sctn.sequences from subtypes other than D included as an outgroup to root the clade C phylogeny.

Comparisons of the MRCA, Least Squares Method ("LSCOT") and Minimum of Means COT ("MMCOT") reconstructions for the Clade B gag, env (encoding gp160), nef, pol, rev, tat, vif, vpr, and vpu genes are shown in FIGS. 9 to 17. Comparisons of the MRCA, Least Squares Method ("LSCOT") and Minimum of Means COT ("MMCOT") reconstructions for the Clade B gag, env (gp160), nef, pol, rev, tat, vif, vpr, and vpu proteins are shown in FIGS. 18 to 26.

Comparisons of the MRCA, Least Squares Method ("LSCOT") and Minimum of Means COT ("MMCOT") reconstructions for the Clade C gag, env (encoding gp160), nef, pol, rev, tat, vif, vpr, and vpu genes are shown in FIGS. 27 to 35. Comparisons of the MRCA, Least Squares Method ("LSCOT") and Minimum of Means COT ("MMCOT") reconstructions for the Clade C gag, env (gp160), nef, pol, rev, tat, vif, vpr, and vpu proteins are shown in FIGS. 36 to 44.

Example 8

HIV-1 has high replication and mutation rates that permit rapid generation of viruses that can escape immune recognition. Within an infected host, the HIV-1 population diversifies over time, producing mostly defective viruses but nonetheless persisting and accumulating mutations at a rate of up to 1% per year in its env gene. HIV sequences sampled from a population of infected individuals recapitulate a star-like phylogeny, i.e., most of the variants sampled at the same time are positioned roughly equidistant from the center of the tree. Thus, any given variant is approximately twice this distance from any other circulating strain. A primary concern in designing protective AIDS vaccines, then, is the choice of strains likely to best provide protection against the expanding population of HIV-1 variants.

Several methods for choosing a vaccine candidate on the basis of genetic or protein sequence data have been put forth recently. First, and the approach followed in current clinical trials, is to choose one or a small number of laboratory-grown or primary viral isolates, typically chosen to approximate a "circulating" strain or to simply match the HIV-1 subtype(s) in the targeted population. An advantage of this approach is that it typically employs viral genes derived from a viable virus and thus produces antigens likely to adopt "native" conformations. However, as a result of HIV-1 mutational radiation, any given "circulating" strain will be genetically, and presumably antigenically, dissimilar to other strains likely to be encountered by the vaccinee, with the degree of dissimilarity proportional to the length of time the virus has been circulating within the population. Thus, vaccines based on specific viral isolates are unlikely to be effective against a broad range of circulating viruses. The results of a first Phase III AIDS vaccine trial suggest that monomeric envelope proteins that are derived from such isolates are insufficient to provide protective immunity, although it remains an open question whether more native presentations of envelope protein might be an effective vaccine component.

To enhance the breadth of the elicited immune response, a second approach is to include as many diverse HIV-1 isolates as possible in the vaccine recipe, with the intention of inducing multiple responses against different Env proteins. This approach has been explored to a limited degree, without clear success. A third approach is to build a consensus sequence based on either circulating strains or strains in the HIV database. The consensus sequence will be genetically closer to circulating strains than any given natural virus isolate, but its sequence may be biased by sampling and may link polymorphisms in combinations not found in any natural or viable virus, thus potentially resulting in inappropriate structural conformations. Consequently, there is a need for new, effective methods of identifying candidate sequences for vaccine development to treat and/or prevent HIV infection.

To this end, the use of an HIV population ancestral sequence can be used as a vaccine candidate. Such a vaccine might correspond to an ancestor of all known HIV strains, an HIV sequence subtype, or viruses circulating in a given geographic region or risk group. The ancestral viral sequence is reconstructed from a phylogenetic tree describing the historical relationships of sequences sampled from the population of interest, and is thus expected to correspond to the most recent common ancestor (MRCA) of the viral strains sampled from the targeted population. It is also likely that such an ancestral sequence will encompass elements conserved within the sampled virus population. To maximize the opportunity for native Envelope protein expression in vivo, DNA vaccines expressing full-length Env gp160 or TM-truncated Env gp140 have been used. These vaccines have been shown are effective in both priming and boosting in macaques. Here, a predicted ancestral env sequence for subtype B HIV-1 encodes a functional protein that elicits neutralizing antibodies to primary isolates in rabbits. Boosting with gp120 protein did not significantly enhance the neutralization capacity of the sera.

Methods

Ancestral state simulations. To assess the accuracy of the ancestral state reconstruction method, the following in silico study was performed. Using the program Seq-Gen (Rambaut and Grassly, Comput. Appl. Biosci. 13:235-8 (1997)), the evolution of sequences was simulated, given an ancestral sequence and following the phylogenetic histories depicted in the trees in FIG. 45A and the model of evolution shown in Table 11. One hundred replicate data sets were generated for both tree shapes. The PAUP* (Swofford, D. 1999. PAUP*, Sinauer Associates, Inc.) to generate a maximum-likelihood tree and an ancestral sequence for all 200 datasets (100 simulations on both the star- and caterpillar-shaped phylogenies). To be conservative, the simulations were performed on trees with very long external branches (having a genetic distance of 20%), and the caterpillar trees had internal branch lengths of 1% genetic distance.

Ancestral state reconstruction of the SIVmacBK28 env sequence: SIVmac sequences obtained from a series of experimental infections of rhesus macaques (Edmonson et al., J. Virol. 72:405-14 (1998)) were used to reconstruct an ancestral sequence to compare to the plasmid-derived SIV clone used to inoculate these monkeys. A region of env from position 8,265 to 8,827 of the SIVmacBK28 genome was amplified by nested-PCR from uncultured PBMC DNA from the SIV infected macaques. First round primers were UP-3, positions (8,117 to 8,130), AGACTGCAGATGTGAAGAGGTACAC (SEQ ID NO: 122) and PEXTM6 (8,977 to 8,953), GGATCTGGTATGCTCATAGCAA (SEQ ID NO: 123). Second round primers were PEXTM7 (8,265 to 8,286), GATACTGCAGCAACAGCAACAGCTG (SEQ ID NO: 124) and UP-5 (8,827 to 8,810), GCAAAGCTTCTCTGGTTGGCAGTG (SEQ ID NO: 125). Amplified products were then cloned and sequenced. The GenBank accession numbers for these sequences are AY169007-AY169163. Methods for SIVmacBK28 ancestor reconstruction were identical to those outlined below. The model of evolution is given in Table 12 and the reconstructed sequence is provided in FIG. 46.

Ancestral state reconstruction of an HIV-1-B env sequence: Thirty-eight sequences representing env genes of HIV-1 subtype B were selected from GenBank and the Los Alamos sequence databases, with three additional sequences from clade D used as an outgroup for rooting (Kuiken et al., HIV Sequence Compendium 2001, Theoretical Biology and Biophysics Group, Los Alamos National Laboratory, Los Alamos, N.Mex.) (Table 11 and FIG. 45B). Sequences were aligned using CLUSTALW (Thompson et al., Nucleic Acids Res. 22:4673-80 (1994) and the alignment was refined using GDE (Smith et al., Comput Appl. Biosci. 10:671-5 (1994)). Inferred amino acid sequences were used to guide the introduction of alignment gaps such that they were inserted between codons. These alignments were then modified for phylogenetic analysis such that regions that could not be unambiguously aligned were removed (Learn et al., J. Virol. 70:5720-30 (1996)).

An appropriate evolutionary model for phylogeny and ancestral state reconstructions was selected using the Akaike Information Criterion (AIC) as implemented in Modeltest 3.0 (Posada and Crandall, Bioinformatics 14:817-8 (1998)). (See Table 12). Evolutionary trees for the sequences (B alignments) were inferred using maximum-likelihood estimation methods as implemented in PAUP* version 4.0b10 (Swofford, D. 1999. PAUP*, Sinauer Associates, Inc.). Ten different subtree-pruning-regrafting (SPR) heuristic searches were performed using a different random-addition order each time and using a neighbor-joining tree of the subtype B sequences as a backbone constraint. The ancestral nucleotide sequence was inferred as the sequence at the basal node of the clade using the inferred phylogenies. The nucleotide at each site of the ancestral sequence had the highest likelihood, integrated over all state assignments at other nodes. This derived sequence is provided in FIG. 46.

To predict the amino acid sequences for the complete gp160, including the highly variable regions, the inferred ancestral sequences were visually aligned to the alignments prior to gap-stripping and translated using GDE (Smith et al., Comput. Appl. Biosci. 10:671-5 (1994)). Since the highly variable regions were deleted as complete codon triplets, the translations into amino acids were in the correct reading frame and codons were properly maintained. The ancestral amino acid sequences for the regions deleted from the gap-striped alignment were predicted visually and refined using parsimony-based sequence reconstruction for these sites using the program MacClade (Maddison and Maddison, MacClade--Analysis of Phylogeny and Character Evolution, 3rd ed., Sinauer Associates, Inc, Sunderland, Mass. (1992)). The complete gp160 amino acid sequences were converted to DNA sequences optimized for expression in human cells using the BACKTRANSLATE program of the Wisconsin Sequence Analysis Package (GCG), version 10 and a human gene codon table from a Codon Usage Database (Nakamura et al., Nucleic Acids Res. 28:292 (2000)). The gene was then chemically synthesized, sequence-verified, and inserted into a mammalian expression vector to produce the plasmid pVR1012-AN1env (Vical, Inc). To generate a gp140 expression plasmid, pVR112-AN1env was subjected to site-directed mutagenesis (Quik-change kit, Stratagene) to introduce two stop codons after the codon for amino acid 711 (nucleotides 2134-9)

Fusion assay. GHOST-CCR5 or GHOST-CXCR4 cells were seeded in two-chamber culture slides (Falcon, Franklin Lakes, N.J.) at 5.times.10.sup.4 cells/well, and transfected the next day with 0.5 .mu.g env plasmid using Fugene-6 (Roche, Indianapolis, Ind.). Transfected cells were allowed to fuse for two days and then were fixed and stained for immunofluorescence using the monoclonal antibody b12 (gift of Dr. Dennis Burton) and 1:2500 FITC-conjugated goat anti-human IgG (Cappel). The presence of large multinucleated cells indicated that cell-cell fusion had occurred.

Western blot analysis. 293T cells were transfected in 6-well dishes with 1 .mu.g env plasmid and Fugene-6, and medium and cell lysates were collected 48 hours later. Twenty one .mu.l of each sample were used for SDS-PAGE, transferred to a PVDF membrane, probed with heat-inactivated pooled human HIV.sup.+ serum (gift of Dr. Lisa M. Frenkel) and horseradish peroxidase-conjugated goat anti-human IgG (Sigma, St Louis, Mo.), and then developed with the color substrate 3-amino-9-ethylcarbazole (AEC).

Recombinant protein production. Recombinant gp120 from HIV-1 SF162 was provided by Chiron Corp and prepared as described in (Barnett et al., Vaccine 15:869-73 (1997)).

Rabbit immunizations. New Zealand White rabbits were vaccinated by Gene Gun (Bio-Rad, Hercules, Calif.). Eighteen shots of 2 .mu.g DNA each were given for each dose. Animals were vaccinated at weeks 1, 5, 10, 19, 23, and 46. Protein boosts were given at weeks 68 and 78; these consisted of 44 .mu.g recombinant gp120 mixed with an equal volume of Incomplete Freund's adjuvant, injected intramuscularly. Blood was collected the day before and 2 weeks after each immunization. Animals were housed at R+R Rabbitry, Marysville, Wash. and procedures followed IACUC-approved protocols.

Neutralization assays. cMAGI assay. Assays were performed as described by (Doria-Rose et al., J. Virol. 77:11563-77 (2003). Briefly, serial dilutions of sera were incubated with virus for one hour, then added to duplicate wells of cMAGI cells. After two days, cells were fixed and stained for .beta.-galactosidase expression (indicating infection). The percent neutralization at a given titer is calculated by the equation (Vo-Vn)/Vo.times.100, where Vn is the number of infected cells in the virus+antibody wells and Vo is the number of positive cells in virus-alone wells. Titers were normalized to the titer of a standard HIV.sup.+ human serum pool that was included on each assay plate.

Luciferase reduction assay (M7-luc assay). 500 TCID.sub.50 of virus was mixed and incubated with serial dilutions of serum in triplicate for 1 hr at 37.degree. C. in 96-well microculture plates. 5.25.EGFP.Luc.M7 cells (kindly provided by Dr. Nathaniel Landau (Brandt et al., J. Biol. Chem. 277:17291-9 (2002)) suspended in RPMI with 12% fetal bovine serum and 25 .mu.g/ml DEAE-dextran at 7.5.times.10.sup.4 cells per well were then added and incubated for 3-4 days. These cells express luciferase and GFP upon HIV infection. Luciferase was measured using a commercial kit (Bright-Glo, Promega, Madison, Wis.) and a Fluoroskan luminometer. The titer was calculated as the dilution at which relative light units (RLU) were 50% that of virus-only wells. For the large panel of viruses, sera were assayed at a 1:10 dilution and the percent reduction in relative light units (RLU) was calculated in relation to a pre-immunization serum sample for each rabbit. A reduction of 50% is considered significant in this assay.

Results

Method validation: First, the ability of likelihood-based phylogenetic methods to accurately predict ancestral sequences was evaluated using both a simulation and an experimental approach. HIV-1 is widely considered to have a star-like phylogeny with all external branches radiating from the same central point on the tree. However, there is at least some level of substructure in the phylogeny (i.e., some short internal branches at the base of the tree). In terms of tree space, the realized HIV-1 tree (see FIG. 45B) is somewhere between a true star and what can be called a caterpillar tree (i.e., long external branches with short internal branches) (FIG. 45a). To estimate the accuracy of ancestral state reconstruction, 100 simulations were performed with a known ancestor at each of the two extremes of the star-caterpillar continuum. For the caterpillar tree, the MRCA was estimated with 95.4% accuracy. For the star phylogeny, the MRCA was estimated with 98.2% accuracy. Since the true HIV phylogeny falls somewhere between the caterpillar and star structures, the MRCA reconstruction accuracy was estimated to be in the range of 95-98%.

Viral gene sequences taken from 4 rhesus macaques infected with a molecularly defined strain of SIV, SIVmac251-BK28 (Kornfeld et al., Nature 326:610-3 (1987) were used in an effort to assess the reconstruction of the infecting viral sequence. From a phylogenetic tree of 1.1 kb env gene fragments taken between 1-3 years post infection, 99.8% of the infecting viral sequence (373 of 376 amino acids), including 98.2% of the 170 variable sites, were accurately predicted.

Derivation of subtype B ancestor: Using the same procedures, an ancestral nucleotide sequence of the envelope gene of HIV-1 subtype B, the most extensively evaluated HIV-1 subtype to date, was derived using the phylogeny shown in FIG. 45b. This ancestral sequence (AN1-EnvB) produced an open reading frame encoding a complete, 884-amino acid gp160 gene product. The amino acid distances between the ancestral sequence and the natural subtype B strains used to estimate it were 12.3% on average (range: 8.0-21.0%) while these sequences were 17.3% different from each other (range: 13.3-23.2%). At 884 amino acids, the encoded protein is long relative to most sequences; much of the additional length is in the variable loops. Also unusual is the number of putative sites for N-linked glycosylation, also called sequins: the average is 25, while AN1-EnvB has 35. Based on features V3 sequence features, AN1-EnvB is predicted to use CCR5 as a coreceptor

In vitro validation of ancestor protein expression. The functional characteristics of the ancestral env sequence were evaluated. The deduced sequence was re-engineered to reflect the dominant patterns of human codon usage, without changing any of the encoded amino acids, and then chemically synthesized the gene and cloned it into a mammalian expression vector pVR1012. Transfection of this plasmid into COS-7 or 293T cells resulted in the production of high levels of viral protein gp160 and its cleavage products, gp120 and gp41--comparable to levels detected following transfection of the widely employed, humanized Env-gp160 gene of SHIV89.6P (Barouch et al., Science 290:486-92 (2000)). Control of viremia and prevention of clinical AIDS in rhesus monkey and higher than that of non-optimized HIV-1 89.6 Env. A truncated gp140 form, which is expected to retain the oligomeric properties of gp160 but is secreted, was also generated and expressed and shown to bind soluble CD4 in an ELISA assay. Western blot analysis of 293T cells transfected with either the gp160 or gp140 form showed that the expressed proteins were of the predicted sizes and were recognized by anti-HIV antibodies. Cell lysates contained both gp140 and gp160, while only the gp140 form appeared in the medium, showing that it was secreted as expected. Lysates of cells transfected with gp160 showed bands for gp41 and gp120, indicating that Env was cleaved to its mature form intracellularly. The cleavage was not complete, as full-length gp160 was also visible. The apparent molecular weight and somewhat diffuse nature of the bands suggested that the AN1-EnvB proteins were highly glycosylated.

Expression of gp160 in transfected cells was confirmed by an immunofluorescence assay. Cell-cell fusion occurred when GHOST-CCR5 cells (Cecilia et al., J. Virol. 72:6988-96 (1998)) expressing the HIV-1 receptors CD4 and CCR5 were transfected with AN1-EnvB gp160. In contrast, no fusion occurred when GHOST-CXCR4 cells expressing CD4 plus CXCR4 were transfected, although Env was expressed in these cells. These results indicated that AN1-EnvB used only CCR5, as predicted by its sequence, and that it was folded properly and fully functional for directing membrane fusion. To confirm the functionality of AN1-EnvB, pVR1012AN1env (encoding full-length gp160) was used to complement the env-defective genome Q23.DELTA.env(Long et al., AIDS Res. Hum. Retroviruses 18:567-76 (2002)). The resulting pseudotyped virus particles were able to infect permissive cMAGI cells. Concentrated virions had a titer of 1.1.times.10.sup.4 infectious particles/ml on cMAGI cells.

Immunogenicity of AN1-EnvB: To assess the humoral immune response potentially elicited by An1-EnvB, rabbits were immunized with DNA encoding gp160 and gp140 versions of the protein via Gene Gun followed by boosting with recombinant AN1-EnvB gp120 protein made in CHO cells. As controls, additional groups were immunized with a codon-optimized Env gp140 from a primary HIV-1 isolate, HIV-SF162 and recombinant SF162 gp120, or with the empty vector and adjuvant. All Envelope plasmids elicited Envelope-specific antibody binding titers in rabbits up to 1:1,000,000. Titers increased with protein boosting.

Virus neutralization: Antibody neutralization was measured using the cMAGI assay (Chackerian et al., J. Virol. 71:3932-9 (1997); Kimpton et al., J. Virol. 66:2232-9 (1992)) with five heterologous subtype B, primary R5 HIV-1 isolates SF162, 92US657, 92TH014, 91US056, and 92HT593, and one subtype C primary isolate, 93IN101 (Table 13). As a positive control, HIV.sup.+ sera pooled from several local subtype B-infected patients was included. All isolates tested were neutralized by this pool, but with differing sensitivities. After four DNA immunizations, all gp140-SF162-immunized rabbit sera had neutralizing activity against the homologous virus. Six of eight AN1-EnvB immunized rabbit sera also neutralized HIV-1 SF162, which in this case was heterologous. Seven of these eight rabbits had 50% neutralization titers of at least 1:8 against 92US657; all eight achieved 90% neutralization against 92TH014 after five immunizations (geometric mean titer=16). In contrast, sera against SF162 protein were less broadly reactive, with neutralization titers of at least 1:8 detected against two of five heterologous viruses (in one of four animals against 93IN101 and in two of four animals against 92US657). Furthermore, HIV 92TH014 and 92US657 were more effectively neutralized by the AN-1-EnvB-immunized rabbit sera compared with Env SF162 sera, with high titers at the 90% cutoff against 92TH014. Two of the six HIV-1 isolates tested were only very weakly or not neutralized by sera from any of the Env-immunized rabbits (92HT593 and 91US056), although the human HIV-positive serum control neutralized both. Subtype C 93IN101 was weakly neutralized by sera from at most one animal in each group.

These data were extended in a second neutralization assay using 5.25.EGFP.Luc.M7 cells. Strong neutralizing activity against HIV-1 SF162 was found in all Env-immunized rabbits, confirming the results in the cMAGI assay. This activity increased over the course of vaccinations. Titers increased with each of the first two immunizations and then declined, as has been seen with vaccinations in primates, while subsequent boosts resulted in sustained titers at a threshold level that was not additionally boosted. Levels of neutralizing antibody against HIV-SF162 were similar in each of the groups receiving homologous Env-SF162, or heterologous AN1-Env-B gp140 or AN1-Env-B-gp160. Inhibition of primary isolate Bx08 was found in four of eight AN1-EnvB- and in one of four SF162-immunized rabbits. However, none of the AN1-Env B-immunized rabbit sera neutralized Bal and JR-FL, whereas sera raised against Env-SF162 neutralized Bal and in one case, JR-FL. In summary, at least five of the eight primary (all heterologous) clade B viruses tested were neutralized by AN1-Env B sera (Table 14).

Discussion

Artificial HIV gene sequences, such as ancestors and consensus can be more effective vaccines and reagents than natural isolates. Such sequences can be engineered to contain more of the conserved features and epitopes found in primary isolates than any one isolate would have. This concept has been put into practice by predicting, synthesizing, and evaluating an ancestor of the HIV-1 clade B env gene. The AN1-EnvB protein can be stably expressed in mammalian cell lines, was glycosylated, bound CD4, directed cell-cell fusion, used CCR5 but not CXCR4, and complemented env-defective genomes. This functional protein elicited high titers of Env-binding antibodies in vaccinated mice and rabbits. After DNA priming and protein boosting, immunized rabbit sera neutralized a variety of primary isolates at a low level. gp140 constructs gave slightly higher titers (binding and neutralizing) than gp160, but this difference was not statistically significant.

The neutralization profile of AN1-EnvB-vaccinated rabbit sera supports the use of ancestral viral sequences as immunogenic compositions. In an effort to examine breadth of neutralization in an impartial manner, coded sera were tested using two different assays, performed in two different laboratories, using panels of laboratory and primary viruses with a single isolate in common, HIV-SF162. (No standard panel of HIV-1 primary isolates has been established for comparisons between laboratories to date.) Results in both laboratories indicated that sera from the AN1-EnvB-immunized rabbits could neutralize heterologous HIV-1 strains to a significant degree. Three of five subtype B viruses were neutralized by a majority of sera using the cMAGI assay and three of five subtype B viruses were neutralized by at least some of the sera in the M7-luc assay. Several rabbits showed low-level responses against one virus from subtype C, but none neutralized another C or the E subtype viruses tested. This profile was similar to that of sera from rabbits immunized with Env from HIV-1 SF162. Indeed, the neutralization of HIV-1 SF162 itself was similar for sera from rabbits given either immunogen, even though this isolate is homologous to the SF162 immunogen but heterologous to the AN1-EnvB immunogen. These data imply that the artificial sequence was at least as immunogenic as this natural isolate.

Several recent vaccine experiments have shown that DNA expressing unmodified HIV-1 Envelopes can elicit low-level antibodies that neutralize the homologous primary isolate at the 50% level. Low titer cross-reactive neutralizing antibodies were elicited using modified Envelope DNA vaccines or recombinant proteins. The codon-optimized vectors used here elicited high titers of binding antibodies with DNA vaccination alone. Responses to DNA vaccines can be effectively boosted with recombinant viral vectors or recombinant glycoproteins. The results reported here support the use of DNA vaccines alone in demonstrating low-level cross-clade neutralizing antibodies. Immunization of the DNA-primed rabbits with homologous gp120 subunit protein in adjuvant resulted in boosting of binding antibody titers against HIV-SF162. However, gp120 failed to boost heterologous (i.e., from a different HIV subtype) neutralizing antibody responses significantly. The gp140 and gp160 DNA vaccines presented native envelope conformations in vivo that were boosted poorly or not at all by adjuvated gp120 monomer proteins, raising responses only to the most neutralization-sensitive primary virus, HIV-SF162. Although native gp120 can bind conformation-dependent broad neutralizing antibodies, elicit neutralizing antibodies and boost DNA priming, there is evidence that use of oligomeric envelope proteins may be more effective in boosting broader responses.

TABLE-US-00014 TABLE 11 Sequences used for phylogenetic reconstruction Accession Sequence name number Subtype HIVELICG K03454 D HIVNDK M27323 D HIVZ2Z6 M22639 D 87USSG3X L02317 B 88USWR27 U26546 B 89SP061 AJ006287 B AUC18 AF042102 B AUC18MBC U37270 B AUMBC200 AF042100 B AUMBC925 AF042101 B AUMBCC18B AF042106 B AUMBCC54 AF042103 B AUMBCC98 AF042104 B AUMBCD36 AF042105 B CNRL42CG U71182 B D31 U43096 B HIVBH102 M15654 B HIVCAM1 D10112 B HIVF12CG Z11530 B HIVHAN2 U43141 B HIVJC16 AF049494 B HIVJRCSF M38429 B HIVJRFL U63632 B HIVLAICG K02013 B HIVMCK1 D86068 B HIVMN M17449 B HIVNL43 M19921 B HIVNY5CG M38431 B HIVOYI M26727 B HIVPV22 K02083 B HIVRF M17451 B HIVSF2CG K02007 B HIVWEAU160 U21135 B HIVYU10X M93259 B HIVYU2X M93258 B MANC U23487 B NLACH320A U34603 B NLACH320B U34604 B US89.6 U39362 B USAD8 U19647 B USDH123 AF069140 B

TABLE-US-00015 TABLE 12 Parameters for evolutionary models used for phylogenetic reconstructions. Evolutionary HIV-1-B ENV SIV env Model Class.sup.a GTR + I + G GTR + I + G RC A.sup.b 1.971 1.8418 RG A 4.315 10.9127 RT A 0.8539 0.5803 RC G 1.021 0.9283 RC T 3.826 6.8499 fA.sup.c 0.34857 0.3516 fC 0.17125 0.1642 fG 0.23694 0.2296 fT 0.24324 0.2546 Proportion of 0.26035 0.471 assumed invariant sites 0.50434 0.9477 .alpha..sup.d .sup.aGTR + I + G: General time-reversible model with a proportion of invariant sites and gamma-distributed site-to-site rate variation. .sup.bRate parameters of the symmetric substitution rate matrix: RX Y is the rate of substitution of nucleotide X by nucleotide Y (or Y by X) scaled to RG T = 1. .sup.cfX: Equilibrium frequency of nucleotide X. .sup.d.alpha.: Shape parameter of the gamma distribution.

TABLE-US-00016 TABLE 13 Neutralization of HIV-1 primary isolates in the cMA GI assay HIV isolate SF162 92US657 92TH014 92HT593 91US056 93IN101 Immunization 4 4 5 5 5 5 5 5 5 5 5 5 Group No. 50% 75% 50% 75% 50% 75% 50% 90% 50% 50% 75% 50% SF162 32N 22 15 16 <8 <8 <8 <8 <8 <4 <4 <4 <8 Gp140 91N 22 18 <8 <8 <8 <8 *55 <8 <4 <4 <4 <8 9335L 9 9 <8 <8 9 <8 <8 <8 <4 <4 <4 <8 9578L 40 13 18 9 14 <8 nt nt <4 <4 <4 8 Ancestral 168N <4 <4 <8 <8 20 12 40 16 <4 16 <4 <8 Gp140 245N 11 <4 <8 <8 32 16 88 48 <4 <4 <4 <8 247N 5 <4 <8 <8 10 <8 32 20 <4 <4 <4 <8 9331L 6 <4 8 <8 28 13 *52 *32 <4 <4 <4 8 Ancestral 93N 6 4 <8 <8 18 8 40 16 <4 4 <4 <8 Gp160 175M 6 4 8 <8 12 <8 24 10 <4 4 <4 <8 9599L <4 <4 <8 <8 9 <8 44 24 <4 <4 <4 <8 9896L 8 4 <8 <8 <8 <8 40 16 <4 <4 <4 nt Control 9827L <4 <4 <8 <8 8 <8 15 <8 <4 <4 <4 &- lt;8 pcDNA 8848L <4 <4 <8 <8 <8 <8 8 <8 <4 <4 <4 - <8 HIV + serum nt nt 2500 500 1100 300 300 100 200 500 300 400 pool Individual pre-immunization sera were tested for isolates SF162, 92US657, 92TH014, and 93IN101; pooled sera were tested for 92HT593 and 91US056. All were below 50% at the indicated dilution, except as noted; for samples marked * the pre-bleed titer was subtracted. nt, not tested.

Individual pre-immunization sera were tested for isolates SF162, 92US657, 92TH014, and 93IN101; pooled sera were tested for 92HT593 and 91US056. All were below 50% at the Indicated dilution, except as noted; for samples marked * the pre-bleed titer was subtracted. nt, not tested.

TABLE-US-00017 TABLE 14 Neutralization of HIV-1 by sera from immunized rabbits - Week 80 (post-8.sup.th vaccination) % Reduction in RLU of: Bal JR-FL Bx08 6101 0692 S080 CM244 Rabbit Immunogen clade B clade B clade B clade B clade B clade C clade E 91N SF162 62 32 68 43 53 24 0 9335L SF162 66 38 66 25 32 0 13 32N SF162 0 0 65 13 35 0 0 175N AN-gp160 71 36 61 1 62 0 27 93N AN-gp160 36 0 45 0 35 0 0 9599L AN-gp160 35 0 59 0 44 0 0 9896L AN-gp160 0 0 0 5 0 0 0 245N AN-gp160 30 3 45 8 40 0 2 247N AN-gp160 0 0 25 0 41 0 33 9331L AN-gp160 0 0 14 0 1 0 0 168N AN-gp160 69 34 73 0 65 0 0 8848L Control 0 16 29 26 0 0 0 9827L Control 51 0 19 10 0 0 0 .sup.1Samples were assayed at a 1:10 dilution in triplicate. Values are the % reduction in relative luciferase units compared to the corresponding week 5 sample (05-16-02). Values .gtoreq.50% neutralization are shown in boldface type.

From the foregoing, it will be appreciated that, although specific embodiments of the invention have been described herein for the purpose of illustration, various modifications may be made without deviating from the spirit and scope of the invention. All publications and patent applications cited in this specification are herein incorporated by reference as if each individual publication or patent application were specifically and individually indicated to be incorporated by reference. Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be readily apparent to one of ordinary skill in the art in light of the teachings of this invention that certain changes and modifications may be made thereto without departing from the spirit or scope of the appended claims.

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2DNAArtificial SequenceAncestral HIV- M, subtype B, env sequence gtga agggcatccg caagaactac cagcacctgt ggcgctgggg caccatgctg 6atgc tgatgatctg ctccgcggcc gagaagctgt gggtgaccgt gtactacggc ccgtgt ggaaggaggc caccaccacc ctgttctgcg ccagcgacgc caaggcttac ccgagg tccacaacgt gtgggccacc cacgcctgcg tgcccaccga ccccaacccc 24gtgg tgctggagaa cgtgaccgag aacttcaaca tgtggaagaa caacatggtg 3gatgc acgaggacat catcagcctg tgggaccaga gcctgaagcc ctgcgtgaag 36cccc tgtgcgtgac cctgaactgc accgacgacc tgcgcaccaa cgccaccaac 42aaca gcagcgccac caccaacacc accagcagcg gcggcggcac gatggagggc 48ggcg agatcaagaa ctgcagcttc aacgtgacca ccagcatccg cgacaagatg 54gagt acgccctgtt ctacaagctg gacgtggtgc ccatcgacaa cgacaacaac 6caaca acaacaccag ctaccgcctc atcaactgca acaccagcgt gatcacccag 66ccca aggtgagctt cgagcccatc cccatccact actgcacccc cgccggcttc 72ctga agtgcaacga caagaagttc aacggcaccg gcccctgcac caacgtgagc 78cagt gcacccacgg catccgcccc gtggtgagca cccagctgct gctgaacggc 84gccg aggaggaggt ggtgatccgc agcgagaact tcaccgacaa cgccaagacc 9cgtgc agctgaacga gagcgtggag atcaactgca cgcgtcccaa caacaacacc 96agca tccccatcgg ccctggccgc gccctgtacg ccaccggcaa gatcatcggc atccgcc aggcccactg caacctgtcg cgagccaagt ggaacaacac cctgaagcag gtgacca agctgcgcga gcagttcggc aacaacaaga ccaccatcgt gttcaaccag agcggcg gcgaccccga gatcgtgatg cacagcttca actgcggcgg cgaattcttc tgcaaca gcacccagct gttcaacagc acctggcact tcaacggcac ctggggcaac aacaccg agcgcagcaa caacgccgcc gacgacaacg acaccatcac cctgccctgc atcaagc agatcatcaa catgtggcag gaggtgggca aggccatgta cgcccccccc agcggcc agatccgctg cagcagcaac atcaccggcc tgctgctgac tcgagacggc aacaacg agaacaccaa caacaccgac accgagatct tccgccccgg gggcggcgac cgcgaca actggcgcag cgagctgtac aagtacaagg tggtgaagat cgagcccctg gtggccc ccaccaaggc caagcgccgc gtggtgcagc gcgagaagcg cgccgtgggc ctgggcg ccatgttcct gggcttcctg ggcgccgccg gcagcaccat gggcgccgcc atgaccc tgaccgtgca ggcccgccag ctgctgagcg gcatcgtgca gcagcagaac ctgctgc gcgccatcga ggcccagcag cacctgctgc agctgaccgt gtggggcatc cagctgc aggcccgcgt gctggccgtg gagcggtacc tgaaggacca gcagctgctg atctggg gctgcagcgg caagctgatc tgcaccaccg cggtgccctg gaacgccagc agcaaca agagcctgga caagatctgg aacaacatga cctggatgga gtgggagcgc atcgaca actacaccgg cctgatctac accctgatcg aggagagcca gaaccagcag 2agaacg agcaggagct gctggagctg gacaagtggg ccagcctgtg gaactggttc 2tcacca actggctgtg gtacatcaag atcttcatca tgatcgtggg cggcctggtg 2tgcgca tcgtgttcgc cgtgctgagc atcgtgaacc gcgtgcgcca gggctacagc 222agct tccagacccg cctgcccgcc ccccgcggcc ccgaccgccc cgagggcatc 228gagg gcggcgagcg cgaccgcgac cgcagcgggc gcctggtgaa cggcttcctg 234atct gggacgacct gcgcagcctg tgcctgttca gctaccaccg cctgcgcgac 24gctga tcgtggcccg catcgtggag ctgctgggcc ggcgcggctg ggaggccctg 246tggt ggaacctgct gcagtactgg agccaggagc tgaagaacag cgccgtgagc 252aacg ccaccgccat cgccgtggcc gagggcaccg accgcgtgat cgaggtggtg 258gcct gccgcgccat cctgcacatc ccccgccgca tccgccaggg cctggagcgc 264ctgt ga 26522883PRTArtificial SequenceAncestral HIV- M, subtype B, env sequence 2Met Arg Val Lys Gly Ile Arg Lys Asn Tyr Gln His Leu Trp Arg Trphr Met Leu Leu Gly Met Leu Met Ile Cys Ser Ala Ala Glu Lys 2Leu Trp Val Thr Val Tyr Tyr Gly Val Pro Val Trp Lys Glu Ala Thr 35 4 Thr Leu Phe Cys Ala Ser Asp Ala Lys Ala Tyr Asp Thr Glu Val 5His Asn Val Trp Ala Thr His Ala Cys Val Pro Thr Asp Pro Asn Pro65 7Gln Glu Val Val Leu Glu Asn Val Thr Glu Asn Phe Asn Met Trp Lys 85 9 Asn Met Val Glu Gln Met His Glu Asp Ile Ile Ser Leu Trp Asp Ser Leu Lys Pro Cys Val Lys Leu Thr Pro Leu Cys Val Thr Leu Cys Thr Asp Asp Leu Arg Thr Asn Ala Thr Asn Thr Thr Asn Ser Ala Thr Thr Asn Thr Thr Ser Ser Gly Gly Gly Thr Met Glu Gly Glu Lys Gly Glu Ile Lys Asn Cys Ser Phe Asn Val Thr Thr Ser Ile Asp Lys Met Gln Lys Glu Tyr Ala Leu Phe Tyr Lys Leu Asp Val Pro Ile Asp Asn Asp Asn Asn Asn Thr Asn Asn Asn Thr Ser Tyr 2eu Ile Asn Cys Asn Thr Ser Val Ile Thr Gln Ala Cys Pro Lys 222r Phe Glu Pro Ile Pro Ile His Tyr Cys Thr Pro Ala Gly Phe225 234e Leu Lys Cys Asn Asp Lys Lys Phe Asn Gly Thr Gly Pro Cys 245 25r Asn Val Ser Thr Val Gln Cys Thr His Gly Ile Arg Pro Val Val 267r Gln Leu Leu Leu Asn Gly Ser Leu Ala Glu Glu Glu Val Val 275 28e Arg Ser Glu Asn Phe Thr Asp Asn Ala Lys Thr Ile Ile Val Gln 29sn Glu Ser Val Glu Ile Asn Cys Thr Arg Pro Asn Asn Asn Thr33rg Lys Ser Ile Pro Ile Gly Pro Gly Arg Ala Leu Tyr Ala Thr Gly 325 33s Ile Ile Gly Asp Ile Arg Gln Ala His Cys Asn Leu Ser Arg Ala 345p Asn Asn Thr Leu Lys Gln Ile Val Thr Lys Leu Arg Glu Gln 355 36e Gly Asn Asn Lys Thr Thr Ile Val Phe Asn Gln Ser Ser Gly Gly 378o Glu Ile Val Met His Ser Phe Asn Cys Gly Gly Glu Phe Phe385 39ys Asn Ser Thr Gln Leu Phe Asn Ser Thr Trp His Phe Asn Gly 44rp Gly Asn Asn Asn Thr Glu Arg Ser Asn Asn Ala Ala Asp Asp 423p Thr Ile Thr Leu Pro Cys Arg Ile Lys Gln Ile Ile Asn Met 435 44p Gln Glu Val Gly Lys Ala Met Tyr Ala Pro Pro Ile Ser Gly Gln 456g Cys Ser Ser Asn Ile Thr Gly Leu Leu Leu Thr Arg Asp Gly465 478n Asn Glu Asn Thr Asn Asn Thr Asp Thr Glu Ile Phe Arg Pro 485 49y Gly Gly Asp Met Arg Asp Asn Trp Arg Ser Glu Leu Tyr Lys Tyr 55al Val Lys Ile Glu Pro Leu Gly Val Ala Pro Thr Lys Ala Lys 5525Arg Arg Val Val Gln Arg Glu Lys Arg Ala Val Gly Met Leu Gly Ala 534e Leu Gly Phe Leu Gly Ala Ala Gly Ser Thr Met Gly Ala Ala545 556t Thr Leu Thr Val Gln Ala Arg Gln Leu Leu Ser Gly Ile Val 565 57n Gln Gln Asn Asn Leu Leu Arg Ala Ile Glu Ala Gln Gln His Leu 589n Leu Thr Val Trp Gly Ile Lys Gln Leu Gln Ala Arg Val Leu 595 6la Val Glu Arg Tyr Leu Lys Asp Gln Gln Leu Leu Gly Ile Trp Gly 662r Gly Lys Leu Ile Cys Thr Thr Ala Val Pro Trp Asn Ala Ser625 634r Asn Lys Ser Leu Asp Lys Ile Trp Asn Asn Met Thr Trp Met 645 65u Trp Glu Arg Glu Ile Asp Asn Tyr Thr Gly Leu Ile Tyr Thr Leu 667u Glu Ser Gln Asn Gln Gln Glu Lys Asn Glu Gln Glu Leu Leu 675 68u Leu Asp Lys Trp Ala Ser Leu Trp Asn Trp Phe Asp Ile Thr Asn 69eu Trp Tyr Ile Lys Ile Phe Ile Met Ile Val Gly Gly Leu Val77ly Leu Arg Ile Val Phe Ala Val Leu Ser Ile Val Asn Arg Val Arg 725 73n Gly Tyr Ser Pro Leu Ser Phe Gln Thr Arg Leu Pro Ala Pro Arg 745o Asp Arg Pro Glu Gly Ile Glu Glu Glu Gly Gly Glu Arg Asp 755 76g Asp Arg Ser Gly Arg Leu Val Asn Gly Phe Leu Ala Leu Ile Trp 778p Leu Arg Ser Leu Cys Leu Phe Ser Tyr His Arg Leu Arg Asp785 79eu Leu Ile Val Ala Arg Ile Val Glu Leu Leu Gly Arg Arg Gly 88lu Ala Leu Lys Tyr Trp Trp Asn Leu Leu Gln Tyr Trp Ser Gln 823u Lys Asn Ser Ala Val Ser Leu Leu Asn Ala Thr Ala Ile Ala 835 84l Ala Glu Gly Thr Asp Arg Val Ile Glu Val Val Gln Arg Ala Cys 856a Ile Leu His Ile Pro Arg Arg Ile Arg Gln Gly Leu Glu Arg865 878u Leu32562DNAArtificial sequenceAncestral HIV- M, subtype C, env sequence 3atgcgggtga tgggcatcct gcggaactgc cagcagtggt ggatctgggg catcctgggc 6atgc tgatgatctg cagcgtgatg ggcaacctgt gggtgaccgt gtactacggc ccgtgt ggaaggaggc caagaccacc ctgttctgcg ccagcgacgc caaggcctac gggagg tgcacaacgt gtgggccacc cacgcctgcg tgcccaccga ccccaacccc 24atgg tgctggagaa cgtgaccgag aacttcaaca tgtggaagaa cgacatggtg 3gatgc acgaggacat catcagcctg tgggaccaga gcctgaagcc ctgcgtgaag 36cccc tgtgcgtgac cctgaactgc accaacgtga ccaacaccaa caacaacaac 42agca tgggcggcga gatcaagaac tgcagcttca acatcaccac cgagctgcgg 48aagc agaaggtgta cgccctgttc taccggctgg acatcgtgcc cctgaacgag 54aaca gcaacagcag cgagtaccgg ctgatcaact gcaacaccag cgccatcacc 6ctgcc ccaaggtgag cttcgacccc atccccatcc actactgcgc ccccgccggc 66atcc tgaagtgcaa caacaagacc ttcaacggca ccggcccctg caacaacgtg 72gtgc agtgcaccca cggcatcaag cccgtggtga gcacccagct gctgctgaac 78ctgg ccgaggagga gatcatcatc cggagcgaga acctgaccaa caacgccaag 84atcg tgcacctgaa cgagagcgtg gagatcgtgt gcacccggcc caacaacaac 9gaaga gcatccggat cggccccggc cagaccttct acgccaccgg cgacatcatc 96atcc ggcaggccca ctgcaacatc agcgagaagg agtggaacaa gaccctgcag gtgggca agaagctgaa ggagcacttc cccaacaaga ccatcaagtt cgagcccagc ggcggcg acctggagat caccacccac agcttcaact gccggggcga gttcttctac aacacca gcaagctgtt caacagcacc tacaacagca ccaacaacgg caccaccagc agcacca tcaccctgcc ctgccggatc aagcagatca tcaacatgtg gcagggcgtg cgggcca tgtacgcccc ccccatcgcc ggcaacatca cctgcaagag caacatcacc ctgctgc tgacccggga cggcggcaac accaacaaca ccaccgagac cttccggccc ggcggcg acatgcggga caactggcgg agcgagctgt acaagtacaa ggtggtggag aagcccc tgggcgtggc ccccaccgag gccaagcggc gggtggtgga gcgggagaag gccgtgg gcatcggcgc cgtgttcctg ggcttcctgg gcgccgccgg cagcaccatg gccgcca gcatcaccct gaccgtgcag gcccggcagc tgctgagcgg catcgtgcag cagagca acctgctgcg ggccatcgag gcccagcagc acatgctgca gctgaccgtg ggcatca agcagctgca gacccgggtg ctggccatcg agcggtacct gaaggaccag ctgctgg gcatctgggg ctgcagcggc aagctgatct gcaccaccgc cgtgccctgg agcagct ggagcaacaa gagccaggac gacatctggg acaacatgac ctggatgcag gaccggg agatcagcaa ctacaccgac accatctacc ggctgctgga ggacagccag cagcagg agaagaacga gaaggacctg ctggccctgg acagctggaa gaacctgtgg tggttcg acatcaccaa ctggctgtgg tacatcaaga tcttcatcat gatcgtgggc 2tgatcg gcctgcggat catcttcgcc gtgctgagca tcgtgaaccg ggtgcggcag 2acagcc ccctgagctt ccagaccctg acccccaacc cccggggccc cgaccggctg 2gcatcg aggaggaggg cggcgagcag gaccgggacc ggagcatccg gctggtgagc 222ctgg ccctggcctg ggacgacctg cggagcctgt gcctgttcag ctaccaccgg 228gact tcatcctgat cgccgcccgg ggcgtgaacc tgctgggccg gagcagcctg 234ctgc agcggggctg ggaggccctg aagtacctgg gcagcctggt gcagtactgg 24ggagc tgaagaagag cgccatcagc ctgctggaca ccatcgccat cgccgtggcc 246accg accggatcat cgagctggtg cagcggatct gccgggccat ccggaacatc 252cgga tccggcaggg cttcgaggcc gccctgcagt ga 25624853PRTArtificial sequenceAncestral HIV- M, subtype C, env sequence. 4Met Arg Val Met Gly Ile Leu Arg Asn Cys Gln Gln Trp Trp Ile Trple Leu Gly Phe Trp Met Leu Met Ile Cys Ser Val Met Gly Asn 2Leu Trp Val Thr Val Tyr Tyr Gly Val Pro Val Trp Lys Glu Ala Lys 35 4 Thr Leu Phe Cys Ala Ser Asp Ala Lys Ala Tyr Glu Arg Glu Val 5His Asn Val Trp Ala Thr His Ala Cys Val Pro Thr Asp Pro Asn Pro65 7Gln Glu Met Val Leu Glu Asn Val Thr Glu Asn Phe Asn Met Trp Lys 85 9 Asp Met Val Asp Gln Met His Glu Asp Ile Ile Ser Leu Trp Asp Ser Leu Lys Pro Cys Val Lys Leu Thr Pro Leu Cys Val Thr Leu Cys Thr Asn Val Thr Asn Thr Asn Asn Asn Asn Asn Thr Ser Met Gly Glu Ile Lys Asn Cys Ser Phe Asn Ile Thr Thr Glu Leu Arg Asp Lys Lys Gln Lys Val Tyr Ala Leu Phe Tyr Arg Leu Asp Ile Val Leu Asn Glu Asn Ser Asn Ser Asn Ser Ser Glu Tyr Arg Leu Ile Cys Asn Thr Ser Ala Ile Thr Gln Ala Cys Pro Lys Val Ser Phe 2ro Ile Pro Ile His Tyr Cys Ala Pro Ala Gly Tyr Ala Ile Leu 222s Asn Asn Lys Thr Phe Asn Gly Thr Gly Pro Cys Asn Asn Val225 234r Val Gln Cys Thr His Gly Ile Lys Pro Val Val Ser Thr Gln 245 25u Leu Leu Asn Gly Ser Leu Ala Glu Glu Glu Ile Ile Ile Arg Ser 267n Leu Thr Asn Asn Ala Lys Thr Ile Ile Val His Leu Asn Glu 275 28r Val Glu Ile Val Cys Thr Arg Pro Asn Asn Asn Thr Arg Lys Ser 29rg Ile Gly Pro Gly Gln Thr Phe Tyr Ala Thr Gly Asp Ile Ile33ly Asp Ile Arg Gln Ala His Cys Asn Ile Ser Glu Lys Glu Trp Asn 325 33s Thr Leu Gln Arg Val Gly Lys Lys Leu Lys Glu His Phe Pro Asn 345r Ile Lys Phe Glu Pro Ser Ser Gly Gly Asp Leu Glu Ile Thr 355 36r His Ser Phe Asn Cys Arg Gly Glu Phe Phe Tyr Cys Asn Thr Ser 378u Phe Asn Ser Thr Tyr Asn Ser Thr Asn Asn Gly Thr Thr Ser385 39er Thr Ile Thr Leu Pro Cys Arg Ile Lys Gln Ile Ile Asn Met 44ln Gly Val Gly Arg Ala Met Tyr Ala Pro Pro Ile Ala Gly Asn 423r Cys Lys Ser Asn Ile Thr Gly Leu Leu Leu Thr Arg Asp Gly 435 44y Asn Thr Asn Asn Thr Thr Glu Thr Phe Arg Pro Gly Gly Gly Asp 456g Asp Asn Trp Arg Ser Glu Leu Tyr Lys Tyr Lys Val Val Glu465 478s Pro Leu Gly Val Ala Pro Thr Glu Ala Lys Arg Arg Val Val 485 49u Arg Glu Lys Arg Ala Val Gly Ile Gly Ala Val Phe Leu Gly Phe 55ly Ala Ala Gly Ser Thr Met Gly Ala Ala Ser Ile Thr Leu Thr 5525Val Gln Ala Arg Gln Leu Leu Ser Gly Ile Val Gln Gln Gln Ser Asn 534u Arg Ala Ile Glu Ala Gln Gln His Met Leu Gln Leu Thr Val545 556y Ile Lys Gln Leu Gln Thr Arg Val Leu Ala Ile Glu Arg Tyr 565 57u Lys Asp Gln Gln Leu Leu Gly Ile Trp Gly Cys Ser Gly Lys Leu 589s Thr Thr Ala Val Pro Trp Asn Ser Ser Trp Ser Asn Lys Ser 595 6ln Asp Asp Ile Trp Asp Asn Met Thr Trp Met Gln Trp Asp Arg Glu 662r Asn Tyr Thr Asp Thr Ile Tyr Arg Leu Leu Glu Asp Ser Gln625 634n Gln Glu Lys Asn Glu Lys Asp Leu Leu Ala Leu Asp Ser Trp 645 65s Asn Leu Trp Asn Trp Phe Asp Ile Thr Asn Trp Leu Trp Tyr Ile 667e Phe Ile Met Ile Val Gly Gly Leu Ile

Gly Leu Arg Ile Ile 675 68e Ala Val Leu Ser Ile Val Asn Arg Val Arg Gln Gly Tyr Ser Pro 69er Phe Gln Thr Leu Thr Pro Asn Pro Arg Gly Pro Asp Arg Leu77ly Gly Ile Glu Glu Glu Gly Gly Glu Gln Asp Arg Asp Arg Ser Ile 725 73g Leu Val Ser Gly Phe Leu Ala Leu Ala Trp Asp Asp Leu Arg Ser 745s Leu Phe Ser Tyr His Arg Leu Arg Asp Phe Ile Leu Ile Ala 755 76a Arg Gly Val Asn Leu Leu Gly Arg Ser Ser Leu Arg Gly Leu Gln 778y Trp Glu Ala Leu Lys Tyr Leu Gly Ser Leu Val Gln Tyr Trp785 79eu Glu Leu Lys Lys Ser Ala Ile Ser Leu Leu Asp Thr Ile Ala 88la Val Ala Glu Gly Thr Asp Arg Ile Ile Glu Leu Val Gln Arg 823s Arg Ala Ile Arg Asn Ile Pro Arg Arg Ile Arg Gln Gly Phe 835 84u Ala Ala Leu Gln 85NAArtificial sequenceSemi-optimized ancestral viral sequences for HIV-pes B and C 5atgagagtga aggggatcag gaagaactat cagcacttgt ggagatgggg caccatgctc 6atgt tgatgatctg tagcgccgcc gagaagctgt gggtgaccgt gtactacggc ccgtgt ggaaggaggc caccaccacc ctgttctgcg ccagcgacgc caaggcttac ccgagg tccacaacgt gtgggccacc cacgcctgcg tgcccaccga ccccaacccc 24gtgg tgctggagaa cgtgaccgag aacttcaaca tgtggaagaa caacatggtg 3gatgc acgaggacat catcagcctg tgggaccaga gcctgaagcc ctgcgtgaag 36cccc tgtgcgtgac cctgaactgc accgacgacc tgcgcaccaa cgccaccaac 42aaca gcagcgccac caccaacacc accagcagcg gcggcggcac gatggagggc 48ggcg agatcaagaa ctgcagcttc aacgtgacca ccagcatccg cgacaagatg 54gagt acgccctgtt ctacaagctg gacgtggtgc ccatcgacaa cgacaacaac 6caaca acaacaccag ctaccgcctc atcaactgca acaccagcgt gatcacccag 66ccca aggtgagctt cgagcccatc cccatccact actgcacccc cgccggcttc 72ctga agtgcaacga caagaagttc aacggcaccg gcccctgcac caacgtgagc 78cagt gcacccacgg catccgcccc gtggtgagca cccagctgct gctgaacggc 84gccg aggaggaggt ggtgatccgc agcgagaact tcaccgacaa cgccaagacc 9cgtgc agctgaacga gagcgtggag atcaactgca cgcgtcccaa caacaacacc 96agca tccccatcgg ccctggccgc gccctgtacg ccaccggcaa gatcatcggc atccgcc aggcccactg caacctgtcg cgagccaagt ggaacaacac cctgaagcag gtgacca agctgcgcga gcagttcggc aacaacaaga ccaccatcgt gttcaaccag agcggcg gcgaccccga gatcgtgatg cacagcttca actgcggcgg cgaattcttc tgcaaca gcacccagct gttcaacagc acctggcact tcaacggcac ctggggcaac aacaccg agcgcagcaa caacgccgcc gacgacaacg acaccatcac cctgccctgc atcaagc agatcatcaa catgtggcag gaggtgggca aggccatgta cgcccccccc agcggcc agatccgctg cagcagcaac atcaccggcc tgctgctgac tcgagacggc aacaacg agaacaccaa caacaccgac accgagatct tccgccccgg gggcggcgac cgcgaca actggcgcag cgagctgtac aagtacaagg tggtgaagat cgagcccctg gtagcac ccaccaaggc aaagagaaga gtggtgcaga gagaaaaaag cgcagtggga ctaggag ctatgttcct tgggttcttg ggagcagcag gaagcactat gggcgcagcg atgacgc tgaccgtaca ggccagacaa ttattgtctg gtatagtgca gcagcagaac ctgctga gggctattga ggcgcaacag catctgttgc aactcacagt ctggggcatc cagctcc aggcaagagt cctggctgtg gaaagatacc taaaggatca gcagctcctg atttggg gttgctctgg aaaactcatc tgcaccactg ctgtgccttg gaatgctagc agcaaca agagcctgga caagatctgg aacaacatga cctggatgga gtgggagcgc atcgaca actacaccgg cctgatctac accctgatcg aggagagcca gaaccagcag 2agaacg agcaggagct gctggagctg gacaagtggg ccagcctgtg gaactggttc 2tcacca actggctgtg gtacatcaag atcttcatca tgatcgtggg cggcctggtg 2tgcgca tcgtgttcgc cgtgctgagc atcgtgaacc gcgtgcgcca gggctacagc 222agct tccagaccca cctgccagcc ccgaggggac ccgacaggcc cgaaggaatc 228gaag gtggagagag agacagagac agatccggtc gattagtgaa tggattctta 234atct gggacgacct gcggagcctg tgcctcttca gctaccaccg cttgagcgac 24cttga ttgtagcgag gattgtggaa cttctgggac gcagggggtg ggaggccctc 246tggt ggaatctcct gcagtactgg agtcaggaac taaagaatag cgccgtgagc 252aacg ccaccgccat cgccgtggcc gagggcaccg accgcgtgat cgaggtggtg 258gcct gccgcgccat cctgcacatc ccccgccgca tccgccaggg cctggagcgc 264ctgt ga 265262562DNAArtificial sequenceSemi-optimized ancestral viral sequences for HIV-pes B and C 6atgagagtga tggggatact gaggaattgt caacaatggt ggatatgggg catcctaggc 6atgc taatgatttg tgacgtgatg ggcaacctgt gggtgaccgt gtactacggc ccgtgt ggaaggaggc caagaccacc ctgttctgcg ccagcgacgc caaggcctac gggagg tgcacaacgt gtgggccacc cacgcctgcg tgcccaccga ccccaacccc 24atgg tgctggagaa cgtgaccgag aacttcaaca tgtggaagaa cgacatggtg 3gatgc acgaggacat catcagcctg tgggaccaga gcctgaagcc ctgcgtgaag 36cccc tgtgcgtgac cctgaactgc accaacgtga ccaacaccaa caacaacaac 42agca tgggcggcga gatcaagaac tgcagcttca acatcaccac cgagctgcgg 48aagc agaaggtgta cgccctgttc taccggctgg acatcgtgcc cctgaacgag 54aaca gcaacagcag cgagtaccgg ctgatcaact gcaacaccag cgccatcacc 6ctgcc ccaaggtgag cttcgacccc atccccatcc actactgcgc ccccgccggc 66atcc tgaagtgcaa caacaagacc ttcaacggca ccggcccctg caacaacgtg 72gtgc agtgcaccca cggcatcaag cccgtggtga gcacccagct gctgctgaac 78ctgg ccgaggagga gatcatcatc cggagcgaga acctgaccaa caacgccaag 84atcg tgcacctgaa cgagagcgtg gagatcgtgt gcacccggcc caacaacaac 9gaaga gcatccggat cggccccggc cagaccttct acgccaccgg cgacatcatc 96atcc ggcaggccca ctgcaacatc agcgagaagg agtggaacaa gaccctgcag gtgggca agaagctgaa ggagcacttc cccaacaaga ccatcaagtt cgagcccagc ggcggcg acctggagat caccacccac agcttcaact gccggggcga gttcttctac aacacca gcaagctgtt caacagcacc tacaacagca ccaacaacgg caccaccagc agcacca tcaccctgcc ctgccggatc aagcagatca tcaacatgtg gcagggcgtg cgggcca tgtacgcccc ccccatcgcc ggcaacatca cctgcaagag caacatcacc ctgctgc tgacccggga cggcggcaac accaacaaca ccaccgagac cttccggccc ggcggcg acatgcggga caactggcgg agcgagctgt acaagtacaa ggtggtggag aagcccc tgggcgtagc acccactgag gcaaaaagga gagtggtgga gagagaaaaa gcagtgg gaataggagc tgtgttcctt gggttcttgg gagcagcagg aagcactatg gcggcgt caataacgct gacggtacag gccagacaat tattgtctgg tatagtgcaa caaagca atttgctgag ggctatagag gcgcaacagc atatgttgca actcacggtc ggcatta agcagctcca gacaagagtc ctggctatag aaagatacct aaaggatcag ctcctgg gcatttgggg ctgctctgga aaactcatct gcaccactgc tgtgccttgg tctagct ggagcaacaa gagccaggac gacatctggg acaacatgac ctggatgcag gaccggg agatcagcaa ctacaccgac accatctacc ggctgctgga ggacagccag cagcagg agaagaacga gaaggacctg ctggccctgg acagctggaa gaacctgtgg tggttcg acatcaccaa ctggctgtgg tacatcaaga tcttcatcat gatcgtgggc 2tgatcg gcctgcggat catcttcgcc gtgctgagca tcgtgaaccg ggtgcggcag 2acagcc ccctgagctt ccagaccctt accccaaacc cgaggggacc cgacaggctc 2gaatcg aagaagaagg tggagagcaa gacagagaca gatccattcg attagtgagc 222ttag cactggcctg ggacgacctg cggagcctgt gcctcttcag ctaccaccga 228gact tcatattgat tgcagccaga gggtgggaac ttctgggacg cagcagtctc 234ctgc agagggggtg ggaagccctt aagtatctgg gaagtcttgt gcagtattgg 24ggagc taaaaaagag tgctattagc ctgctggaca ccatcgccat cgccgtggcc 246accg accggatcat cgagctggtg cagcggatct gccgggccat ccggaacatc 252cgga tccggcaggg cttcgaggcc gccctgcagt ga 256277DNAArtificial sequenceConsensus sequence-maximum likelihood reconstruction of determined ancestral node. 7gatcctg 787DNAArtificial sequenceConsensus sequence, most parsimonious reconstruction of determined ancestral node 8gawcctg 797DNAArtificial sequenceConsensus sequence, maximum likelihood reconstruction of determined ancestral node. 9gaacctg 7rtificial sequenceConsensus sequence, maximum likelihood reconstruction of determined ancestral node. tc 7rtificial sequenceConsensus sequence, maximum likelihood reconstruction of determined ancestral node. tc 7rtificial sequenceConsensus sequence, most parsimonious reconstruction of determined ancestral node. tc 7rtificial sequenceConsensus sequence, maximum likelihood reconstruction of determined ancestral node. tc 7rtificial sequenceConsensus sequence, maximum likelihood reconstruction of determined ancestral node. tt 7rtificial sequenceConsensus sequence, maximum likelihood reconstruction of determined ancestral node. ta 7rtificial sequenceConsensus sequence, maximum likelihood reconstruction of determined ancestral node. tg 7rtificial sequenceConsensus sequence, most parsimonious reconstruction of determined ancestral node. tv 7rtificial sequenceConsensus sequence, maximum likelihood reconstruction of determined ancestral node. tg 7rtificial sequenceConsensus sequence, maximum likelihood reconstruction of determined ancestral node. tg 72tificial sequenceConsensus sequence, maximum likelihood reconstruction of determined ancestral node. 2g 72tificial sequenceConsensus sequence, maximum likelihood reconstruction of determined ancestral node. 2g 7227DNAArtificial sequenceConsensus sequence, maximum likelihood reconstruction of determined ancestral node. 22cttgctg 7237DNAArtificial sequenceConsensus sequence, maximum likelihood reconstruction of determined ancestral node. 23cttgctt 724Artificial sequenceMost recent common ancestor of reconstruction clade B gag gene sequence 24atgggtgcga gagcgtcagt attaagcggg ggagaattag ataaatggga aaaaattcgg 6ccag ggggaaagaa aaaatataaa ttaaaacata tagtatgggc aagcagggag aacgat tcgcagttaa tcctggcctt ttagaaacat cagaaggctg tagacaaata gacagc tacaaccatc ccttcagaca ggatcagaag aacttagatc attatataat 24gcag tcctctattg tgtgcatcaa aagatagagg taaaagacac caaggaagct 3taaga tagaggaaga gcaaaacaaa agtaagaaaa aggcacagca agcagcagct 36ggaa acagcagcca ggtcagccaa aattacccta tagtgcagaa cctacagggg 42gtac atcaggccct atcacctaga actttaaatg catgggtaaa agtaatagaa 48gctt tcagcccaga agtaataccc atgttttcag cattatcaga aggagccacc 54gatt taaacaccat gctaaacaca gtggggggac atcaagcagc catgcaaatg 6agaga ccatcaatga ggaagctgca gaatgggata gattgcatcc agtgcatgca 66attg caccaggcca gatgagagaa ccaaggggaa gtgacatagc aggaactact 72cttc aggaacaaat agcatggatg acaaataatc cacctatccc agtaggagaa 78aaaa gatggataat cctgggatta aataaaatag taagaatgta tagccctgtc 84ctgg acataagaca aggaccaaag gaacccttta gagactatgt agaccggttc 9aactc taagagccga gcaagcttca caggaggtaa aaaattggat gacagaaacc 96gtcc aaaatgcgaa cccagattgt aagactatct taaaagcatt gggaccagga acactag aagaaatgat gacagcatgt cagggagtgg ggggacccgg ccataaagca gttttgg ctgaagcaat gagccaagta acaaattcag ctaccataat gatgcagaga aatttta ggaacccaag aaagactgtt aagtgtttca attgtggcaa agaagggcac gccagaa attgcagggc ccctaggaaa aagggctgtt ggaaatgtgg aaaggaagga caaatga aagattgtac tgagagacag gctaattttt tagggaaaat ctggccttcc aagggaa ggccagggaa ttttcttcag agcagaccag agccaacagc cccaccagaa agcttca ggtttgggga agagacaaca actccctctc agaagcagga gcagaaagac gaactgt atcctttagc ttccctcaaa tcactctttg gcaacgaccc ctcgtcacaa 5tificial sequenceLeast squares center of tree reconstruction of clade B gag gene sequence 25atgggtgcga gagcgtcagt attaagcggg ggagaattag atagatggga aaaaattcgg 6ccag ggggaaagaa aaaatataga ttaaaacata tagtatgggc aagcagggag aacgat tcgcagttaa tcctggcctg ttagaaacat cagaaggctg tagacaaata gacagc tacaaccatc ccttcagaca ggatcagaag aacttagatc attatataat 24gcaa ccctctattg tgtgcatcaa aggatagagg taaaagacac caaggaagct 3gaaga tagaggaaga gcaaaacaaa agtaagaaaa aggcacagca agcagcagct 36ggaa acagcagcca ggtcagccaa aattacccta tagtgcagaa cctccagggg 42gtac atcaggccat atcacctaga actttaaatg catgggtaaa agtagtagag 48gctt tcagcccaga agtaataccc atgttttcag cattatcaga aggagccacc 54gatt taaacaccat gctaaacaca gtggggggac atcaagcagc catgcaaatg 6agaga ccatcaatga ggaagctgca gaatgggata gattgcatcc agtgcatgca 66attg caccaggcca gatgagagaa ccaaggggaa gtgacatagc aggaactact 72cttc aggaacaaat aggatggatg acaaataatc cacctatccc agtaggagaa 78aaaa gatggataat cctgggatta aataaaatag taagaatgta tagccctacc 84ctgg acataagaca aggaccaaag gaacccttta gagactatgt agaccggttc 9aactc taagagccga gcaagcttca caggaggtaa aaaattggat gacagaaacc 96gtcc aaaatgcgaa cccagattgt aagactattt taaaagcatt gggaccagca acactag aagaaatgat gacagcatgt cagggagtgg ggggacccgg ccataaagca gttttgg ctgaagcaat gagccaagta acaaattcag ctaccataat gatgcagaga aatttta ggaaccaaag aaagactgtt aagtgtttca attgtggcaa agaagggcac gccaaaa attgcagggc ccctaggaaa aagggctgtt ggaaatgtgg aaaggaagga caaatga aagattgtac tgagagacag gctaattttt tagggaagat ctggccttcc aagggaa ggccagggaa ttttcttcag agcagaccag agccaacagc cccaccagaa agcttca ggtttgggga agagacaaca actccctctc agaagcagga gccgatagac gaactgt atcctttagc ttccctcaga tcactctttg gcaacgaccc ctcgtcacaa 5tificial sequenceMinimum of means center of tree reconstruction of clade B gag gene sequence 26atgggtgcgg gagcgtcggt attaagcggg ggaaaattag ataggtggga aaaaattcgg 6ccag ggggaaagaa aaaatataaa ttaaaacata tagtatgggc aagcagggag aacgat ttgcagtcaa tcctggcctg ttagaaacat cagaaggctg cagacgaata aacagc tacatccatc ccttcagaca ggatcagaag aacttaaatc attatataat 24gcaa ccctctattg tgtgcatcaa aatatagagg taagagacac caaggatgct 3aaaaa tagaggaaga acaaaacaaa attaagaaaa gggcacagca agcagcagct 36ggaa acagcaaccc ggtcagccaa aattacccta tagtgcagaa tatgcagggg 42gtac atcaggccat atcacctaga actttaaatg catgggtaaa agtagtagaa 48gctt tcagccccga agtaataccc atgttttcag cattatcaga aggagccacc 54gatt taaacaccat gctaaacaca gtggggggac atcaagcagc catgcaaatg 6agaaa ccatcaatga ggaagctgca gaatgggata gattgcaccc agtgcatgca 66attg caccaggcca gatgagagaa ccaaggggaa gtgacatagc aggaactact 72cttc aggaacaaat aggatggatg acacataatc cacctatccc agtaggagaa 78aaaa gatggataat catgggatta aataaaatag taagaatgta tagccctacc 84ctgg acataagaca aggaccaaag gaacccttta gagattatgt tgaccggttc 9aactc taagagccga gcaagcttca caggaggtaa aaaattggat gacagaaacc 96gtcc aaaatgcgaa cccagattgt aagaccattt taaaagcatt aggaccagca acactag aagaaatgat gacagcatgt cagggagtgg gagggcccag ccataaagca gttttgg cagaagcaat gagccaagca acaaattcag ctaccataat gatgcagagg aatttta agggccaaag aaagactgtt aaatgtttca attgtggcaa agaagggcac gccagaa attgcagggc ccctagaaaa aagggctgtt ggaaatgtgg aaaggaagga caaatga aagattgtac tgagagacag gctaattttt tagggaagat ctggccttcc aagggaa ggccagggaa ttttctccaa agcaggccag agccaacagc cccaccagaa agcttca ggtttgggga ggagacaaca actccccctc agaagcagga gccgagggac gaacagt atcccttgac ttccctcaga tcactctttg gcaacgaccc atcgtcacaa 589DNAArtificial sequenceMost recent common ancestor reconstruction of clade B env gene sequence 27atgagagtga aggggatcag gaagaattgt cagcacttgt ggaaatgggg caccatgctc 6atgt tgatgatctg tagtgctgca gaaaacttgt gggtcacagt ctattatggg ctgtgt ggaaagaagc aaccaccact ctattttgtg catcagatgc taaagcatat cagagg tacataatgt ctgggccaca catgcctgtg tacccacaga ccccaaccca 24gtag tattggaaaa tgtgacagaa aattttaaca tgtggaaaaa taacatggta 3gatgc atgaggatat aatcagttta tgggatcaaa gcctaaagcc atgtgtaaaa 36ccac tctgtgttac tttaaattgc actgatgcga acaagaatgc tactaatacc 42agta gtgggggaac aatggagaaa ggagaaatga aaaactgctc tttcaatatc 48agca taagagataa gatgcagaaa gaatatgcac ttttttataa acttgatgta 54atag ataatgataa taatagtaat aataatacca actataggtt gataaattgt 6ctcag tcattacaca ggcctgtcca aaggtatcct ttgagccaat tcccatacat 66accc cggctggttt tgcgattcta aagtgtaatg ataagaagtt caatggaaca 72tgta aaaatgtcag

cacagtacaa tgtacacatg gaattaggcc agtagtgtca 78ctgc tgttaaatgg cagtctagca gaagaagagg tagtaattag atctgaaaat 84gaca atgctaaaac cataatagta cagctgaatg aatctgtaga aattaattgt 9accca acaacaatac aagaaaaagt atacctatag gaccagggag agcactttat 96ggag aaataatagg agatataaga caagcacatt gtaacattag tagagcaaaa aataaca ctttaaaaca ggtagttaca aaattaagag aacaatttgg gaataataaa atagtct ttaatccatc ctcaggaggg gacccagaaa ttgtaatgca cagttttaat ggagggg aatttttcta ctgtaataca acacaactgt ttaatagtac ttggaatagt gaagggt caaataaaac tacagggtca aataacactg gaggagaaac tatcacactc tgcagaa taaaacaaat tataaacatg tggcaggaag taggaaaagc aatgtatgcc cccatca gaggacaaat taaatgttca tcaaatatta cagggctact attaacaaga ggtggtg aaaatagtac caatgagacc gagatcttca gacctggagg aggagatatg gacaatt ggagaagtga attatataaa tataaagtag taaaaattga accattagga gcaccca ccaaggcaaa gagaagagtg gtgcaaagag aaaaaagagc agtgggaata ggagcta tgttccttgg gttcttggga gcagcaggaa gcactatggg cgcagcgtca acgctga cggtacaggc cagacaatta ttgtctggta tagtgcaaca gcaaaacaat ctgaggg ctattgaggc gcaacagcat ctgttgcaac tcacggtctg gggcatcaaa ctccagg caagagtcct ggctgtggaa agatacctaa gggatcaaca gctcctagga tggggtt gctctggaaa actcatttgc accactactg tgccttggaa tgctagttgg aataaat ctctggataa gatttggaat aacatgacct ggatggagtg ggaaagagaa gacaatt acacaggctt aatatacaac ttaattgaag aatcgcagaa ccagcaagaa aatgaac aagaattatt ggaattggat aagtgggcaa gtttgtggaa ttggtttgac 2cacaat ggctgtggta tataaaaata ttcataatga tagtaggagg cttggtaggt 2gaatag tttttgctgt gctttctata gtgaatagag ttaggcaggg atactcacca 2catttc agacccgcct cccagccccg aggggacccg acaggcccga aggaatcgaa 222ggtg gagagagaga cagagacaga tccggtcgat tagtgaatgg attcttagca 228tggg acgatctgcg gagcctgtgc ctcttcagct accaccgctt gagagactta 234attg tagcgaggat tgtggaactt ctgggacgca gggggtggga agccctcaaa 24gtgga atctcctgca gtattggagt caggaactaa agaatagtgc tgttagcttg 246gcca cagcaatagc agtagctgag gggacagata gggttataga agtagtacaa 252tgta gagctattct tcacatacct agaagaataa gacagggctt agaaagggct 258taa 2589282589DNAArtificial sequenceLeast square center of tree and minimum of means center of tree reconstruction of clade B env gene sequence 28atgagagtga aggggatcag gaagaattat cagcacttgt ggagatgggg caccatgctc 6atgt tgatgatctg tagtgctgca gaaaaattgt gggtcacagt ctattatggg ctgtgt ggaaagaagc aaccaccact ctattttgtg catcagatgc taaagcatat cagagg tacataatgt ttgggccaca catgcctgtg tacccacaga ccccaaccca 24gtag tattggaaaa tgtgacagaa aattttaaca tgtggaaaaa taacatggta 3gatgc atgaggatat aatcagttta tgggatcaaa gcctaaagcc atgtgtaaaa 36ccac tctgtgttac tttaaattgc actgatttga ataagaatgc tactaatacc 42agta gcggggaaat gatggagaaa ggagaaataa aaaactgctc tttcaatatc 48agca taagagataa ggtgcagaaa gaatatgcac ttttttataa acttgatgta 54atag ataatgataa taatactaat aatactacca gctataggtt gataagttgt 6ctcag tcattacaca ggcctgtcca aaggtatcct ttgagccaat tcccatacat 66gccc cggctggttt tgcgattcta aagtgtaatg ataagaagtt caatggaaca 72tgta caaatgtcag cacagtacaa tgtacacatg gaattaggcc agtagtatca 78ctgc tgttaaatgg cagtctagca gaagaagagg tagtaattag atctgacaat 84gaca atgctaaaac cataatagta cagctgaatg aatctgtaga aattaattgt 9accca acaacaatac aagaaaaagt atacatatag gaccagggag agcattttat 96ggag aaataatagg agatataaga caagcacatt gtaacattag tagagcaaaa aataaca ctttaaaaca gatagttaaa aaattaagag aacaatttgg gaataataaa atagtct ttaatcaatc ctcaggaggg gacccagaaa ttgtaatgca cagttttaat ggagggg aatttttcta ctgtaattca acacaactgt ttaatagtac ttggaatggt tggactt ggaatactac tgaagggtca aatgacactg aaggagacac tatcacactc tgcagaa taaaacaaat tataaacatg tggcaggaag taggaaaagc aatgtatgcc cccatca gaggacaaat tagatgttca tcaaatatta cagggctgct attaacaaga ggtggta ataataacac caacgagacc gagatcttca gacctggagg aggagatatg gacaatt ggagaagtga attatataaa tataaagtag taaaaattga accattagga gcaccca ccaaggcaaa gagaagagtg gtgcagagag aaaaaagagc agtgggaata ggagctg tgttccttgg gttcttggga gcagcaggaa gcactatggg cgcagcgtca acgctga cggtacaggc cagacaatta ttgtctggta tagtgcaaca gcagaacaat ctgaggg ctattgaggc gcaacagcat ctgttgcaac tcacagtctg gggcatcaag ctccagg caagagtcct ggctgtggaa agatacctaa gggatcaaca gctcctgggg tggggtt gctctggaaa actcatttgc accactgctg tgccttggaa tgctagttgg aataaat ctctggatga gatttggaat aacatgacct ggatggagtg ggaaagagaa gacaatt acacaagctt aatatacacc ttaattgaag aatcgcaaaa ccaacaagaa aatgaac aagaattatt ggaattagat aaatgggcaa gtttgtggaa ttggtttgac 2caaact ggctgtggta tataaaaata ttcataatga tagtaggagg cttggtaggt 2gaatag tttttgctgt actttctata gtgaatagag ttaggcaggg atactcacca 2cgtttc agacccgcct cccagccccg aggggacccg acaggcccga aggaatcgaa 222ggtg gagagagaga cagagacaga tccggtcgat tagtgaacgg attcttagca 228tggg acgacctgcg gagcctgtgc ctcttcagct accaccgctt gagagactta 234attg taacgaggat tgtggaactt ctgggacgca gggggtggga agccctcaaa 24gtgga atctcctaca gtattggagt caggaactaa agaatagtgc tgttagcttg 246gcca cagccatagc agtagctgag gggacagata gggttataga agtagtacaa 252tgta gagctattct ccacatacct acaagaataa gacagggctt ggaaagggct 258taa 25892962ificial sequenceMost recent common ancestor reconstruction of clade B nef gene sequence 29atgggtggca agtggtcaaa acgtagtgtg gttggatggc ctgctgtaag ggaaagaatg 6gctg agccagcagc agatggggtg ggagcagtat ctcgagacct ggaaaaacat caatca caagtagcaa tacagcagct actaatgctg cttgtgcctg gctagaagca aggagg aggaggtggg ttttccagtc agacctcagg tacctttaag accaatgact 24gcag ctgtagatct tagccacttt ttaaaagaaa aggggggact ggaagggcta 3ctccc aaaaaagaca agatatcctt gatctgtggg tctaccacac acaaggctac 36gatt ggcagaacta cacaccaggg ccagggacca gatatccact gacctttgga 42ttca agctagtacc agttgagcca gagaaggtag aagaggccac tgaaggagag 48agct tgttacaccc tatgagcctg catggaatgg atgacccgga gagagaagtg 54tgga ggtttgacag ccgcctagca tttcatcaca tggcccgaga gaagcatccg 6ctaca aggactgctg a 62NAArtificial sequenceLeast squares center of tree reconstruction of clade B nef gene sequence 3ggca agtggtcaaa acgtagtgtg gttggatggc ctgctgtaag ggaaagaatg 6gctg agccagcagc agatggggtg ggagcagtat ctcgagacct ggaaaaacat caatca caagtagcaa tacagcagct actaatgctg attgtgcctg gctagaagca aggagg aggaggtggg ttttccagtc agacctcagg tacctttaag accaatgact 24gcag ctttagatct tagccacttt ttaaaagaaa aggggggact ggaagggcta 3ctccc aaaaaagaca agatatcctt gatctgtggg tctaccacac acaaggctac 36gatt ggcagaacta cacaccaggg ccagggatca gatatccact gacctttgga 42ttca agctagtacc agttgagcca gagaaggtag aagaggccaa tgaaggagag 48agct tgttacaccc tatgagcctg catgggatgg atgacccgga gaaagaagtg 54tgga agtttgacag ccgcctagca tttcatcaca tggcccgaga gctgcatccg 6ctaca aggactgctg a 62NAArtificial sequenceMinimum of means center of tree reconstruction of clade B nef gene sequence 3ggca agtggtcaaa acgtagtgtg gttggatggc ctgctgtaag ggaaagaatg 6gctg agccagcagc agatggggtg ggagcagtat ctcgagacct ggaaaaacat caatca caagtagcaa tacagcagct actaatgctg attgtgcctg gctagaagca aggagg aggaggtggg ttttccagtc agacctcagg tacctttaag accaatgact 24gcag ctttagatct tagccacttt ttaaaagaaa aggggggact ggaagggcta 3ctccc aaaaaagaca agatatcctt gatctgtggg tctaccacac acaaggctac 36gatt ggcagaacta cacaccaggg ccagggatca gatatccact gacctttgga 42ttca agctagtacc agttgagcca gagaaggtag aagaggccaa tgaaggagag 48tgct tgttacaccc tatgagccag catgggatgg atgacccgga gaaagaagtg 54tgga agtttgacag ccgcctagca tttcatcaca tggcccgaga gctgcatccg 6ctaca aggactgctg a 62DNAArtificial SequenceMost recent common ancestor reconstruction of clade B pol gene sequence 32ttttttaggg aaaatctggc cttcccacaa gggaaggcca gggaactttc ttcagagcag 6gcca acagccccac cagaagagag cttcaggttt ggggaagaga caacaactcc cagaag caggagcaga tagacaagga actgtatcct ttagcttccc tcaaatcact ggcaac gacccctcgt cacaataaag ataggggggc aactaaagga agctctatta 24ggag cagatgatac agtattagaa gaaatgaatt tgccaggaaa atggaaacca 3gatag ggggaattgg aggttttatc aaagtaagac agtatgatca aatacccata 36tgtg gacataaagc tataggtaca gtattagtag gacctacacc tgtcaacata 42agaa atctgttgac tcagattggt tgcactttaa attttcccat tagtcctatt 48gtac cagtaaaatt aaagccagga atggatggcc caaaagttaa acaatggcca 54gaag aaaaaataaa agcattagta gaaatttgta cagaaatgga aaaggaagga 6ttcaa aaattgggcc tgaaaatcca tacaatactc cagtatttgc cataaagaaa 66agta ctaaatggag aaaattagta gatttcagag aacttaataa gagaactcaa 72tggg aagttcaatt aggaatacca catcctgcag ggttaaaaaa gaaaaaatca 78gtac tggatgtggg tgatgcatat ttttcagttc ccttagatga agacttcagg 84actg catttaccat acctagtata aacaatgaga caccagggat tagatatcag 9tgtgc ttccacaggg atggaaagga tcaccagcaa tattccaaag tagcatgaca 96ttag agccttttag aaaacaaaat ccagaaatag ttatctatca atacatggat ttgtatg taggatctga cttagaaata gggcagcata gaacaaaaat agaggaactg gaacatc tgttgaggtg gggatttacc acaccagaca aaaaacatca gaaagaacct tttcttt ggatgggtta tgaactccat cctgataaat ggacagtaca gcctatagtg ccagaaa aagacagctg gactgtcaat gacatacaga agttagtggg aaaattgaat gcaagtc agatttatgc agggattaaa gtaaagcaat tatgtaaact ccttagggga aaagcac taacagaagt agtaccacta acagaagaag cagagctaga actggcagaa agggaga ttctaaaaga accagtacat ggagtgtatt atgacccatc aaaagactta gcagaaa tacagaagca ggggcaaggc caatggacat atcaaattta tcaagagcca aaaaatc tgaaaacagg aaagtatgca agaatgaggg gtgcccacac taatgatgta caattaa cagaggcagt gcaaaaaata gccacagaaa gcatagtaat atggggaaag cctaaat ttaaactacc catacaaaag gaaacatggg aagcatggtg gacagagtat caagcca cctggattcc tgagtgggag tttgtcaata cccctccctt agtaaaatta taccagt tagagaaaga acccatagta ggagcagaaa ctttctatgt agatggggca aatagag agactaaatt aggaaaagca ggatatgtta ctgacagagg aagacaaaaa gtctccc taactgacac aacaaatcag aagactgagt tacaagcaat tcatctagct caggatt cgggattaga agtaaacata gtaacagact cacaatatgc attaggaatc caagcac aaccagataa gagtgaatca gagttagtca gtcaaataat agagcagtta 2aaaagg aaaaggtcta cctggcatgg gtaccagcac acaaaggaat tggaggaaat 2aagtag ataaattagt cagtactgga atcaggaaag tactattttt ggatggaata 2aggccc aagaagaaca tgagaaatat cacagtaatt ggagagcaat ggctagtgat 222ctgc cacctgtagt agcaaaagaa atagtagcca gctgtgataa atgtcagcta 228gaag ccatgcatgg acaagtagac tgtagtccag gaatatggca actagattgt 234ttag aaggaaaagt tatcctggta gcagttcatg tagccagtgg ctatatagaa 24agtta ttccagcaga aacagggcag gaaacagcat actttctctt aaaattagca 246tggc cagtaaaagt aatacataca gacaatggca gcaatttcac cagtactaca 252gccg cctgttggtg ggcagggatc aagcaggaat ttggcattcc ctacaatccc 258caag gagtagtaga atctatgaat aaagaattaa agaaaattat aggacaggta 264cagg ctgaacatct taagacagca gtacaaatgg cagtattcat ccacaatttt 27aaaag gggggattgg ggggtacagt gcaggggaaa gaatagtaga cataatagca 276atac aaactaaaga actacaaaaa caaattacaa aaattcaaaa ttttcgggtt 282aggg acagcagaga tccactttgg aaaggaccag caaagcttct ctggaaaggt 288gcag tagtaataca agataatagt gacataaaag tagtgccaag aagaaaagca 294atta gggattatgg aaaacagatg gcaggtgatg attgtgtggc aagtagacag 3aggatt ag 3tificial SequenceLeast squares center of tree reconstruction of clade B pol gene sequence 33ttttttaggg aagatctggc cttcccacaa gggaaggcca gggaattttc ttcagagcag 6gcca acagccccac cagaagagag cttcaggttt ggggaagaga caacaactcc cagaag caggagccga tagacaagga actgtatcct ttagcttccc tcagatcact ggcaac gacccctcgt cacaataaag ataggggggc aactaaagga agctctatta 24ggag cagatgatac agtattagaa gaaatgaatt tgccaggaag atggaaacca 3gatag ggggaattgg aggttttatc aaagtaagac agtatgatca gatacccata 36tgtg gacataaagc tataggtaca gtattagtag gacctacacc tgtcaacata 42agaa atctgttgac tcagattggt tgcactttaa attttcccat tagtcctatt 48gtac cagtaaaatt aaagccagga atggatggcc caaaagttaa acaatggcca 54gaag aaaaaataaa agcattagta gaaatttgta cagaaatgga aaaggaaggg 6ttcaa aaattgggcc tgaaaatcca tacaatactc cagtatttgc cataaagaaa 66agta ctaaatggag aaaattagta gatttcagag aacttaataa gagaactcaa 72tggg aagttcaatt aggaatacca catcccgcag ggttaaaaaa gaaaaaatca 78gtac tggatgtggg tgatgcatat ttttcagttc ccttagatga agacttcagg 84actg catttaccat acctagtata aacaatgaga caccagggat tagatatcag 9tgtgc ttccacaggg atggaaagga tcaccagcaa tattccaaag tagcatgaca 96ttag agccttttag aaaacaaaat ccagacatag ttatctatca atacatggat ttgtatg taggatctga cttagaaata gggcagcata gaacaaaaat agaggaactg caacatc tgttgaggtg gggatttacc acaccagaca aaaaacatca gaaagaacct ttccttt ggatgggtta tgaactccat cctgataaat ggacagtaca gcctatagtg ccagaaa aagacagctg gactgtcaat gacatacaga agttagtggg aaaattgaat gcaagtc agatttatgc agggattaaa gtaaagcaat tatgtaaact ccttagggga aaagcac taacagaagt aataccacta acagaagaag cagagctaga actggcagaa agggaga ttctaaaaga accagtacat ggagtgtatt atgacccatc aaaagactta gcagaaa tacagaagca ggggcaaggc caatggacat atcaaattta tcaagagcca aaaaatc tgaaaacagg aaagtatgca agaatgaggg gtgcccacac taatgatgta caattaa cagaggcagt gcaaaaaata gccacagaaa gcatagtaat atggggaaag cctaaat ttaaactacc catacaaaaa gaaacatggg aagcatggtg gacagagtat caagcca cctggattcc tgagtgggag tttgtcaata cccctccctt agtgaaatta taccagt tagagaaaga acccatagta ggagcagaaa ctttctatgt agatggggca aataggg agactaaatt aggaaaagca ggatatgtta ctgacagagg aagacaaaaa gtctccc taactgacac aacaaatcag aagactgagt tacaagcaat tcatctagct caggatt cgggattaga agtaaacata gtaacagact cacaatatgc attaggaatc caagcac aaccagataa gagtgaatca gagttagtca gtcaaataat agagcagtta 2aaaagg aaaaggtcta cctggcatgg gtaccagcac acaaaggaat tggaggaaat 2aagtag ataaattagt cagtgctgga atcaggaaag tactattttt ggatggaata 2aggccc aagaagaaca tgagaaatat cacagtaatt ggagagcaat ggctagtgat 222ctgc cacctgtagt agcaaaagaa atagtagcca gctgtgataa atgtcagcta 228gaag ccatgcatgg acaagtagac tgtagtccag gaatatggca actagattgt 234ttag aaggaaaagt tatcctggta gcagttcatg tagccagtgg atatatagaa 24agtta ttccagcaga gacagggcag gaaacagcat actttctctt aaaattagca 246tggc cagtaaaaac aatacataca gacaatggca gcaatttcac cagtactacg 252gccg cctgttggtg ggcagggatc aagcaggaat ttggcattcc ctacaatccc 258caag gagtagtaga atctatgaat aaagaattaa agaaaattat aggacaggta 264cagg ctgaacatct taagacagca gtacaaatgg cagtattcat ccacaatttt 27aaaag gggggattgg ggggtacagt gcaggggaaa gaatagtaga cataatagca 276atac aaactaaaga attacaaaaa caaattacaa aaattcaaaa ttttcgggtt 282aggg acagcagaga tccactttgg aaaggaccag caaagcttct ctggaaaggt 288gcag tagtaataca agataatagt gacataaaag tagtgccaag aagaaaagca 294atta gggattatgg aaaacagatg gcaggtgatg attgtgtggc aagtagacag 3aggatt ag 3tificial sequenceMinimum of means center of tree reconstruction of clade B pol gene sequence 34ttttttaggg aagatctggc cttcccacaa gggaaggcca gggaattttc ttcagagcag 6gcca acagccccac cagaagagag cttcaggttt ggggaagaga caacaactcc cagaag caggagccga tagacaagga actgtatcct ttagcttccc tcagatcact ggcaac gacccctcgt cacaataaag ataggggggc aactaaagga agctctatta 24ggag cagatgatac agtattagaa gaaatgaatt tgccaggaag atggaaacca 3gatag ggggaattgg aggttttatc aaagtaagac agtatgatca gatactcata 36tgtg gacataaagc tataggtaca gtattagtag gacctacacc tgtcaacata 42agaa atctgttgac tcagattggt tgcactttaa attttcccat tagtcctatt 48gtac cagtaaaatt aaagccagga atggatggcc caaaagttaa acaatggcca 54gaag aaaaaataaa agcattagta gaaatttgta cagaaatgga aaaggaaggg 6ttcaa aaattgggcc tgaaaatcca tacaatactc cagtatttgc cataaagaaa 66agta ctaaatggag aaaattagta gatttcagag aacttaataa gagaactcaa 72tggg aagttcaatt aggaatacca catcccgcag ggttaaaaaa gaaaaaatca 78gtac tggatgtggg tgatgcatat ttttcagttc ccttagatga agacttcagg 84actg catttaccat acctagtata aacaatgaga caccagggat tagatatcag 9tgtgc ttccacaggg atggaaagga tcaccagcaa tattccaaag tagcatgaca 96ttag agccttttag aaaacaaaat ccagacatag ttatctatca atacatggat ttgtatg taggatctga cttagaaata gggcagcata gaacaaaaat agaggaactg caacatc tgttgaggtg gggatttacc acaccagaca aaaaacatca gaaagaacct ttccttt ggatgggtta tgaactccat cctgataaat ggacagtaca gcctatagtg ccagaaa aagacagctg gactgtcaat gacatacaga agttagtggg aaaattgaat gcaagtc agatttaccc agggattaaa gtaaagcaat tatgtaaact ccttagggga aaagcac taacagaagt aataccacta acagaagaag cagagctaga actggcagaa agggaaa ttctaaaaga accagtacat ggagtgtatt atgacccatc aaaagactta gcagaaa tacagaagca ggggcaaggc caatggacat atcaaattta tcaagagcca aaaaatc tgaaaacagg aaaatatgca agaatgaggg gtgcccacac taatgatgta caattaa cagaggcagt gcaaaaaata gccacagaaa gcatagtaat atggggaaag cctaaat ttaaactacc catacaaaaa gaaacatggg aaacatggtg gacagagtat caagcca cctggattcc tgagtgggag tttgtcaata

cccctccctt agtgaaatta taccagt tagagaaaga acccatagta ggagcagaaa ctttctatgt agatggggca aacaggg agactaaatt aggaaaagca ggatatgtta ctaacagagg aagacaaaaa gtctccc taactgacac aacaaatcag aagactgagt tacaagcaat tcatctagct caggatt cgggattaga agtaaacata gtaacagact cacaatatgc attaggaatc caagcac aaccagataa aagtgaatca gagttagtca gtcaaataat agagcagtta 2aaaagg aaaaggtcta cctggcatgg gtaccagcac acaaaggaat tggaggaaat 2aagtag ataaattagt cagtgctgga atcaggaaag tactattttt agatggaata 2aggccc aagaagaaca tgagaaatat cacagtaatt ggagagcaat ggctagtgat 222ctgc cacctgtagt agcaaaagaa atagtagcca gctgtgataa atgtcagcta 228gaag ccatgcatgg acaagtagac tgtagtccag gaatatggca actagattgt 234ttag aaggaaaagt tatcctggta gcagttcatg tagccagtgg atatatagaa 24agtta ttccagcaga gacagggcag gaaacagcat actttctctt aaaattagca 246tggc cagtaaaaac aatacataca gacaatggca gcaatttcac cagtactacg 252gccg cctgttggtg ggcggggatc aagcaggaat ttggcattcc ctacaatccc 258caag gagtagtaga atctatgaat aaagaattaa agaaaattat aggacaggta 264cagg ctgaacatct taagacagca gtacaaatgg cagtattcat ccacaatttt 27aaaag gggggattgg ggggtacagt gcaggggaaa gaatagtaga cataatagca 276atac aaactaaaga attacaaaaa caaattacaa aaattcaaaa ttttcgggtt 282aggg acagcagaga tccactttgg aaaggaccag caaagcttct ctggaaaggt 288gcag tagtaataca agataatagt gacataaaag tagtgccaag aagaaaagca 294atta gggattatgg aaaacagatg gcaggtgatg attgtgtggc aagtagacag 3aggatt ag 3ificial sequenceMost recent common ancestor reconstruction of clade B rev gene sequence 35atggcaggaa gaagcggaga cagcgacgaa gagctcctca agacagtcag actcatcaag 6tatc aaagcaaccc gcctcccagc cccgagggga cccgacaggc ccgaaggaat gaagaa ggtggagaga gagacagaga cagatccgtt cgattagtga acggattctt cttatc tgggacgatc tgcggagcct gtgcctcttc agctaccacc gcttgagaga 24cttg attgtagcga ggattgtgga acttctggga cgcagggggt gggaagtcct 3attgg tggaatctcc tgcagtattg gagtcaggaa ctaaagaata gtgctgttag 36NAArtificial sequenceLeast squares center of tree reconstruction of clade B rev gene sequence 36atggcaggaa gaagcggaga cagcgacgaa gagctcctca agacagtcag actcatcaag 6tatc aaagcaaccc gcctcccagc cccgagggga cccgacaggc ccgaaggaat gaagaa ggtggagaga gagacagaga cagatccggt cgattagtga atggattctt cttatc tgggtcgacc tgcggagcct gtgcctcttc agctaccacc gcttgagaga 24cttg attgtaacga ggattgtgga acttctggga cgcagggggt gggaagtcct 3attgg tggaatctcc tacagtattg gagtcaggaa ctaaagaata gtgctgttag 36NAArtificial sequenceMinimum of means center of tree reconstruction of clade B rev gene sequence 37atggcaggaa gaagcggaga cagcgacgaa gagctcctca agacagtcag actcatcaag 6tatc aaagcaaccc gcctcccagc cccgagggga cccgacaggc ccgaaggaat gaagaa ggtggagaga gagacagaga cagatccggt cgattagtga atggattctt cttatc tgggacgacc tgcggagcct gtgcctcttc agctaccacc gcttgagaga 24cttg attgtagcga ggattgtgga acttctggga cgcagggggt gggaagtcct 3attgg tggaatctcc tgcagtattg gagtcaggaa ctaaagaata gtgctgttag 36NAArtificial sequenceMost recent common ancestor reconstruction of clade B tat gene sequence 38atggagccag tagatcctag actagagccc tggaagcatc caggaagtca gcctaagact 6acca attgctattg taaaaagtgt tgctatcatt gccaagtttg cttcataaca gcttag gcatctccta tggcaggaag aagcggagac agcgacgaag acctcctcaa gtcaga ctcatcaagt ttctctatca aagcaacccg cctcccagcc ccgaggggac 24ggcc cgaaggaatc gaagaagaag gtggagagag agacagagac agatccggtc 3gtgaa tggattctta g 32NAArtificial sequenceLeast squares center of tree reconstruction of clade B tat gene sequence 39atggagccag tagatcctag actagagccc tggaagcatc caggaagtca gcctaagact 6acca attgctattg taaaaagtgt tgctttcatt gccaagtttg tttcataaca gcttag gcatctccta tggcaggaag aagcggagac agcgacgaag agctcctcaa gtcaga ctcatcaagt ttctctatca aagcaacccg cctcccagcc ccgaggggac 24ggcc cgaaggaatc gaagaagaag gtggagagag agacagagac agatccggtc 3gtgaa tggattctta g 32NAArtificial sequenceMinimum of means center of tree reconstruction of clade B tat gene sequence 4ccag tagatcctag actagagccc tggaagcatc caggaagtca gcctaagact 6acca attgctattg taaaaagtgt tgctttcatt gccaagtttg tttcataaca gcttag gcatctccta tggcaggaag aagcggagac agcgacgaag agctcctcaa gtcaga ctcatcaagt ttctctatca aagcaacccg cctcccagcc ccgaggggac 24ggcc cgaaggaatc gaagaagaag gtggagagag agacagagac agatccggtc 3gtgga tggattctta t 32NAArtificial sequenceMost recent common ancestor reconstruction of clade B vif gene sequence 4aaca gatggcaggt gatgattgtg tggcaagtag acaggatgag gattagaaca 6agtt tagtaaaaca ccatatgtat atttcaaaga aagctaaggg atggttttat atcact atgaaagcac tcatccaaga ataagttcag aagtacacat cccactagga ctagat tggtaataaa aacatattgg ggtctgcata caggagaaag agaatggcat 24cagg gagtctccat agaatggagg aaaaggagat atagcacaca agtagaccct 3agcag accaactaat tcatctgtat tattttgatt gtttttcaga atctgctata 36gcca tattaggaca tatagttagt cctaggtgtg aatatcaagc aggacataac 42ggat ctctacagta cttggcacta acagcattaa taacaccaaa aaagataaag 48ttgc ctagtgttag gaaactgaca gaggatagat ggaacaagcc ccagaagacc 54caca gagggagcca tacaatgaat ggacactag 57942579DNAArtificial sequenceLeast squares and minimum of means center of tree reconstructions of clade B vif gene sequence 42atggaaaaca gatggcaggt gatgattgtg tggcaagtag acaggatgag gattagaaca 6agtt tagtaaaaca ccatatgtat atttcaagga aagctaaggg atggttttat atcact atgaaagcac tcatccaaga ataagttcag aagtacacat cccactaggg ctagat tggtaataac aacatattgg ggtctgcata caggagaaag agactggcat 24cagg gagtctccat agaatggagg aaaaagagat atagcacaca agtagaccct 3agcag accaactaat tcatctgtat tactttgatt gtttttcaga atctgctata 36gcca tattaggaca tatagttagt cctaggtgtg aatatcaagc aggacataac 42ggat ctctacagta cttggcacta gcagcattaa taacaccaaa aaagataaag 48ttgc ctagtgttac gaaactgaca gaggatagat ggaacaagcc ccagaagacc 54caca gagggagcca tacaatgaat ggacactag 5794329ificial sequenceMost recent common ancestor reconstruction of clade b vpr gene sequence 43atggaacaag ccccagaaga ccaagggcca cagagggagc catacaatga atggacacta 6ttag aggagcttaa gagtgaagct gttagacatt ttcctaggct atggctccat taggac aacatatcta tgaaacttat ggggatacct gggcaggagt ggaagctata gaattc tgcaacaact gctgtttatt catttcagaa ttgggtgtca acatagcaga 24atta ctcgacagag aagagcaaga aatggagcca gtagatccta g 29NAArtificial sequenceLeast squares and minimum of means center of tree reconstruction of clade B vpr gene sequence 44atggaacaag ccccagaaga ccaagggcca cagagggagc catacaatga atggacacta 6ttag aggagcttaa gagtgaagct gttagacatt ttcctaggat atggctccat taggac aacatatcta tgaaacttat ggggatactt gggcaggagt ggaagccata gaattc tgcaacaact gctgtttatt catttcagaa ttgggtgtcg acatagcaga 24atta ctcgacagag gagagcaaga aatggagcca gtagatccta g 29NAArtificial sequenceMost recent common ancestor reconstruction of clade B vpu gene sequence 45atgcaacctt tagaaatatt agcaatagta gcattagtag tagcagcaat actagcaata 6tgga ccatagtatt catagaatat aggaaaatat taaggcaaag aaaaatagac taattg atagaataag agaaagagca gaagacagtg gcaatgagag tgaaggggat aagaat tatcagcact tgtggaaatg gggcaccatg ctccttggga tgttgatgat 24 24646246DNAArtificial sequenceLeast squares and minimum of means center of tree reconstructions of clade B vpu gene sequence 46atgcaacctt tacaaatatt agcaatagta gcattagtag tagcagcaat aatagcaata 6tgga ccatagtatt catagaatat aggaaaatat taagacaaag aaaaatagac taattg atagaataag agaaagagca gaagacagtg gcaatgagag tgaaggggat aagaat tatcagcact tgtggagatg gggcaccatg ctccttggga tgttgatgat 24 246475tificial sequenceMost recent common ancestor reconstruction of clade B gag protein sequence 47Met Gly Ala Arg Ala Ser Val Leu Ser Gly Gly Glu Leu Asp Lys Trpys Ile Arg Leu Arg Pro Gly Gly Lys Lys Lys Tyr Lys Leu Lys 2His Ile Val Trp Ala Ser Arg Glu Leu Glu Arg Phe Ala Val Asn Pro 35 4 Leu Leu Glu Thr Ser Glu Gly Cys Arg Gln Ile Leu Gly Gln Leu 5Gln Pro Ser Leu Gln Thr Gly Ser Glu Glu Leu Arg Ser Leu Tyr Asn65 7Thr Val Ala Val Leu Tyr Cys Val His Gln Lys Ile Glu Val Lys Asp 85 9 Lys Glu Ala Leu Asp Lys Ile Glu Glu Glu Gln Asn Lys Ser Lys Lys Ala Gln Gln Ala Ala Ala Asp Thr Gly Asn Ser Ser Gln Val Gln Asn Tyr Pro Ile Val Gln Asn Leu Gln Gly Gln Met Val His Ala Leu Ser Pro Arg Thr Leu Asn Ala Trp Val Lys Val Ile Glu Glu Lys Ala Phe Ser Pro Glu Val Ile Pro Met Phe Ser Ala Leu Ser Gly Ala Thr Pro Gln Asp Leu Asn Thr Met Leu Asn Thr Val Gly His Gln Ala Ala Met Gln Met Leu Lys Glu Thr Ile Asn Glu Glu 2la Glu Trp Asp Arg Leu His Pro Val His Ala Gly Pro Ile Ala 222y Gln Met Arg Glu Pro Arg Gly Ser Asp Ile Ala Gly Thr Thr225 234r Leu Gln Glu Gln Ile Ala Trp Met Thr Asn Asn Pro Pro Ile 245 25o Val Gly Glu Ile Tyr Lys Arg Trp Ile Ile Leu Gly Leu Asn Lys 267l Arg Met Tyr Ser Pro Val Ser Ile Leu Asp Ile Arg Gln Gly 275 28o Lys Glu Pro Phe Arg Asp Tyr Val Asp Arg Phe Tyr Lys Thr Leu 29la Glu Gln Ala Ser Gln Glu Val Lys Asn Trp Met Thr Glu Thr33eu Leu Val Gln Asn Ala Asn Pro Asp Cys Lys Thr Ile Leu Lys Ala 325 33u Gly Pro Gly Ala Thr Leu Glu Glu Met Met Thr Ala Cys Gln Gly 345y Gly Pro Gly His Lys Ala Arg Val Leu Ala Glu Ala Met Ser 355 36n Val Thr Asn Ser Ala Thr Ile Met Met Gln Arg Gly Asn Phe Arg 378o Arg Lys Thr Val Lys Cys Phe Asn Cys Gly Lys Glu Gly His385 39la Arg Asn Cys Arg Ala Pro Arg Lys Lys Gly Cys Trp Lys Cys 44ys Glu Gly His Gln Met Lys Asp Cys Thr Glu Arg Gln Ala Asn 423u Gly Lys Ile Trp Pro Ser His Lys Gly Arg Pro Gly Asn Phe 435 44u Gln Ser Arg Pro Glu Pro Thr Ala Pro Pro Glu Glu Ser Phe Arg 456y Glu Glu Thr Thr Thr Pro Ser Gln Lys Gln Glu Gln Lys Asp465 478u Leu Tyr Pro Leu Ala Ser Leu Lys Ser Leu Phe Gly Asn Asp 485 49o Ser Ser Gln 5PRTArtificial sequenceLeast squares center of tree reconstruction of clade B gag protein sequence 48Met Gly Ala Arg Ala Ser Val Leu Ser Gly Gly Glu Leu Asp Arg Trpys Ile Arg Leu Arg Pro Gly Gly Lys Lys Lys Tyr Arg Leu Lys 2His Ile Val Trp Ala Ser Arg Glu Leu Glu Arg Phe Ala Val Asn Pro 35 4 Leu Leu Glu Thr Ser Glu Gly Cys Arg Gln Ile Leu Gly Gln Leu 5Gln Pro Ser Leu Gln Thr Gly Ser Glu Glu Leu Arg Ser Leu Tyr Asn65 7Thr Val Ala Thr Leu Tyr Cys Val His Gln Arg Ile Glu Val Lys Asp 85 9 Lys Glu Ala Leu Glu Lys Ile Glu Glu Glu Gln Asn Lys Ser Lys Lys Ala Gln Gln Ala Ala Ala Asp Thr Gly Asn Ser Ser Gln Val Gln Asn Tyr Pro Ile Val Gln Asn Leu Gln Gly Gln Met Val His Ala Ile Ser Pro Arg Thr Leu Asn Ala Trp Val Lys Val Val Glu Glu Lys Ala Phe Ser Pro Glu Val Ile Pro Met Phe Ser Ala Leu Ser Gly Ala Thr Pro Gln Asp Leu Asn Thr Met Leu Asn Thr Val Gly His Gln Ala Ala Met Gln Met Leu Lys Glu Thr Ile Asn Glu Glu 2la Glu Trp Asp Arg Leu His Pro Val His Ala Gly Pro Ile Ala 222y Gln Met Arg Glu Pro Arg Gly Ser Asp Ile Ala Gly Thr Thr225 234r Leu Gln Glu Gln Ile Gly Trp Met Thr Asn Asn Pro Pro Ile 245 25o Val Gly Glu Ile Tyr Lys Arg Trp Ile Ile Leu Gly Leu Asn Lys 267l Arg Met Tyr Ser Pro Thr Ser Ile Leu Asp Ile Arg Gln Gly 275 28o Lys Glu Pro Phe Arg Asp Tyr Val Asp Arg Phe Tyr Lys Thr Leu 29la Glu Gln Ala Ser Gln Glu Val Lys Asn Trp Met Thr Glu Thr33eu Leu Val Gln Asn Ala Asn Pro Asp Cys Lys Thr Ile Leu Lys Ala 325 33u Gly Pro Ala Ala Thr Leu Glu Glu Met Met Thr Ala Cys Gln Gly 345y Gly Pro Gly His Lys Ala Arg Val Leu Ala Glu Ala Met Ser 355 36n Val Thr Asn Ser Ala Thr Ile Met Met Gln Arg Gly Asn Phe Arg 378n Arg Lys Thr Val Lys Cys Phe Asn Cys Gly Lys Glu Gly His385 39la Lys Asn Cys Arg Ala Pro Arg Lys Lys Gly Cys Trp Lys Cys 44ys Glu Gly His Gln Met Lys Asp Cys Thr Glu Arg Gln Ala Asn 423u Gly Lys Ile Trp Pro Ser His Lys Gly Arg Pro Gly Asn Phe 435 44u Gln Ser Arg Pro Glu Pro Thr Ala Pro Pro Glu Glu Ser Phe Arg 456y Glu Glu Thr Thr Thr Pro Ser Gln Lys Gln Glu Pro Ile Asp465 478u Leu Tyr Pro Leu Ala Ser Leu Arg Ser Leu Phe Gly Asn Asp 485 49o Ser Ser Gln 5PRTArtificial sequenceMinimum of means center of tree reconstruction of clade B gag protein sequence 49Met Gly Ala Gly Ala Ser Val Leu Ser Gly Gly Lys Leu Asp Arg Trpys Ile Arg Leu Arg Pro Gly Gly Lys Lys Lys Tyr Lys Leu Lys 2His Ile Val Trp Ala Ser Arg Glu Leu Glu Arg Phe Ala Val Asn Pro 35 4 Leu Leu Glu Thr Ser Glu Gly Cys Arg Arg Ile Leu Glu Gln Leu 5His Pro Ser Leu Gln Thr Gly Ser Glu Glu Leu Lys Ser Leu Tyr Asn65 7Thr Val Ala Thr Leu Tyr Cys Val His Gln Asn Ile Glu Val Arg Asp 85 9 Lys Asp Ala Leu Glu Lys Ile Glu Glu Glu Gln Asn Lys Ile Lys Arg Ala Gln Gln Ala Ala Ala Asp Thr Gly Asn Ser Asn Pro Val Gln Asn Tyr Pro Ile Val Gln Asn Met Gln Gly Gln Met Val His Ala Ile Ser Pro Arg Thr Leu Asn Ala Trp Val Lys Val Val Glu Glu Lys Ala Phe Ser Pro Glu Val Ile Pro Met Phe Ser Ala Leu Ser Gly Ala Thr Pro Gln Asp Leu Asn Thr Met Leu Asn Thr Val Gly His Gln Ala Ala Met Gln Met Leu Lys Glu Thr Ile Asn Glu Glu 2la Glu Trp Asp Arg Leu His Pro Val His Ala Gly Pro Ile Ala 222y Gln Met Arg Glu Pro Arg Gly Ser Asp Ile Ala Gly Thr Thr225 234r Leu Gln Glu Gln Ile Gly Trp Met Thr His Asn Pro Pro Ile 245 25o Val Gly Glu Ile Tyr Lys Arg Trp Ile Ile Met Gly Leu Asn Lys 26BR> 265 27l Arg Met Tyr Ser Pro Thr Ser Ile Leu Asp Ile Arg Gln Gly 275 28o Lys Glu Pro Phe Arg Asp Tyr Val Asp Arg Phe Tyr Lys Thr Leu 29la Glu Gln Ala Ser Gln Glu Val Lys Asn Trp Met Thr Glu Thr33eu Leu Val Gln Asn Ala Asn Pro Asp Cys Lys Thr Ile Leu Lys Ala 325 33u Gly Pro Ala Ala Thr Leu Glu Glu Met Met Thr Ala Cys Gln Gly 345y Gly Pro Ser His Lys Ala Arg Val Leu Ala Glu Ala Met Ser 355 36n Ala Thr Asn Ser Ala Thr Ile Met Met Gln Arg Gly Asn Phe Lys 378n Arg Lys Thr Val Lys Cys Phe Asn Cys Gly Lys Glu Gly His385 39la Arg Asn Cys Arg Ala Pro Arg Lys Lys Gly Cys Trp Lys Cys 44ys Glu Gly His Gln Met Lys Asp Cys Thr Glu Arg Gln Ala Asn 423u Gly Lys Ile Trp Pro Ser His Lys Gly Arg Pro Gly Asn Phe 435 44u Gln Ser Arg Pro Glu Pro Thr Ala Pro Pro Glu Glu Ser Phe Arg 456y Glu Glu Thr Thr Thr Pro Pro Gln Lys Gln Glu Pro Arg Asp465 478u Gln Tyr Pro Leu Thr Ser Leu Arg Ser Leu Phe Gly Asn Asp 485 49o Ser Ser Gln 5PRTArtificial sequenceMost recent common ancestor reconstruction of clade B gp tein sequence 5g Val Lys Gly Ile Arg Lys Asn Cys Gln His Leu Trp Lys Trphr Met Leu Leu Gly Met Leu Met Ile Cys Ser Ala Ala Glu Asn 2Leu Trp Val Thr Val Tyr Tyr Gly Val Pro Val Trp Lys Glu Ala Thr 35 4 Thr Leu Phe Cys Ala Ser Asp Ala Lys Ala Tyr Lys Thr Glu Val 5His Asn Val Trp Ala Thr His Ala Cys Val Pro Thr Asp Pro Asn Pro65 7Gln Glu Val Val Leu Glu Asn Val Thr Glu Asn Phe Asn Met Trp Lys 85 9 Asn Met Val Glu Gln Met His Glu Asp Ile Ile Ser Leu Trp Asp Ser Leu Lys Pro Cys Val Lys Leu Thr Pro Leu Cys Val Thr Leu Cys Thr Asp Ala Asn Lys Asn Ala Thr Asn Thr Asn Ser Ser Ser Gly Thr Met Glu Lys Gly Glu Met Lys Asn Cys Ser Phe Asn Ile Thr Thr Ser Ile Arg Asp Lys Met Gln Lys Glu Tyr Ala Leu Phe Tyr Leu Asp Val Val Pro Ile Asp Asn Asp Asn Asn Ser Asn Asn Asn Asn Tyr Arg Leu Ile Asn Cys Asn Thr Ser Val Ile Thr Gln Ala 2ro Lys Val Ser Phe Glu Pro Ile Pro Ile His Tyr Cys Thr Pro 222y Phe Ala Ile Leu Lys Cys Asn Asp Lys Lys Phe Asn Gly Thr225 234o Cys Lys Asn Val Ser Thr Val Gln Cys Thr His Gly Ile Arg 245 25o Val Val Ser Thr Gln Leu Leu Leu Asn Gly Ser Leu Ala Glu Glu 267l Val Ile Arg Ser Glu Asn Phe Thr Asp Asn Ala Lys Thr Ile 275 28e Val Gln Leu Asn Glu Ser Val Glu Ile Asn Cys Thr Arg Pro Asn 29sn Thr Arg Lys Ser Ile Pro Ile Gly Pro Gly Arg Ala Leu Tyr33hr Thr Gly Glu Ile Ile Gly Asp Ile Arg Gln Ala His Cys Asn Ile 325 33r Arg Ala Lys Trp Asn Asn Thr Leu Lys Gln Val Val Thr Lys Leu 345u Gln Phe Gly Asn Asn Lys Thr Ile Val Phe Asn Pro Ser Ser 355 36y Gly Asp Pro Glu Ile Val Met His Ser Phe Asn Cys Gly Gly Glu 378e Tyr Cys Asn Thr Thr Gln Leu Phe Asn Ser Thr Trp Asn Ser385 39lu Gly Ser Asn Lys Thr Thr Gly Ser Asn Asn Thr Gly Gly Glu 44le Thr Leu Pro Cys Arg Ile Lys Gln Ile Ile Asn Met Trp Gln 423l Gly Lys Ala Met Tyr Ala Pro Pro Ile Arg Gly Gln Ile Lys 435 44s Ser Ser Asn Ile Thr Gly Leu Leu Leu Thr Arg Asp Gly Gly Glu 456r Thr Asn Glu Thr Glu Ile Phe Arg Pro Gly Gly Gly Asp Met465 478p Asn Trp Arg Ser Glu Leu Tyr Lys Tyr Lys Val Val Lys Ile 485 49u Pro Leu Gly Val Ala Pro Thr Lys Ala Lys Arg Arg Val Val Gln 55lu Lys Arg Ala Val Gly Ile Ile Gly Ala Met Phe Leu Gly Phe 5525Leu Gly Ala Ala Gly Ser Thr Met Gly Ala Ala Ser Met Thr Leu Thr 534n Ala Arg Gln Leu Leu Ser Gly Ile Val Gln Gln Gln Asn Asn545 556u Arg Ala Ile Glu Ala Gln Gln His Leu Leu Gln Leu Thr Val 565 57p Gly Ile Lys Gln Leu Gln Ala Arg Val Leu Ala Val Glu Arg Tyr 589g Asp Gln Gln Leu Leu Gly Ile Trp Gly Cys Ser Gly Lys Leu 595 6le Cys Thr Thr Thr Val Pro Trp Asn Ala Ser Trp Ser Asn Lys Ser 662p Lys Ile Trp Asn Asn Met Thr Trp Met Glu Trp Glu Arg Glu625 634p Asn Tyr Thr Gly Leu Ile Tyr Asn Leu Ile Glu Glu Ser Gln 645 65n Gln Gln Glu Lys Asn Glu Gln Glu Leu Leu Glu Leu Asp Lys Trp 667r Leu Trp Asn Trp Phe Asp Ile Thr Gln Trp Leu Trp Tyr Ile 675 68s Ile Phe Ile Met Ile Val Gly Gly Leu Val Gly Leu Arg Ile Val 69la Val Leu Ser Ile Val Asn Arg Val Arg Gln Gly Tyr Ser Pro77eu Ser Phe Gln Thr Arg Leu Pro Ala Pro Arg Gly Pro Asp Arg Pro 725 73u Gly Ile Glu Glu Glu Gly Gly Glu Arg Asp Arg Asp Arg Ser Gly 745u Val Asn Gly Phe Leu Ala Leu Ile Trp Asp Asp Leu Arg Ser 755 76u Cys Leu Phe Ser Tyr His Arg Leu Arg Asp Leu Leu Leu Ile Val 778g Ile Val Glu Leu Leu Gly Arg Arg Gly Trp Glu Ala Leu Lys785 79rp Trp Asn Leu Leu Gln Tyr Trp Ser Gln Glu Leu Lys Asn Ser 88al Ser Leu Leu Asn Ala Thr Ala Ile Ala Val Ala Glu Gly Thr 823g Val Ile Glu Val Val Gln Arg Ala Cys Arg Ala Ile Leu His 835 84e Pro Arg Arg Ile Arg Gln Gly Leu Glu Arg Ala Leu Leu 856RTArtificial sequenceLeast squares and minimum of means center of tree reconstruction of clade B gp tein sequence 5g Val Lys Gly Ile Arg Lys Asn Tyr Gln His Leu Trp Arg Trphr Met Leu Leu Gly Met Leu Met Ile Cys Ser Ala Ala Glu Lys 2Leu Trp Val Thr Val Tyr Tyr Gly Val Pro Val Trp Lys Glu Ala Thr 35 4 Thr Leu Phe Cys Ala Ser Asp Ala Lys Ala Tyr Asp Thr Glu Val 5His Asn Val Trp Ala Thr His Ala Cys Val Pro Thr Asp Pro Asn Pro65 7Gln Glu Val Val Leu Glu Asn Val Thr Glu Asn Phe Asn Met Trp Lys 85 9 Asn Met Val Glu Gln Met His Glu Asp Ile Ile Ser Leu Trp Asp Ser Leu Lys Pro Cys Val Lys Leu Thr Pro Leu Cys Val Thr Leu Cys Thr Asp Leu Asn Lys Asn Ala Thr Asn Thr Asn Ser Ser Ser Glu Met Met Glu Lys Gly Glu Ile Lys Asn Cys Ser Phe Asn Ile Thr Thr Ser Ile Arg Asp Lys Val Gln Lys Glu Tyr Ala Leu Phe Tyr Leu Asp Val Val Pro Ile Asp Asn Asp Asn Asn Thr Asn Asn Thr Ser Tyr Arg Leu Ile Ser Cys Asn Thr Ser Val Ile Thr Gln Ala 2ro Lys Val Ser Phe Glu Pro Ile Pro Ile His Tyr Cys Ala Pro 222y Phe Ala Ile Leu Lys Cys Asn Asp Lys Lys Phe Asn Gly Thr225 234o Cys Thr Asn Val Ser Thr Val Gln Cys Thr His Gly Ile Arg 245 25o Val Val Ser Thr Gln Leu Leu Leu Asn Gly Ser Leu Ala Glu Glu 267l Val Ile Arg Ser Asp Asn Phe Thr Asp Asn Ala Lys Thr Ile 275 28e Val Gln Leu Asn Glu Ser Val Glu Ile Asn Cys Thr Arg Pro Asn 29sn Thr Arg Lys Ser Ile His Ile Gly Pro Gly Arg Ala Phe Tyr33hr Thr Gly Glu Ile Ile Gly Asp Ile Arg Gln Ala His Cys Asn Ile 325 33r Arg Ala Lys Trp Asn Asn Thr Leu Lys Gln Ile Val Lys Lys Leu 345u Gln Phe Gly Asn Asn Lys Thr Ile Val Phe Asn Gln Ser Ser 355 36y Gly Asp Pro Glu Ile Val Met His Ser Phe Asn Cys Gly Gly Glu 378e Tyr Cys Asn Ser Thr Gln Leu Phe Asn Ser Thr Trp Asn Gly385 39rp Thr Trp Asn Thr Thr Glu Gly Ser Asn Asp Thr Glu Gly Asp 44le Thr Leu Pro Cys Arg Ile Lys Gln Ile Ile Asn Met Trp Gln 423l Gly Lys Ala Met Tyr Ala Pro Pro Ile Arg Gly Gln Ile Arg 435 44s Ser Ser Asn Ile Thr Gly Leu Leu Leu Thr Arg Asp Gly Gly Asn 456n Thr Asn Glu Thr Glu Ile Phe Arg Pro Gly Gly Gly Asp Met465 478p Asn Trp Arg Ser Glu Leu Tyr Lys Tyr Lys Val Val Lys Ile 485 49u Pro Leu Gly Val Ala Pro Thr Lys Ala Lys Arg Arg Val Val Gln 55lu Lys Arg Ala Val Gly Ile Ile Gly Ala Val Phe Leu Gly Phe 5525Leu Gly Ala Ala Gly Ser Thr Met Gly Ala Ala Ser Met Thr Leu Thr 534n Ala Arg Gln Leu Leu Ser Gly Ile Val Gln Gln Gln Asn Asn545 556u Arg Ala Ile Glu Ala Gln Gln His Leu Leu Gln Leu Thr Val 565 57p Gly Ile Lys Gln Leu Gln Ala Arg Val Leu Ala Val Glu Arg Tyr 589g Asp Gln Gln Leu Leu Gly Ile Trp Gly Cys Ser Gly Lys Leu 595 6le Cys Thr Thr Ala Val Pro Trp Asn Ala Ser Trp Ser Asn Lys Ser 662p Glu Ile Trp Asn Asn Met Thr Trp Met Glu Trp Glu Arg Glu625 634p Asn Tyr Thr Ser Leu Ile Tyr Thr Leu Ile Glu Glu Ser Gln 645 65n Gln Gln Glu Lys Asn Glu Gln Glu Leu Leu Glu Leu Asp Lys Trp 667r Leu Trp Asn Trp Phe Asp Ile Thr Asn Trp Leu Trp Tyr Ile 675 68s Ile Phe Ile Met Ile Val Gly Gly Leu Val Gly Leu Arg Ile Val 69la Val Leu Ser Ile Val Asn Arg Val Arg Gln Gly Tyr Ser Pro77eu Ser Phe Gln Thr Arg Leu Pro Ala Pro Arg Gly Pro Asp Arg Pro 725 73u Gly Ile Glu Glu Glu Gly Gly Glu Arg Asp Arg Asp Arg Ser Gly 745u Val Asn Gly Phe Leu Ala Leu Ile Trp Asp Asp Leu Arg Ser 755 76u Cys Leu Phe Ser Tyr His Arg Leu Arg Asp Leu Leu Leu Ile Val 778g Ile Val Glu Leu Leu Gly Arg Arg Gly Trp Glu Ala Leu Lys785 79rp Trp Asn Leu Leu Gln Tyr Trp Ser Gln Glu Leu Lys Asn Ser 88al Ser Leu Leu Asn Ala Thr Ala Ile Ala Val Ala Glu Gly Thr 823g Val Ile Glu Val Val Gln Arg Ala Cys Arg Ala Ile Leu His 835 84e Pro Thr Arg Ile Arg Gln Gly Leu Glu Arg Ala Leu Leu 856RTArtificial sequenceMost recent common ancestor reconstruction of clade B nef protein sequence 52Met Gly Gly Lys Trp Ser Lys Arg Ser Val Val Gly Trp Pro Ala Vallu Arg Met Arg Arg Ala Glu Pro Ala Ala Asp Gly Val Gly Ala 2Val Ser Arg Asp Leu Glu Lys His Gly Ala Ile Thr Ser Ser Asn Thr 35 4 Ala Thr Asn Ala Ala Cys Ala Trp Leu Glu Ala Gln Glu Glu Glu 5Glu Val Gly Phe Pro Val Arg Pro Gln Val Pro Leu Arg Pro Met Thr65 7Tyr Lys Ala Ala Val Asp Leu Ser His Phe Leu Lys Glu Lys Gly Gly 85 9 Glu Gly Leu Val Tyr Ser Gln Lys Arg Gln Asp Ile Leu Asp Leu Val Tyr His Thr Gln Gly Tyr Phe Pro Asp Trp Gln Asn Tyr Thr Gly Pro Gly Thr Arg Tyr Pro Leu Thr Phe Gly Trp Cys Phe Lys Val Pro Val Glu Pro Glu Lys Val Glu Glu Ala Thr Glu Gly Glu Asn Asn Ser Leu Leu His Pro Met Ser Leu His Gly Met Asp Asp Pro Arg Glu Val Leu Val Trp Arg Phe Asp Ser Arg Leu Ala Phe His Met Ala Arg Glu Lys His Pro Glu Tyr Tyr Lys Asp Cys 2PRTArtificial sequenceLeast squares center of tree reconstruction of clade B nef protein sequence 53Met Gly Gly Lys Trp Ser Lys Arg Ser Val Val Gly Trp Pro Ala Vallu Arg Met Arg Arg Ala Glu Pro Ala Ala Asp Gly Val Gly Ala 2Val Ser Arg Asp Leu Glu Lys His Gly Ala Ile Thr Ser Ser Asn Thr 35 4 Ala Thr Asn Ala Asp Cys Ala Trp Leu Glu Ala Gln Glu Glu Glu 5Glu Val Gly Phe Pro Val Arg Pro Gln Val Pro Leu Arg Pro Met Thr65 7Tyr Lys Ala Ala Leu Asp Leu Ser His Phe Leu Lys Glu Lys Gly Gly 85 9 Glu Gly Leu Ile Tyr Ser Gln Lys Arg Gln Asp Ile Leu Asp Leu Val Tyr His Thr Gln Gly Tyr Phe Pro Asp Trp Gln Asn Tyr Thr Gly Pro Gly Ile Arg Tyr Pro Leu Thr Phe Gly Trp Cys Phe Lys Val Pro Val Glu Pro Glu Lys Val Glu Glu Ala Asn Glu Gly Glu Asn Asn Ser Leu Leu His Pro Met Ser Leu His Gly Met Asp Asp Pro Lys Glu Val Leu Val Trp Lys Phe Asp Ser Arg Leu Ala Phe His Met Ala Arg Glu Leu His Pro Glu Tyr Tyr Lys Asp Cys 2PRTArtificial sequenceMinimum of means center of tree reconstruction of clade B nef protein sequence 54Met Gly Gly Lys Trp Ser Lys Arg Ser Val Val Gly Trp Pro Ala Vallu Arg Met Arg Arg Ala Glu Pro Ala Ala

Asp Gly Val Gly Ala 2Val Ser Arg Asp Leu Glu Lys His Gly Ala Ile Thr Ser Ser Asn Thr 35 4 Ala Thr Asn Ala Asp Cys Ala Trp Leu Glu Ala Gln Glu Glu Glu 5Glu Val Gly Phe Pro Val Arg Pro Gln Val Pro Leu Arg Pro Met Thr65 7Tyr Lys Ala Ala Leu Asp Leu Ser His Phe Leu Lys Glu Lys Gly Gly 85 9 Glu Gly Leu Ile Tyr Ser Gln Lys Arg Gln Asp Ile Leu Asp Leu Val Tyr His Thr Gln Gly Tyr Phe Pro Asp Trp Gln Asn Tyr Thr Gly Pro Gly Ile Arg Tyr Pro Leu Thr Phe Gly Trp Cys Phe Lys Val Pro Val Glu Pro Glu Lys Val Glu Glu Ala Asn Glu Gly Glu Asn Asn Cys Leu Leu His Pro Met Ser Gln His Gly Met Asp Asp Pro Lys Glu Val Leu Val Trp Lys Phe Asp Ser Arg Leu Ala Phe His Met Ala Arg Glu Leu His Pro Glu Tyr Tyr Lys Asp Cys 23PRTArtificial sequenceMost recent common ancestor reconstruction of clade B pol protein sequence 55Phe Phe Arg Glu Asn Leu Ala Phe Pro Gln Gly Lys Ala Arg Glu Leuer Glu Gln Thr Arg Ala Asn Ser Pro Thr Arg Arg Glu Leu Gln 2Val Trp Gly Arg Asp Asn Asn Ser Leu Ser Glu Ala Gly Ala Asp Arg 35 4 Gly Thr Val Ser Phe Ser Phe Pro Gln Ile Thr Leu Trp Gln Arg 5Pro Leu Val Thr Ile Lys Ile Gly Gly Gln Leu Lys Glu Ala Leu Leu65 7Asp Thr Gly Ala Asp Asp Thr Val Leu Glu Glu Met Asn Leu Pro Gly 85 9 Trp Lys Pro Lys Met Ile Gly Gly Ile Gly Gly Phe Ile Lys Val Gln Tyr Asp Gln Ile Pro Ile Glu Ile Cys Gly His Lys Ala Ile Thr Val Leu Val Gly Pro Thr Pro Val Asn Ile Ile Gly Arg Asn Leu Thr Gln Ile Gly Cys Thr Leu Asn Phe Pro Ile Ser Pro Ile Glu Thr Val Pro Val Lys Leu Lys Pro Gly Met Asp Gly Pro Lys Val Gln Trp Pro Leu Thr Glu Glu Lys Ile Lys Ala Leu Val Glu Ile Thr Glu Met Glu Lys Glu Gly Lys Ile Ser Lys Ile Gly Pro Glu 2ro Tyr Asn Thr Pro Val Phe Ala Ile Lys Lys Lys Asp Ser Thr 222p Arg Lys Leu Val Asp Phe Arg Glu Leu Asn Lys Arg Thr Gln225 234e Trp Glu Val Gln Leu Gly Ile Pro His Pro Ala Gly Leu Lys 245 25s Lys Lys Ser Val Thr Val Leu Asp Val Gly Asp Ala Tyr Phe Ser 267o Leu Asp Glu Asp Phe Arg Lys Tyr Thr Ala Phe Thr Ile Pro 275 28r Ile Asn Asn Glu Thr Pro Gly Ile Arg Tyr Gln Tyr Asn Val Leu 29ln Gly Trp Lys Gly Ser Pro Ala Ile Phe Gln Ser Ser Met Thr33ys Ile Leu Glu Pro Phe Arg Lys Gln Asn Pro Glu Ile Val Ile Tyr 325 33n Tyr Met Asp Asp Leu Tyr Val Gly Ser Asp Leu Glu Ile Gly Gln 345g Thr Lys Ile Glu Glu Leu Arg Glu His Leu Leu Arg Trp Gly 355 36e Thr Thr Pro Asp Lys Lys His Gln Lys Glu Pro Pro Phe Leu Trp 378y Tyr Glu Leu His Pro Asp Lys Trp Thr Val Gln Pro Ile Val385 39ro Glu Lys Asp Ser Trp Thr Val Asn Asp Ile Gln Lys Leu Val 44ys Leu Asn Trp Ala Ser Gln Ile Tyr Ala Gly Ile Lys Val Lys 423u Cys Lys Leu Leu Arg Gly Thr Lys Ala Leu Thr Glu Val Val 435 44o Leu Thr Glu Glu Ala Glu Leu Glu Leu Ala Glu Asn Arg Glu Ile 456s Glu Pro Val His Gly Val Tyr Tyr Asp Pro Ser Lys Asp Leu465 478a Glu Ile Gln Lys Gln Gly Gln Gly Gln Trp Thr Tyr Gln Ile 485 49r Gln Glu Pro Phe Lys Asn Leu Lys Thr Gly Lys Tyr Ala Arg Met 55ly Ala His Thr Asn Asp Val Lys Gln Leu Thr Glu Ala Val Gln 5525Lys Ile Ala Thr Glu Ser Ile Val Ile Trp Gly Lys Thr Pro Lys Phe 534u Pro Ile Gln Lys Glu Thr Trp Glu Ala Trp Trp Thr Glu Tyr545 556n Ala Thr Trp Ile Pro Glu Trp Glu Phe Val Asn Thr Pro Pro 565 57u Val Lys Leu Trp Tyr Gln Leu Glu Lys Glu Pro Ile Val Gly Ala 589r Phe Tyr Val Asp Gly Ala Ala Asn Arg Glu Thr Lys Leu Gly 595 6ys Ala Gly Tyr Val Thr Asp Arg Gly Arg Gln Lys Val Val Ser Leu 662p Thr Thr Asn Gln Lys Thr Glu Leu Gln Ala Ile His Leu Ala625 634n Asp Ser Gly Leu Glu Val Asn Ile Val Thr Asp Ser Gln Tyr 645 65a Leu Gly Ile Ile Gln Ala Gln Pro Asp Lys Ser Glu Ser Glu Leu 667r Gln Ile Ile Glu Gln Leu Ile Lys Lys Glu Lys Val Tyr Leu 675 68a Trp Val Pro Ala His Lys Gly Ile Gly Gly Asn Glu Gln Val Asp 69eu Val Ser Thr Gly Ile Arg Lys Val Leu Phe Leu Asp Gly Ile77sp Lys Ala Gln Glu Glu His Glu Lys Tyr His Ser Asn Trp Arg Ala 725 73t Ala Ser Asp Phe Asn Leu Pro Pro Val Val Ala Lys Glu Ile Val 745r Cys Asp Lys Cys Gln Leu Lys Gly Glu Ala Met His Gly Gln 755 76l Asp Cys Ser Pro Gly Ile Trp Gln Leu Asp Cys Thr His Leu Glu 778s Val Ile Leu Val Ala Val His Val Ala Ser Gly Tyr Ile Glu785 79lu Val Ile Pro Ala Glu Thr Gly Gln Glu Thr Ala Tyr Phe Leu 88ys Leu Ala Gly Arg Trp Pro Val Lys Val Ile His Thr Asp Asn 823r Asn Phe Thr Ser Thr Thr Val Lys Ala Ala Cys Trp Trp Ala 835 84y Ile Lys Gln Glu Phe Gly Ile Pro Tyr Asn Pro Gln Ser Gln Gly 856l Glu Ser Met Asn Lys Glu Leu Lys Lys Ile Ile Gly Gln Val865 878p Gln Ala Glu His Leu Lys Thr Ala Val Gln Met Ala Val Phe 885 89e His Asn Phe Lys Arg Lys Gly Gly Ile Gly Gly Tyr Ser Ala Gly 99rg Ile Val Asp Ile Ile Ala Thr Asp Ile Gln Thr Lys Glu Leu 9925Gln Lys Gln Ile Thr Lys Ile Gln Asn Phe Arg Val Tyr Tyr Arg Asp 934g Asp Pro Leu Trp Lys Gly Pro Ala Lys Leu Leu Trp Lys Gly945 956y Ala Val Val Ile Gln Asp Asn Ser Asp Ile Lys Val Val Pro 965 97g Arg Lys Ala Lys Ile Ile Arg Asp Tyr Gly Lys Gln Met Ala Gly 989p Cys Val Ala Ser Arg Gln Asp Glu Asp 995 rtificial sequenceLeast squares center of tree reconstruction of clade B pol protein sequence 56Phe Phe Arg Glu Asp Leu Ala Phe Pro Gln Gly Lys Ala Arg Glu Pheer Glu Gln Thr Arg Ala Asn Ser Pro Thr Arg Arg Glu Leu Gln 2Val Trp Gly Arg Asp Asn Asn Ser Leu Ser Glu Ala Gly Ala Asp Arg 35 4 Gly Thr Val Ser Phe Ser Phe Pro Gln Ile Thr Leu Trp Gln Arg 5Pro Leu Val Thr Ile Lys Ile Gly Gly Gln Leu Lys Glu Ala Leu Leu65 7Asp Thr Gly Ala Asp Asp Thr Val Leu Glu Glu Met Asn Leu Pro Gly 85 9 Trp Lys Pro Lys Met Ile Gly Gly Ile Gly Gly Phe Ile Lys Val Gln Tyr Asp Gln Ile Pro Ile Glu Ile Cys Gly His Lys Ala Ile Thr Val Leu Val Gly Pro Thr Pro Val Asn Ile Ile Gly Arg Asn Leu Thr Gln Ile Gly Cys Thr Leu Asn Phe Pro Ile Ser Pro Ile Glu Thr Val Pro Val Lys Leu Lys Pro Gly Met Asp Gly Pro Lys Val Gln Trp Pro Leu Thr Glu Glu Lys Ile Lys Ala Leu Val Glu Ile Thr Glu Met Glu Lys Glu Gly Lys Ile Ser Lys Ile Gly Pro Glu 2ro Tyr Asn Thr Pro Val Phe Ala Ile Lys Lys Lys Asp Ser Thr 222p Arg Lys Leu Val Asp Phe Arg Glu Leu Asn Lys Arg Thr Gln225 234e Trp Glu Val Gln Leu Gly Ile Pro His Pro Ala Gly Leu Lys 245 25s Lys Lys Ser Val Thr Val Leu Asp Val Gly Asp Ala Tyr Phe Ser 267o Leu Asp Glu Asp Phe Arg Lys Tyr Thr Ala Phe Thr Ile Pro 275 28r Ile Asn Asn Glu Thr Pro Gly Ile Arg Tyr Gln Tyr Asn Val Leu 29ln Gly Trp Lys Gly Ser Pro Ala Ile Phe Gln Ser Ser Met Thr33ys Ile Leu Glu Pro Phe Arg Lys Gln Asn Pro Asp Ile Val Ile Tyr 325 33n Tyr Met Asp Asp Leu Tyr Val Gly Ser Asp Leu Glu Ile Gly Gln 345g Thr Lys Ile Glu Glu Leu Arg Gln His Leu Leu Arg Trp Gly 355 36e Thr Thr Pro Asp Lys Lys His Gln Lys Glu Pro Pro Phe Leu Trp 378y Tyr Glu Leu His Pro Asp Lys Trp Thr Val Gln Pro Ile Val385 39ro Glu Lys Asp Ser Trp Thr Val Asn Asp Ile Gln Lys Leu Val 44ys Leu Asn Trp Ala Ser Gln Ile Tyr Ala Gly Ile Lys Val Lys 423u Cys Lys Leu Leu Arg Gly Thr Lys Ala Leu Thr Glu Val Ile 435 44o Leu Thr Glu Glu Ala Glu Leu Glu Leu Ala Glu Asn Arg Glu Ile 456s Glu Pro Val His Gly Val Tyr Tyr Asp Pro Ser Lys Asp Leu465 478a Glu Ile Gln Lys Gln Gly Gln Gly Gln Trp Thr Tyr Gln Ile 485 49r Gln Glu Pro Phe Lys Asn Leu Lys Thr Gly Lys Tyr Ala Arg Met 55ly Ala His Thr Asn Asp Val Lys Gln Leu Thr Glu Ala Val Gln 5525Lys Ile Ala Thr Glu Ser Ile Val Ile Trp Gly Lys Thr Pro Lys Phe 534u Pro Ile Gln Lys Glu Thr Trp Glu Ala Trp Trp Thr Glu Tyr545 556n Ala Thr Trp Ile Pro Glu Trp Glu Phe Val Asn Thr Pro Pro 565 57u Val Lys Leu Trp Tyr Gln Leu Glu Lys Glu Pro Ile Val Gly Ala 589r Phe Tyr Val Asp Gly Ala Ala Asn Arg Glu Thr Lys Leu Gly 595 6ys Ala Gly Tyr Val Thr Asp Arg Gly Arg Gln Lys Val Val Ser Leu 662p Thr Thr Asn Gln Lys Thr Glu Leu Gln Ala Ile His Leu Ala625 634n Asp Ser Gly Leu Glu Val Asn Ile Val Thr Asp Ser Gln Tyr 645 65a Leu Gly Ile Ile Gln Ala Gln Pro Asp Lys Ser Glu Ser Glu Leu 667r Gln Ile Ile Glu Gln Leu Ile Lys Lys Glu Lys Val Tyr Leu 675 68a Trp Val Pro Ala His Lys Gly Ile Gly Gly Asn Glu Gln Val Asp 69eu Val Ser Ala Gly Ile Arg Lys Val Leu Phe Leu Asp Gly Ile77sp Lys Ala Gln Glu Glu His Glu Lys Tyr His Ser Asn Trp Arg Ala 725 73t Ala Ser Asp Phe Asn Leu Pro Pro Val Val Ala Lys Glu Ile Val 745r Cys Asp Lys Cys Gln Leu Lys Gly Glu Ala Met His Gly Gln 755 76l Asp Cys Ser Pro Gly Ile Trp Gln Leu Asp Cys Thr His Leu Glu 778s Val Ile Leu Val Ala Val His Val Ala Ser Gly Tyr Ile Glu785 79lu Val Ile Pro Ala Glu Thr Gly Gln Glu Thr Ala Tyr Phe Leu 88ys Leu Ala Gly Arg Trp Pro Val Lys Thr Ile His Thr Asp Asn 823r Asn Phe Thr Ser Thr Thr Val Lys Ala Ala Cys Trp Trp Ala 835 84y Ile Lys Gln Glu Phe Gly Ile Pro Tyr Asn Pro Gln Ser Gln Gly 856l Glu Ser Met Asn Lys Glu Leu Lys Lys Ile Ile Gly Gln Val865 878p Gln Ala Glu His Leu Lys Thr Ala Val Gln Met Ala Val Phe 885 89e His Asn Phe Lys Arg Lys Gly Gly Ile Gly Gly Tyr Ser Ala Gly 99rg Ile Val Asp Ile Ile Ala Thr Asp Ile Gln Thr Lys Glu Leu 9925Gln Lys Gln Ile Thr Lys Ile Gln Asn Phe Arg Val Tyr Tyr Arg Asp 934g Asp Pro Leu Trp Lys Gly Pro Ala Lys Leu Leu Trp Lys Gly945 956y Ala Val Val Ile Gln Asp Asn Ser Asp Ile Lys Val Val Pro 965 97g Arg Lys Ala Lys Ile Ile Arg Asp Tyr Gly Lys Gln Met Ala Gly 989p Cys Val Ala Ser Arg Gln Asp Glu Asp 995 rtificial sequenceMinimum of means center of tree reconstruction of clade B pol protein sequence 57Phe Phe Arg Glu Asp Leu Ala Phe Pro Gln Gly Lys Ala Arg Glu Pheer Glu Gln Thr Arg Ala Asn Ser Pro Thr Arg Arg Glu Leu Gln 2Val Trp Gly Arg Asp Asn Asn Ser Leu Ser Glu Ala Gly Ala Asp Arg 35 4 Gly Thr Val Ser Phe Ser Phe Pro Gln Ile Thr Leu Trp Gln Arg 5Pro Leu Val Thr Ile Lys Ile Gly Gly Gln Leu Lys Glu Ala Leu Leu65 7Asp Thr Gly Ala Asp Asp Thr Val Leu Glu Glu Met Asn Leu Pro Gly 85 9 Trp Lys Pro Lys Met Ile Gly Gly Ile Gly Gly Phe Ile Lys Val Gln Tyr Asp Gln Ile Leu Ile Glu Ile Cys Gly His Lys Ala Ile Thr Val Leu Val Gly Pro Thr Pro Val Asn Ile Ile Gly Arg Asn Leu Thr Gln Ile Gly Cys Thr Leu Asn Phe Pro Ile Ser Pro Ile Glu Thr Val Pro Val Lys Leu Lys Pro Gly Met Asp Gly Pro Lys Val Gln Trp Pro Leu Thr Glu Glu Lys Ile Lys Ala Leu Val Glu Ile Thr Glu Met Glu Lys Glu Gly Lys Ile Ser Lys Ile Gly Pro Glu 2ro Tyr Asn Thr Pro Val Phe Ala Ile Lys Lys Lys Asp Ser Thr 222p Arg Lys Leu Val Asp Phe Arg Glu Leu Asn Lys Arg Thr Gln225 23BR>
24e Trp Glu Val Gln Leu Gly Ile Pro His Pro Ala Gly Leu Lys 245 25s Lys Lys Ser Val Thr Val Leu Asp Val Gly Asp Ala Tyr Phe Ser 267o Leu Asp Glu Asp Phe Arg Lys Tyr Thr Ala Phe Thr Ile Pro 275 28r Ile Asn Asn Glu Thr Pro Gly Ile Arg Tyr Gln Tyr Asn Val Leu 29ln Gly Trp Lys Gly Ser Pro Ala Ile Phe Gln Ser Ser Met Thr33ys Ile Leu Glu Pro Phe Arg Lys Gln Asn Pro Asp Ile Val Ile Tyr 325 33n Tyr Met Asp Asp Leu Tyr Val Gly Ser Asp Leu Glu Ile Gly Gln 345g Thr Lys Ile Glu Glu Leu Arg Gln His Leu Leu Arg Trp Gly 355 36e Thr Thr Pro Asp Lys Lys His Gln Lys Glu Pro Pro Phe Leu Trp 378y Tyr Glu Leu His Pro Asp Lys Trp Thr Val Gln Pro Ile Val385 39ro Glu Lys Asp Ser Trp Thr Val Asn Asp Ile Gln Lys Leu Val 44ys Leu Asn Trp Ala Ser Gln Ile Tyr Pro Gly Ile Lys Val Lys 423u Cys Lys Leu Leu Arg Gly Thr Lys Ala Leu Thr Glu Val Ile 435 44o Leu Thr Glu Glu Ala Glu Leu Glu Leu Ala Glu Asn Arg Glu Ile 456s Glu Pro Val His Gly Val Tyr Tyr Asp Pro Ser Lys Asp Leu465 478a Glu Ile Gln Lys Gln Gly Gln Gly Gln Trp Thr Tyr Gln Ile 485 49r Gln Glu Pro Phe Lys Asn Leu Lys Thr Gly Lys Tyr Ala Arg Met 55ly Ala His Thr Asn Asp Val Lys Gln Leu Thr Glu Ala Val Gln 5525Lys Ile Ala Thr Glu Ser Ile Val Ile Trp Gly Lys Thr Pro Lys Phe 534u Pro Ile Gln Lys Glu Thr Trp Glu Thr Trp Trp Thr Glu Tyr545 556n Ala Thr Trp Ile Pro Glu Trp Glu Phe Val Asn Thr Pro Pro 565 57u Val Lys Leu Trp Tyr Gln Leu Glu Lys Glu Pro Ile Val Gly Ala 589r Phe Tyr Val Asp Gly Ala Ala Asn Arg Glu Thr Lys Leu Gly 595 6ys Ala Gly Tyr Val Thr Asn Arg Gly Arg Gln Lys Val Val Ser Leu 662p Thr Thr Asn Gln Lys Thr Glu Leu Gln Ala Ile His Leu Ala625 634n Asp Ser Gly Leu Glu Val Asn Ile Val Thr Asp Ser Gln Tyr 645 65a Leu Gly Ile Ile Gln Ala Gln Pro Asp Lys Ser Glu Ser Glu Leu 667r Gln Ile Ile Glu Gln Leu Ile Lys Lys Glu Lys Val Tyr Leu 675 68a Trp Val Pro Ala His Lys Gly Ile Gly Gly Asn Glu Gln Val Asp 69eu Val Ser Ala Gly Ile Arg Lys Val Leu Phe Leu Asp Gly Ile77sp Lys Ala Gln Glu Glu His Glu Lys Tyr His Ser Asn Trp Arg Ala 725 73t Ala Ser Asp Phe Asn Leu Pro Pro Val Val Ala Lys Glu Ile Val 745r Cys Asp Lys Cys Gln Leu Lys Gly Glu Ala Met His Gly Gln 755 76l Asp Cys Ser Pro Gly Ile Trp Gln Leu Asp Cys Thr His Leu Glu 778s Val Ile Leu Val Ala Val His Val Ala Ser Gly Tyr Ile Glu785 79lu Val Ile Pro Ala Glu Thr Gly Gln Glu Thr Ala Tyr Phe Leu 88ys Leu Ala Gly Arg Trp Pro Val Lys Thr Ile His Thr Asp Asn 823r Asn Phe Thr Ser Thr Thr Val Lys Ala Ala Cys Trp Trp Ala 835 84y Ile Lys Gln Glu Phe Gly Ile Pro Tyr Asn Pro Gln Ser Gln Gly 856l Glu Ser Met Asn Lys Glu Leu Lys Lys Ile Ile Gly Gln Val865 878p Gln Ala Glu His Leu Lys Thr Ala Val Gln Met Ala Val Phe 885 89e His Asn Phe Lys Arg Lys Gly Gly Ile Gly Gly Tyr Ser Ala Gly 99rg Ile Val Asp Ile Ile Ala Thr Asp Ile Gln Thr Lys Glu Leu 9925Gln Lys Gln Ile Thr Lys Ile Gln Asn Phe Arg Val Tyr Tyr Arg Asp 934g Asp Pro Leu Trp Lys Gly Pro Ala Lys Leu Leu Trp Lys Gly945 956y Ala Val Val Ile Gln Asp Asn Ser Asp Ile Lys Val Val Pro 965 97g Arg Lys Ala Lys Ile Ile Arg Asp Tyr Gly Lys Gln Met Ala Gly 989p Cys Val Ala Ser Arg Gln Asp Glu Asp 995 tificial sequenceMost recent common ancestor reconstruction of clade B rev protein sequence 58Met Ala Gly Arg Ser Gly Asp Ser Asp Glu Glu Leu Leu Lys Thr Valeu Ile Lys Phe Leu Tyr Gln Ser Asn Pro Pro Pro Ser Pro Glu 2Gly Thr Arg Gln Ala Arg Arg Asn Arg Arg Arg Arg Trp Arg Glu Arg 35 4 Arg Gln Ile Arg Ser Ile Ser Glu Arg Ile Leu Ser Thr Tyr Leu 5Gly Arg Ser Ala Glu Pro Val Pro Leu Gln Leu Pro Pro Leu Glu Arg65 7Leu Thr Leu Asp Cys Ser Glu Asp Cys Gly Thr Ser Gly Thr Gln Gly 85 9 Gly Ser Pro Gln Ile Leu Val Glu Ser Pro Ala Val Leu Glu Ser Thr Lys Glu 6PRT; 59Met Ala Gly Arg Ser Gly Asp Ser Asp Glu Glu Leu Leu Lys Thr Valeu Ile Lys Phe Leu Tyr Gln Ser Asn Pro Pro Pro Ser Pro Glu 2Gly Thr Arg Gln Ala Arg Arg Asn Arg Arg Arg Arg Trp Arg Glu Arg 35 4 Arg Gln Ile Arg Ser Ile Ser Glu Trp Ile Leu Ser Thr Tyr Leu 5Gly Arg Pro Ala Glu Pro Val Pro Leu Gln Leu Pro Pro Leu Glu Arg65 7Leu Thr Leu Asp Cys Asn Glu Asp Cys Gly Thr Ser Gly Thr Gln Gly 85 9 Gly Ser Pro Gln Ile Leu Val Glu Ser Pro Thr Val Leu Glu Ser Thr Lys Glu 6PRTArtificial sequenceMinimum of means center of tree reconstruction of clade B rev protein sequence 6a Gly Arg Ser Gly Asp Ser Asp Glu Glu Leu Leu Lys Thr Valeu Ile Lys Phe Leu Tyr Gln Ser Asn Pro Pro Pro Ser Pro Glu 2Gly Thr Arg Gln Ala Arg Arg Asn Arg Arg Arg Arg Trp Arg Glu Arg 35 4 Arg Gln Ile Arg Ser Ile Ser Glu Trp Ile Leu Ser Thr Tyr Leu 5Gly Arg Pro Ala Glu Pro Val Pro Leu Gln Leu Pro Pro Leu Glu Arg65 7Leu Thr Leu Asp Cys Ser Glu Asp Cys Gly Thr Ser Gly Thr Gln Gly 85 9 Gly Ser Pro Gln Ile Leu Val Glu Ser Pro Ala Val Leu Glu Ser Thr Lys Glu ificial sequenceMost common recent ancestor reconstruction of clade B tat protein sequence 6u Pro Val Asp Pro Arg Leu Glu Pro Trp Lys His Pro Gly Serro Lys Thr Ala Cys Thr Asn Cys Tyr Cys Lys Lys Cys Cys Tyr 2His Cys Gln Val Cys Phe Ile Thr Lys Gly Leu Gly Ile Ser Tyr Gly 35 4 Lys Lys Arg Arg Gln Arg Arg Arg Pro Pro Gln Gly Ser Gln Thr 5His Gln Val Ser Leu Ser Lys Gln Pro Ala Ser Gln Pro Arg Gly Asp65 7Pro Thr Gly Pro Lys Glu Ser Lys Lys Lys Val Glu Arg Glu Thr Glu 85 9 Asp Pro Val Asp ificial sequenceLeast squares and minimum of means reconstruction of clade B tat protein sequence 62Met Glu Pro Val Asp Pro Arg Leu Glu Pro Trp Lys His Pro Gly Serro Lys Thr Ala Cys Thr Asn Cys Tyr Cys Lys Lys Cys Cys Phe 2His Cys Gln Val Cys Phe Ile Thr Lys Gly Leu Gly Ile Ser Tyr Gly 35 4 Lys Lys Arg Arg Gln Arg Arg Arg Ala Pro Gln Asp Ser Gln Thr 5His Gln Val Ser Leu Ser Lys Gln Pro Ala Ser Gln Pro Arg Gly Asp65 7Pro Thr Gly Pro Lys Glu Ser Lys Lys Lys Val Glu Arg Glu Thr Glu 85 9 Asp Pro Val Asp 2PRTArtificial sequenceMost recent common ancestor reconstruction of clade B vif protein sequence 63Met Glu Asn Arg Trp Gln Val Met Ile Val Trp Gln Val Asp Arg Metle Arg Thr Trp Lys Ser Leu Val Lys His His Met Tyr Ile Ser 2Lys Lys Ala Lys Gly Trp Phe Tyr Arg His His Tyr Glu Ser Thr His 35 4 Arg Ile Ser Ser Glu Val His Ile Pro Leu Gly Asp Ala Arg Leu 5Val Ile Lys Thr Tyr Trp Gly Leu His Thr Gly Glu Arg Glu Trp His65 7Leu Gly Gln Gly Val Ser Ile Glu Trp Arg Lys Arg Arg Tyr Ser Thr 85 9 Val Asp Pro Gly Leu Ala Asp Gln Leu Ile His Leu Tyr Tyr Phe Cys Phe Ser Glu Ser Ala Ile Arg Asn Ala Ile Leu Gly His Ile Ser Pro Arg Cys Glu Tyr Gln Ala Gly His Asn Lys Val Gly Ser Gln Tyr Leu Ala Leu Thr Ala Leu Ile Thr Pro Lys Lys Ile Lys Pro Pro Leu Pro Ser Val Arg Lys Leu Thr Glu Asp Arg Trp Asn Lys Gln Lys Thr Lys Gly His Arg Gly Ser His Thr Met Asn Gly His 2PRTArtificial sequenceLeast squares and minimum of means reconstruction of clade B vif protein sequence 64Met Glu Asn Arg Trp Gln Val Met Ile Val Trp Gln Val Asp Arg Metle Arg Thr Trp Lys Ser Leu Val Lys His His Met Tyr Ile Ser 2Arg Lys Ala Lys Gly Trp Phe Tyr Arg His His Tyr Glu Ser Thr His 35 4 Arg Ile Ser Ser Glu Val His Ile Pro Leu Gly Asp Ala Arg Leu 5Val Ile Thr Thr Tyr Trp Gly Leu His Thr Gly Glu Arg Asp Trp His65 7Leu Gly Gln Gly Val Ser Ile Glu Trp Arg Lys Lys Arg Tyr Ser Thr 85 9 Val Asp Pro Asp Leu Ala Asp Gln Leu Ile His Leu Tyr Tyr Phe Cys Phe Ser Glu Ser Ala Ile Arg Asn Ala Ile Leu Gly His Ile Ser Pro Arg Cys Glu Tyr Gln Ala Gly His Asn Lys Val Gly Ser Gln Tyr Leu Ala Leu Ala Ala Leu Ile Thr Pro Lys Lys Ile Lys Pro Pro Leu Pro Ser Val Thr Lys Leu Thr Glu Asp Arg Trp Asn Lys Gln Lys Thr Lys Gly His Arg Gly Ser His Thr Met Asn Gly His PRTArtificial sequenceMost recent common ancestor reconstruction of clade B vpr protein sequence 65Met Glu Gln Ala Pro Glu Asp Gln Gly Pro Gln Arg Glu Pro Tyr Asnrp Thr Leu Glu Leu Leu Glu Glu Leu Lys Ser Glu Ala Val Arg 2His Phe Pro Arg Leu Trp Leu His Ser Leu Gly Gln His Ile Tyr Glu 35 4 Tyr Gly Asp Thr Trp Ala Gly Val Glu Ala Ile Ile Arg Ile Leu 5Gln Gln Leu Leu Phe Ile His Phe Arg Ile Gly Cys Gln His Ser Arg65 7Ile Gly Ile Thr Arg Gln Arg Arg Ala Arg Asn Gly Ala Ser Arg Ser 85 96PRTArtificial sequenceLeast squares and minimum of means reconstruction of clade b vpr protein sequence 66Met Glu Gln Ala Pro Glu Asp Gln Gly Pro Gln Arg Glu Pro Tyr Asnrp Thr Leu Glu Leu Leu Glu Glu Leu Lys Ser Glu Ala Val Arg 2His Phe Pro Arg Ile Trp Leu His Ser Leu Gly Gln His Ile Tyr Glu 35 4 Tyr Gly Asp Thr Trp Ala Gly Val Glu Ala Ile Ile Arg Ile Leu 5Gln Gln Leu Leu Phe Ile His Phe Arg Ile Gly Cys Arg His Ser Arg65 7Ile Gly Ile Thr Arg Gln Arg Arg Ala Arg Asn Gly Ala Ser Arg Ser 85 96PRTArtificial sequenceMinimum of means reconstructions for the clade B of vpr protein sequence 67Met Glu Gln Ala Pro Glu Asp Gln Gly Pro Gln Arg Glu Pro Tyr Asnrp Thr Leu Glu Leu Leu Glu Glu Leu Lys Ser Glu Ala Val Arg 2His Phe Pro Arg Ile Trp Leu His Ser Leu Gly Gln His Ile Tyr Glu 35 4 Tyr Gly Asp Thr Trp Ala Gly Val Glu Ala Ile Ile Arg Ile Leu 5Gln Gln Leu Leu Phe Ile His Phe Arg Ile Gly Cys Gln His Ser Arg65 7Ile Gly Ile Thr Arg Gln Arg Arg Ala Arg Asn Gly Ala Ser Arg Ser 85 9ificial sequenceMost recent common ancestor reconstruction of clade B rev protein sequence 68Met Gln Pro Leu Glu Ile Leu Ala Ile Val Ala Leu Val Val Ala Alaeu Ala Ile Val Val Trp Thr Ile Val Phe Ile Glu Tyr Arg Lys 2Ile Leu Arg Gln Arg Lys Ile Asp Arg Leu Ile Asp Arg Ile Arg Glu 35 4 Ala Glu Asp Ser Gly Asn Glu Ser Glu Gly Asp Gln Glu Glu Leu 5Ser Ala Leu Val Glu Met Gly His His Ala Pro Trp Asp Val Asp Asp65 7Leu698ificial sequenceLeast squares and minimum of means reconstructions for the clade B vpu protein sequences 69Met Gln Pro Leu Gln Ile Leu Ala Ile Val Ala Leu Val Val Ala Alale Ala Ile Val Val Trp Thr Ile Val Phe Ile Glu Tyr Arg Lys 2Ile Leu Arg Gln Arg Lys Ile Asp Arg Leu Ile Asp Arg Ile Arg Glu 35 4 Ala Glu Asp Ser Gly Asn Glu Ser Glu Gly Asp Gln Glu Glu Leu 5Ser Ala Leu Val Glu Met Gly His His Ala Pro Trp Asp Val Asp Asp65 7Leu7AArtificial sequenceMost recent common ancestor reconstruction of clade C gag protein sequence 7gcga gagcgtcaat attaagaggg ggaaaattag atacatggga aaaaattagg 6ccag ggggaaagaa acattatatg ataaaacacc tagtatgggc aagcagggag aaagat ttgcacttaa ccctggcctt ttagagacat cagaaggctg taaacaaata aacagc tacaaccagc tcttcagaca ggaacagagg aacttaaatc attatataac 24gcaa ctctctattg tgtacatcaa aggatagagg tacgagacac caaggaagcc 3caaga tagaggaaga acaaaacaaa agtcagcaaa aaacacagca ggcagaagcg 36ggaa aggtcagtca aaattatcct atagtgcaga atctccaagg gcaaatggta 42gcca tatcacctag aactttgaat gcatgggtaa aagtaataga ggagaaggct 48ccag aggtaatacc catgtttaca gcattatcag aaggagccac cccacaagat 54acca tgttaaatac agtgggggga catcaagcag ccatgcaaat gttaaaagat 6caatg aggaggctgc agaatgggat aggttacatc cagtgcatgc agggcctgtt 66ggcc aaatgagaga accaagggga agtgacatag caggaactac tagtaccctt 72caaa tagcatggat gacaagtaac ccacctatcc cagtgggaga catctataaa 78ataa ttctggggtt aaataaaata gtaagaatgt atagccctgt cagcattttg 84aaac

aagggccaaa ggaacccttt agagactatg tagaccggtt ctttaaaact 9agctg aacaagctac acaagatgta aaaaattgga tgacagacac cttgttggtc 96gcga acccagattg taagaccatt ttaagagcat taggaccagg ggctacacta gaaatga tgacagcatg tcagggagtg ggaggaccta gccataaagc aagagttttg gaggcaa tgagccaagc aaacaataca aacataatga tgcagagagg caattttaag cctagaa gaattgttaa atgtttcaac tgtggcaagg aaggacacat agccagaaat agggccc ctaggaaaaa gggctgttgg aaatgtggaa aggaaggaca ccaaatgaaa tgtactg agaggcaggc taatttttta gggaaaattt ggccttccca caaggggagg gggaatt tccttcagag cagaccagag ccaacagccc caccagcaga gagcttcagg gaggaga caacccccgc tccgaagcag gagccgaaag acagggaacc cttaacttcc aaatcac tctttggcag cgaccccttg tctcaataa 479DNAArtificial sequenceLeast squares center minumum of means reconstructions for clade C gag gene 7gcga gagcgtcaat attaagaggc ggaaaattag atacatggga aaaaattagg 6ccag ggggaaagaa acattatatg ctaaaacacc tagtatgggc aagcagggag aaagat ttgcacttaa ccctggcctt ttagagacat cagaaggctg taaacaaata aacagc tacaaccagc tcttcagaca ggaacagagg aacttagatc attatataac 24gcaa ctctctattg tgtacatgaa aagatagagg tacgagacac caaggaagcc 3caaga tagaggaaga acaaaacaaa agtcagcaaa aaacacagca ggcagaagcg 36ggaa aggtcagtca aaattatcct atagtgcaga atctccaagg gcaaatggta 42gcca tatcacctag aactttgaat gcatgggtaa aagtaataga ggagaaggct 48ccag aggtaatacc catgtttaca gcattatcag aaggagccac cccacaagat 54acca tgttaaatac agtgggggga catcaagcag ccatgcaaat gttaaaagat 6caatg aggaggctgc agaatgggat aggttacatc cagtacatgc agggcctgtt 66ggcc aaatgagaga accaagggga agtgacatag caggaactac tagtaccctt 72caaa tagcatggat gacaagtaac ccacctgttc cagtgggaga catctataaa 78ataa ttctggggtt aaataaaata gtaagaatgt atagccctgt cagcattttg 84aaac aagggccaaa ggaacccttt agagactatg tagaccggtt ctttaaaact 9agctg aacaagctac acaagatgta aaaaattgga tgacagacac cttgttggtc 96gcga acccagattg taagaccatt ttaagagcat taggaccagg ggctacatta gaaatga tgacagcatg tcagggagtg ggaggacctg gccacaaagc aagagtgttg gaggcaa tgagccaagc aaacaataca aacataatga tgcagagaag caattttaaa cctaaaa gaattgttaa atgtttcaac tgtggcaagg aagggcacat agccagaaat agggccc ctaggaaaaa aggctgttgg aaatgtggaa aggaaggaca ccaaatgaaa tgtactg agaggcaggc taatttttta gggaaaattt ggccttccca caaggggagg gggaatt tccttcagag cagaccagag ccaacagccc caccagcaga gagcttcagg gaggaga caacccccgc tccgaagcag gagccgaaag acagggaacc cttaacttcc aaatcac tctttggcag cgaccccttg tctcaataa 482DNAArtificial sequenceMinimum of means reconstructions for clade C gag gene 72atgggtgcga gagcgtcaat attaagaggc ggaaaattag atacatggga aaaaattagg 6ccag ggggaaagaa acattatatg ctaaaacacc tagtatgggc aagcagggag aaagat ttgcacttaa ccctggcctt ttagagacat cagaaggctg taaacaaata aacagc tacaaccagc tcttcagaca ggaacagagg aacttagatc attatataac 24gcaa ctctctattg tgtacatgaa aagatagagg tacgagacac caaggaagcc 3caaga tagaggaaga acaaaacaaa agtcagcaaa aaacacagca ggcagaagcg 36gacg gaaaggtcag tcaaaattat cctatagtgc agaatctcca agggcaaatg 42cagg ccatatcacc tagaactttg aatgcatggg taaaagtaat agaggagaag 48agcc cagaggtaat acccatgttt acagcattat cagaaggagc caccccacaa 54aaca ccatgttaaa tacagtgggg ggacatcaag cagccatgca aatgttaaaa 6catca atgaggaggc tgcagaatgg gataggttac atccagtaca tgcagggcct 66ccag gccaaatgag agaaccaagg ggaagtgaca tagcaggaac tactagtacc 72gaac aaatagcatg gatgacaagt aacccacctg ttccagtggg agacatctat 78tgga taattctggg gttaaataaa atagtaagaa tgtatagccc tgtcagcatt 84ataa aacaagggcc aaaggaaccc tttagagact atgtagaccg gttctttaaa 9aagag ctgaacaagc tacacaagat gtaaaaaatt ggatgacaga caccttgttg 96aatg cgaacccaga ttgtaagacc attttaagag cattaggacc aggggctaca gaagaaa tgatgacagc atgtcaggga gtgggaggac ctggccacaa agcaagagtg gctgagg caatgagcca agcaaacaat acaaacataa tgatgcagag aagcaatttt ggcccta aaagaattgt taaatgtttc aactgtggca aggaagggca catagccaga tgcaggg cccctaggaa aaaaggctgt tggaaatgtg gaaaggaagg acaccaaatg gactgta ctgagaggca ggctaatttt ttagggaaaa tttggccttc ccacaagggg ccaggga atttccttca gagcagacca gagccaacag ccccaccagc agagagcttc ttcgagg agacaacccc cgctccgaag caggagccga aagacaggga acccttaact ctcaaat cactctttgg cagcgacccc ttgtctcaat aa 547DNAArtificial sequenceMost recent common ancestor reconstructions of clade C env gene 73atgagagtga tggggataca gaggaattgt caacaatggt ggatatgggg catcttaggc 6atgt taatgatttg tagtgtggtg gggaacttgt gggtcacagt ctattatggg ctgtgt ggaaagaagc aaaaactact ctattttgtg catcagatgc taaagcatat gagaag tgcataatgt ctgggctaca catgcctgtg tacccacaga ccccaaccca 24atgg ttttggaaaa tgtaacagaa aattttaaca tgtggaaaaa tgacatggtg 3gatgc atgaggatat aatcagttta tgggatcaaa gcctaaagcc atgtgtaaag 36ccac tctgtgtcac tttaaactgt actaatgtta ataatactaa taataccaat 42atga atggagaaat gaaaaattgc tctttcaata taaccacaga aataagagat 48aaga aagaatatgc acttttttat agacttgata tagtaccact taatgaaaat 54aata ctagtgaata tagattaata aattgtaata cctcagccat aacacaagcc 6aaagg tctcttttga cccaattcct atacattatt gtgctccagc tggttatgcg 66aagt gtaataataa gacattcaat ggaacaggac catgcaaaaa tgtcagcaca 72tgta cacatggaat taagccagtg gtatcaactc aactactgtt aaatggtagt 78gaag aagagataat aattagatct gaaaatctga caaacaatgc caaaacaata 84cagc ttaatgaatc tgtagaaatt gtgtgtacaa gacccaacaa taatacaaga 9tatga ggataggacc aggacaaaca ttctatgcaa caggagacat aataggagat 96caag cacattgtaa cattagtgga agggaatgga ataacacttt acaacaggta gaaaaat taagaaaaca cttccctaat aaaacaataa aatttgcacc atcctcagga gacctag aaattacaac acatagcttt aattgtagag gagaattttt ctattgcaat tcaaaac tgtttaatag tacatacaat agtacaaata gtacaaattc aaccatcaca ccatgca gaataaaaca aattataaac atgtggcagg gggtaggaca agcaatgtat cctccca ttgcaggaaa cataacatgt aaatcaaata tcacaggact actattgaca gatggag gaaaaaatga aactaatgaa actgagacat tcagacctgg aggaggagat agggaca attggagaag tgaattatat aaatataaag tagtagaaat taaaccatta gtagcac ccactaaggc aaaaaggaga gtggtggaga gagaaaaaag agcagtggga ggagctg tgttccttgg gttcttggga gcagcaggaa gcactatggg cgcagcgtca acgctga cggtacaggc cagacaatta ttgtctggta tagtgcaaca gcaaagcaat ctgaggg ctatagaggc gcaacagcat atgttgcaac tcacagtctg gggcattaag ctccagg caagagtcct ggctatggaa agatacctaa aggatcaaca gctcctaggg tggggct gctctggaaa actcatctgc accactgctg tgccttggaa ctctagttgg aataaat ctcaagatga tatttgggat aacatgacct ggatggagtg ggatagagaa aacaatt acacagacac aatatacagg ttgcttgaag aatcgcaaaa ccagcaggaa aatgaac aagatttatt ggcattggac agttgggaaa atctgtggaa ttggtttgac tcaaatt ggctgtggta tataaaaata ttcataatga tagtaggagg cttgataggt 2gaataa tttttgctgt gctttctata gtaaatagag ttaggcaggg atactcacct 2cgtttc agacccttac cccaaacccg aggggacccg acaggctcga aagaatcgaa 2aaggtg gagagcaaga cagagacaga tccattcgat tagtgagcgg attcttagca 222tggg acgacctgcg gagcctgtgc ctcttcagct accaccgctt gagagacttc 228attg cagcgaggac tgtggaactt ctgggacgca gcagtctcag gggactacag 234tggg aagcccttaa atatctggga agtcttgtgc agtattgggg tcaggagcta 24gagtg ctattagtct gcttgatacc atagcaatag cagtagctga agggacagat 246atag aagtagtaca aagagcttgt agagctatcc tcaacatacc tagaagaata 252ggct ttgaagcagc tttgcaa 2547742547DNAArtificial sequenceLeast squares and minimum of means reconstructions for clade C env gene 74atgagagtga gggggatact gaggaattgt caacaatggt ggatatgggg catcttaggc 6atgt taatgatttg taatgtggtg gggaacttgt gggtcacagt ctattatggg ctgtgt ggaaagaagc aaaaactact ctattctgtg catcagatgc taaagcatat aagaag tgcataatgt ctgggctaca catgcctgtg tacccacaga ccccaaccca 24atgg ttttggaaaa tgtaacagaa aattttaaca tgtggaaaaa tgacatggtg 3gatgc atgaggatat aatcagttta tgggatcaaa gcctaaagcc atgtgtaaag 36ccac tctgtgtcac tttaaattgt agtaatgtta atgctaccaa tactaccaat 42atga agggagaaat aaaaaattgc tctttcaatg caaccacaga aataagagat 48caga aagtgtatgc acttttttat agacttgata tagtaccact taatgagaat 54aatt ctagtgagta tagattaata aattgtaata cctcagccat aacacaagcc 6aaagg tctcttttga cccaattcct atacattatt gtgctccagc tggttatgcg 66aagt gtaataataa gacattcaat ggaacaggac catgcaataa tgtcagcaca 72tgta cacatggaat taagccagtg gtatcaactc aactactgtt aaatggtagc 78gaag aagagataat aattagatct gaaaatctga caaacaatgt caaaacaata 84catc ttaatgaatc tgtagaaatt gtgtgtacaa gacccaacaa taatacaaga 9tataa ggataggacc aggacaaaca ttctatgcaa caggagacat aataggagac 96caag cacattgtaa cattagtgaa gaggaatgga ataaaacttt acaaagggta aaaaaat tagaagaaca cttccctaat aaaacaataa aatttgaacc atcctcagga gacctag aaattacaac acatagcttt aattgtagag gagaattttt ctattgcaat tcaaaac tgtttaatag tacatacaat ggtacaaata gtacaaatac aaccatcaca ccatgca gaataaaaca aattataaac atgtggcagg aggtaggacg agcaatgtat cctccca ttgcaggaaa cataacatgt aaatcaaata tcacaggact actattggta gatggag gaaaaaataa cacaaataac acagagatat tcagacctgg aggaggagat agggaca attggagaag tgaattatat aaatataaag tggtagaaat taagccattg atagcac ccactaaggc aaaaaggaga gtggtggaga gagaaaaaag agcagtggga ggagctg tgttccttgg gttcttggga gcagcaggaa gcactatggg cgcggcgtca acgctga cggtacaggc cagacaattg ttgtctggta tagtgcaaca gcaaagcaat ctgaggg ctatagaggc gcaacagcat atgttgcaac tcacggtctg gggcattaag ctccaga caagagtcct ggctatagaa agatacctaa aggatcaaca gctcctaggg tggggct gctctggaaa actcatctgc accactgctg tgccttggaa ctctagttgg aataaat ctcaagaaga tatttgggat aacatgacct ggatgcagtg ggatagagaa agtaatt acacagacac aatatacagg ttgcttgaag actcgcaaaa ccagcaggaa aatgaaa aagatttact agcattggac agttggaaaa atctgtggaa ttggtttgac acaaatt ggctgtggta tataaaaata ttcataatga tagtaggagg cttgataggt 2gaataa tttttgctgt gctttctata gtgaatagag ttaggcaggg atactcacct 2cgtttc agacccttac cccaaacccg aggggacccg acaggctcgg aagaatcgaa 2aaggtg gagagcaaga cagagacaga tccattcgat tagtgagcgg attcttagca 222tggg acgacctgcg gagcctgtgc ctcttcagct accaccgatt gagagacttc 228gtgg cagcgagagc ggtggaactt ctgggacgca gcagtctcag gggactacag 234tggg aagcccttaa gtatctggga agtcttgtgc agtattgggg tctggagcta 24gagtg ctattagtct gcttgatacc atagcaatag cagtagctga aggaacagat 246atag aattaataca aagaatttgt agagctatcc gcaacatacc tagaagaata 252ggct ttgaagcagc tttgcaa 254775255ificial sequenceMinimum of means reconstructions for clade C env gene 75atgagagtga gggggatact gaggaattgt caacaatggt ggatatgggg catcttaggc 6atgt taatgatttg taatgtggtg gggaacttgt gggtcacagt ctattatggg ctgtgt ggaaagaagc aaaaactact ctattctgtg catcagatgc taaagcatat aagaag tgcataatgt ctgggctaca catgcctgtg tacccacaga ccccaaccca 24atgg ttttggaaaa tgtaacagaa aattttaaca tgtggaaaaa tgacatggtg 3gatgc atgaggatat aatcagttta tgggatcaaa gcctaaagcc atgtgtaaag 36ccac tctgtgtcac tttaaattgt agtaatgtta atactaccaa tactaccaat 42atga aaggagaaat aaaaaattgc tctttcaatg taaccacaga actaagagat 48aaga aagagtatgc acttttttat agacttgata tagtaccact taatgagaat 54aatt ctagtgagta tagattaata aattgtaata cctcagccat aacacaagcc 6aaagg tctcttttga cccaattcct atacattatt gtgctccagc tggttatgcg 66aagt gtaataataa gacattcaat ggaacaggac catgcaataa tgtcagcaca 72tgta cacatggaat taagccagtg gtatcaactc aactactgtt aaatggtagc 78gaag aagagataat aattagatct gaaaatctga caaacaatgc caaaacaata 84catc ttaatgaatc tgtagaaatt gtgtgtacaa gacccaacaa taatacaaga 9tataa ggataggacc aggacaaaca ttctatgcaa caggagacat aataggagac 96caag cacattgtaa cattagtgaa gaggaatgga ataaaacttt acaaagggta aaaaaat tagaagaaca cttccctaat aaaacaataa aatttgaacc atcctcagga gacctag aaattacaac acatagcttt aattgtagag gagaattttt ctattgcaat tcaaaac tgtttaatag tacatacaat ggtacaaata gtacaaattc aaccatcaca caatgca gaataaaaca aattataaac atgtggcagg aggtaggacg agcaatgtat cctccca ttgcaggaaa cataacatgt aaatcaaata tcacaggact actattggta gatggag gaaaaaatga cacaaatgac acagagatat tcagacctgg aggaggagat agggaca attggagaag tgaattatat aaatataaag tggtagaaat taagccattg atagcac ccactaaggc aaaaaggaga gtggtggaga gagaaaaaag agcagtggga ggagctg tgttccttgg gttcttggga gcagcaggaa gcactatggg cgcagcgtca acgctga cggtacaggc cagacaattg ttgtctggta tagtgcaaca gcaaagcaat ctgaggg ctatagaggc gcaacagcat atgttgcaac tcacggtctg gggcattaag ctccaga caagagtcct ggctatagaa agatacctaa aggatcaaca gctcctaggg tggggct gctctggaaa actcatctgc accactgctg tgccttggaa ctctagttgg aataaat ctcaagagga tatttgggat aacatgacct ggatgcagtg ggatagagaa agtaatt acacagacac aatatacagg ttgcttgaag actcgcaaaa ccagcaggaa aatgaaa aagatttact agcattggac agttggaaaa atctgtggaa ttggtttgac acaaatt ggctgtggta tataaaaata ttcataatga tagtaggagg cttgataggt 2gaataa tttttgctgt gctttctata gtgaatagag ttaggcaggg atactcacct 2cgtttc agacccttac cccaaacccg aggggacccg acaggctcgg aagaatcgaa 2aaggtg gagagcaaga cagagacaga tccattcgat tagtgagcgg attcttagca 222tggg acgacctgcg gagcctgtgc ctcttcagct accaccgatt gagagacttc 228gtgg cagcgagagc ggtggaactt ctgggacgca gcagtctcag gggactacag 234tggg aagcccttaa gtatctggga agtcttgtgc agtattgggg tctggagcta 24gagtg ctattagtct gcttgatacc atagcaatag cagtagctga aggaacagat 246atag aattaataca aagaatttgt agagctatcc gcaacatacc tagaagaata 252ggct ttgaagcagc tttgcaataa 255NAArtificial sequenceMost recent common ancestor reconstructions of clade C nef gene 76atggggggca agtggtcaaa aagcagtata gttggatggc ctgctgtaag agaaagaata 6actg ctccagcagc agaaggagta ggagcagcgt ctcaagactt agataaacat cactta caagcagcaa cacagccgcc actaatgctg attgtgcctg gctggaagca aggagg aagaagtagg ctttccagtc agacctcagg tgcctttaag accaatgact 24ggag cagtcgatct cagcttcttt ttaaaagaaa aggggggact ggaagggtta 3ctcta agaaaaggca agagatcctt gatttgtggg tctatcacac acaaggctac 36gatt ggcaaaacta cacaccggga ccagggatca gatttccact gacctttgga 42ttca agctagtgcc agttgaccca agggaagtag aagaggccaa tgaaggagag 48tgct tgctacaccc tatgagccag catggaatgg aggatgaaga cagagaagta 54tgga agtttgacag tcacctagca cgcagacaca tggcccgcga gctacatccg 6ttaca aagactgc 6DNAArtificial sequenceLeast squares and minimum of means reconstructions of clade C nef gene 77atggggggca agtggtcaaa aagcagtata gttggatggc ctgctgtaag agaaagaata 6actg agccagcagc agagggagta ggagcagcgt ctcaagactt agataaacat cactta caagcagcaa cacagccgcc aataatgctg attgtgcctg gctggaagca aggagg aagaagaagt aggctttcca gtcagacctc aggtgccttt aagaccaatg 24aagg gagcattcga tctcagcttc tttttaaaag aaaagggggg actggaaggg 3ttact ctaagaaaag gcaagagatc cttgatttgt gggtctatca cacacaaggc 36cctg attggcaaaa ctacacaccg ggaccagggg tcagatatcc actgaccttt 42tgct tcaagctagt gccagttgac ccaagggaag tagaagaggc caacgaagga 48aact gtttgctaca ccctatgagc cagcatggaa tggaggatga agacagagaa 54aagt ggaagtttga cagtcaccta gcacgcagac acatggcccg cgagctacat 6gtatt acaaagactg ctga 624783rtificial sequenceMost recent common ancestor reconstructions of clade C pol gene 78ttttttaggg aaaatttggc cttcccacaa ggggaggcca gggaatttcc ttcagagcag 6gcca acagccccac cagcagagag cttcaggttc gaggagacaa cccccgctcc caggag ccgaaagaca gggaaccctt aacttccctc aaatcactct ttggcagcga ttgtct caataaaagt agggggccag ataaaggaag ctctattaga tacaggagca 24acag tattagaaga cataaatttg ccaggaaaat ggaaaccaaa aatgataggg 3tggag gttttatcaa agtaagacag tatgatcaaa tacttataga aatttgtgga 36gcta taggtacagt attagtagga cctacacctg tcaacataat tggaagaaat 42actc agcttggttg cactctaaat tttccaatta gtcctattga aactgtacca 48ttaa agccaggaat ggatggccca aaggttaaac aatggccatt gacagaagag 54aaag cattaacagc aatttgtgaa gaaatggaaa aggaaggaaa aattacaaaa 6gcctg aaaatccata taacactcca gtatttgcca taaaaaagaa ggacagtact 66agaa aattagtaga tttcagagaa ctcaataaaa gaactcaaga cttctgggaa 72ttag gaataccaca cccagcaggg ttaaaaaaga aaaaatcagt aacagtactg 78gggg atgcatattt ttcagttcct ttagatgaag acttcaggaa atatactgca 84atac ctagtataaa caatgaaaca ccagggatta gatatcaata taatgtgctt 9gggat ggaaaggatc accagcaata ttccagagta gcatgacaaa aatcttagag 96aggg cacaaaaccc agaaatagtt atctatcaat acatggatga cttgtatgta tctgact tagaaatagg gcaacataga gcaaaaatag aggagttaag agaacatcta aaatggg gatttaccac accagacaag aaacatcaga aagaaccccc atttctttgg gggtatg aactccatcc tgacaaatgg acagtacagc ctatacagct gccagaaaag agctgga ctgtcaatga tatacagaag ttagtgggaa aattaaactg ggcaagtcag tacccag ggattaaagt aaggcaactg tgtaaactcc ttaggggagc caaagcacta gacatag taccactgac tgaagaagca gaattagaat tggcagagaa cagggaaatt aaagaac cagtacatgg agtatattat gacccatcaa aagacttaat agctgaaata aaacagg ggcatgacca atggacatat caaatttacc aagaaccatt caaaaatctg acaggaa agtatgcaaa aatgaggtct gcccacacta atgatgtaaa acaattaaca gcagtgc aaaaaatagc catggaaagc atagtaatat ggggaaagac tcctaaattt

ctaccca tccaaaaaga aacatgggag acatggtgga cagactattg gcaagccacc attcctg agtgggagtt tgttaatacc cctcccctag taaaattatg gtaccagcta aaagaac ccatagcagg agcagaaact ttctatgtag atggggcagc taatagggaa aaactag gaaaagcagg gtatgttact gacaaaggaa gacagaaagt tgtttctcta gaaacaa caaatcagaa gactgaatta caagcaattc agctagcttt gcaggattca tcagaag taaacatagt aacagactca caatatgcat taggaatcat tcaagcacaa gataaga gtgaatcaga gttagtcaat caaataatag agcagttaat aaaaaaggaa 2tctacc tgtcatgggt accagcacat aaaggaattg gaggaaatga acaagtagat 2tagtaa gttctggaat caggaaagtg ctgtttctag atggaataga taaagctcaa 2aacatg aaaaatatca cagcaattgg agagcaatgg ctagtgagtt taatctgcca 222gtag caaaagaaat agtagctagc tgtgataaat gtcagctaaa aggggaagcc 228ggac aagtagactg tagtccaggg atatggcaat tagattgtac acatttagaa 234gtta tcctggtagc agtccatgta gccagtggct acatagaagc agaagttatc 24agaaa caggacagga aacagcatac tttatattaa aattagcagg aagatggcca 246gtaa tacatacaga caatggcagc aatttcacca gtgctgcagt taaggcagcc 252tggg caggtatcca acaggaattt ggaattccct acaatcccca aagtcaggga 258gaat ccatgaataa agaattaaag aaaatcatag ggcaggtaag agatcaagct 264ctta agacagcagt acaaatggca gtattcattc acaattttaa aagaaaaggg 27tgggg ggtacagtgc aggggaaaga ataatagaca taatagcaac agacatacaa 276gaat tacaaaaaca aattataaaa attcaaaatt ttcgggttta ttacagagac 282gacc ctgtttggaa aggaccagcc aaactactct ggaaaggtga aggggcagta 288caag acaatagtga cataaaggta gtaccaagga ggaaagcaaa gatcattagg 294ggaa aacagatggc aggtgctgat tgtgtggcag gtagacagga tgaagattag 399DNAArtificial sequenceLeast squares reconstruction for clade c pol gene 79ttttttaggg aaaatttggc cttcccacaa ggggaggcca gggaatttcc ttcagagcag 6gcca acagccccac cagcagagag cttcaggttc gaggagacaa cccccgctcc caggag ccgaaagaca gggaaccctt aacttccctc aaatcactct ttggcagcga ttgtct caataaaagt agggggccag ataaaggagg ctctcttaga cacaggagca 24acag tattagaaga aataaatttg ccaggaaaat ggaaaccaaa aatgatagga 3tggag gttttatcaa agtaagacag tatgatcaaa tacttataga aatttgtgga 36gcta taggtacagt attagtagga cctacacctg tcaacataat tggaagaaat 42actc agcttggatg cacactaaat tttccaatta gtcccattga aactgtacca 48ttaa agccaggaat ggatggccca aaggttaaac aatggccatt gacagaagag 54aaac attaacagca atttgtgaag aaatggagaa ggaaggaaaa attacaaaaa 6cctga aaatccatat aacactccag tatttgccat aaaaaagaag gacagtacta 66gaaa attagtagat ttcagggaac tcaataaaag aactcaagac ttttgggaag 72tagg aataccacac ccagcagggt taaaaaagaa aaaatcagtg acagtactgg 78ggga tgcatatttt tcagttcctt tagatgaagg cttcaggaaa tatactgcat 84tacc tagtataaac aatgaaacac cagggattag atatcaatat aatgtgcttc 9ggatg gaaaggatca ccagcaatat tccagagtag catgacaaaa atcttagagc 96gggc acaaaatcca gaaatagtca tctatcaata tatggatgac ttgtatgtag ctgactt agaaataggg caacatagag caaaaataga ggagttaaga gaacatctat agtgggg atttaccaca ccagacaaga aacatcagaa agaaccccca tttctttgga ggtatga actccatcct gacaaatgga cagtacagcc tatacagctg ccagaaaagg gctggac tgtcaatgat atacagaagt tagtgggaaa attaaactgg gcaagtcaga acccagg gattaaagta aggcaacttt gtaaactcct taggggggcc aaagcactaa acatagt accactaact gaagaagcag aattagaatt ggcagagaac agggaaattc aagaacc agtacatgga gtatattatg acccatcaaa agacttgata gctgaaatac aacaggg gcatgaccaa tggacatatc aaatttacca agaaccattc aaaaatctga cagggaa gtatgcaaaa atgaggactg cccacactaa tgatgtaaaa cagttaacag cagtgca aaaaatagcc atggaaagca tagtaatatg gggaaagact cctaaattta tacccat ccaaaaagaa acatgggaga catggtggac agactattgg caagccacct ttcctga gtgggagttt gttaataccc ctcccctagt aaaattatgg taccagctgg aagaacc catagcagga gcagaaactt tctatgtaga tggagcagct aatagggaaa aaatagg aaaagcaggg tatgttactg acagaggaag gcagaaaatt gtttctctaa aaacaac aaatcagaag actgaattac aagcaattca gctagctttg caagattcag cagaagt aaacatagta acagactcac agtatgcatt aggaatcatt caagcacaac ataagag tgaatcagag ttagtcaacc aaataataga acaattaata aaaaaggaaa 2ctacct gtcatgggta ccagcacata aaggaattgg aggaaatgaa caagtagata 2agtaag tagtggaatc aggaaagtgc tgtttctaga tggaatagat aaggctcaag 2gcatga aaagtatcac agcaattgga gagcaatggc tagtgagttt aatctgccac 222tagc aaaagaaata gtagctagct gtgataaatg tcagctaaaa ggggaagcca 228gaca agtagactgt agtccaggga tatggcaatt agattgtaca catttagaag 234tcat cctggtagca gtccatgtag ccagtggcta catagaagca gaggttatcc 24gaaac aggacaagaa acagcatact ttatactaaa attagcagga agatggccag 246taat acatacagac aatggcagta atttcaccag tgctgcagtt aaggcagcct 252gggc aggtatccaa caggaatttg gaattcccta caatccccaa agtcagggag 258aatc catgaataaa gaattaaaga aaatcatagg gcaggtaaga gatcaagctg 264ttaa gacagcagta caaatggcag tattcattca caattttaaa agaaaagggg 27ggggg gtacagtgca ggggaaagaa taatagacat aatagcaaca gacatacaaa 276aatt acaaaaacaa attataaaaa ttcaaaattt tcgggtttat tacagagaca 282accc tatttggaaa ggaccagcca aactactctg gaaaggtgaa ggggcagtag 288aaga taatagtgac ataaaggtag taccaaggag gaaagcaaaa atcattaagg 294gaaa acagatggca ggtgctgatt gtgtggcagg tagacaggat gaagattag 29998AArtificial sequenceMinimum of means reconstructions of clade c pol gene 8aggg aaaatttggc cttcccacaa ggggaggcca gggaatttcc ttcagagcag 6gcca acagccccac cagcagagag cttcaggttc gaggagacaa ccccctctcc caggag ccgaaagaca gggaaccctt aacttccctc aaatcactct ttggcagcga ttgtca caataaaagt agggggacag ctaaaggagg ctctcttaga cacaggagca 24acag tattagaaga aataaatttg ccaggaaaat ggaaaccaaa aatgatagga 3tggag gttttatcaa agtaagacag tatgatcaaa tacttataga aatttgtgga 36gcta taggtacagt actagtagga cctacacctg tcaacataat tggaagaaat 42actc agcttggatg cacactaaat tttccaatta gtcccattga aactgtacca 48ttaa agccaggaat ggatggccca aaggtcaaac aatggccatt gacagaagag 54aaag cattaacagc aatttgtgaa gaaatggaga aggaaggaaa aattacaaaa 6gcctg aaaatccata taacactcca gtatttgcca taaaaaagaa ggacagtact 66agaa aattagtaga tttcagggaa ctcaataaaa gaactcaaga cttttgggaa 72ttag ggataccaca cccagcaggg ttaaaaaaga aaaaatcagt gacagtactg 78gggg atgcatattt ttcagttcct ttagatgaag gcttcaggaa atatactgca 84atac ctagtataaa caatgaaaca ccagggatta gatatcaata taatgtgctt 9gggat ggaaaggatc accagcaata ttccagagta gcatgacaaa aatcttagag 96aggg cacaaaatcc agaaatagtt atctatcaat atatggatga cttgtatgta tctgact tagaaatagg gcaacataga gcaaaaatag aggagttaag agaacatcta aagtggg gatttaccac accagacaag aaacatcaga aagaaccccc atttctttgg gggtatg aactccatcc tgacaaatgg acagtacagc ctatacagct gccagaaaag agctgga ctgtcaatga tatacagaag ttagtgggaa aattaaactg ggcaagtcag tacccag ggattaaagt aaggcaactg tgtaaactcc ttaggggagc caaagcacta gacatag taccactaac tgaagaagca gaattagaat tggcagagaa cagggaaatt aaagaac cagtacatgg agtatattat gacccatcaa aagacttaat agctgaaata aaacagg ggcatgacca atggacatat caaatttacc aagaaccatt caaaaatctg acaggga agtatgcaaa aatgaggact gcccacacta atgatgtaaa acagttaaca gcagtgc aaaaaatagc catggaaagc atagtaatat ggggaaagac tcctaaattt ttaccca tccagaaaga aacatgggag gcatggtgga cagactattg gcaagccacc attcctg agtgggagtt tgttaatacc cctcccctag taaaattatg gtaccagctg aaagaac ccatagcagg agcagaaact ttctatgtag atggagcagc taatagggaa aaaatag gaaaagcagg gtatgttact gacagaggaa ggcagaaaat tgtttctcta gaaacaa caaatcagaa gactgaatta caagcaattc agctagcttt gcaggattca tcagaag taaacatagt aacagactca cagtatgcat taggaatcat tcaagcacaa gataaga gtgaatcaga gttagtcaat caaataatag aacagttaat aaaaaaggaa 2tctacc tgtcatgggt accagcacat aaaggaattg gaggaaatga acaagtagat 2tagtaa gtagtggaat caggaaagtg ctgtttctag atggaataga taaggctcaa 2agcatg aaaaatatca cagcaattgg agagcaatgg ctagtgagtt taatctgcca 222gtag caaaagaaat agtagctagc tgtgataaat gtcagctaaa aggggaagcc 228ggac aagtagactg tagtccaggg atatggcaat tagattgtac acatttagaa 234atca tcctggtagc agtccatgta gccagtggct acatagaagc agaggttatc 24agaaa caggacaaga aacagcatac tttatactaa aattagcagg aagatggcca 246gtaa tacatacaga caatggcagt aatttcacca gtgctgcagt taaagcagcc 252tggg caggtatcca acaggaattt ggaattccct acaatcccca aagtcaggga 258gaat ccatgaataa agaattaaag aaaatcatag ggcaggtaag agatcaagct 264ctta agacagcagt acaaatggca gtattcattc acaattttaa aagaaaaggg 27tgggg ggtacagtgc aggggaaaga ataatagaca taatagcaac agacatacaa 276gaat tacaaaaaca aattataaaa attcaaaatt ttcgggttta ttacagagac 282gacc ctatttggaa aggaccagcc aaactactct ggaaaggtga aggggcagta 288caag ataacagtga cataaaggta gtaccaagga ggaaagcaaa aatcattaag 294ggaa aacagatggc aggtgctgat tgtgtggcag gtagacagga tgaagattag 3ificial sequenceMost common recent ancestor reconstructions of clade c rev gene 8ggaa gaagcggaga cagcgacgaa gcgctcctcc aagcagtgag gatcatcaaa 6tatc aaagcaaccc ttaccccaaa cccgagggga cccgacaggc tcgaaggaat gaagaa ggtggagagc aagacagaga cagatccatt cgattagtga gcggattctt cttgcc tgggacgacc tgcggagcct gtgcctcttc agctaccacc gcttgagaga 24cttg attgcagcga ggactgtgga acttctggga cgcagcagtc tcaggggact 3ggggg tgggaagccc ttaaatatct gggaagcctt gtgcagtatt ggggtcagga 36aaag agtgctatta g 38NAArtificial sequenceLeast squares of reconstructions of clade c rev gene 82atggcaggaa gaagcggaga cagcgacgaa gcgctcctcc aagcagtgag gatcatcaaa 6tatc aaagcaaccc ttaccccaaa cccgagggga cccgacaggc tcggaagaat gaagaa ggtggagagc aagacagaga cagatccatt cgattagtga gcggattctt cttgcc tgggacgacc tgcggagcct gtgcctcttc agctaccacc gattgagaga 24attg gtgacagcga gagcagtgga acttctggga cgcagcagtc tcaggggact 3ggggg tgggaagccc ttaagtatct gggaagtctt gtgcagtatt ggggtctgga 36aaag agtgctatta g 38NAArtificial sequenceMinimum of means reconstruction of clade C rev gene 83atggcaggaa gaagcggaga cagcgacgaa gcgctcctcc aagcagtgag gatcatcaaa 6tatc aaagcaaccc ttaccccaaa cccgagggga cccgacaggc tcggaagaat gaagaa ggtggagagc aagacagaga cagatccatt cgattagtga gcggattctt cttgcc tgggacgacc tgcggagcct gtgccttttc agctaccacc gattgagaga 24attg gtgacagcga gagcagtgga acttctggga cgcagcagtc tcaggggact 3ggggg tgggaagccc ttaagtatct gggaagtctt gtgcagtatt ggggtctgga 36aaag agtgctatta g 38NAArtificial sequenceMost recent common ancestor reconstruction of clade C tat gene sequence 84atggagccag tagatcctaa cctagagccc tggaaccatc caggaagtca gcctaaaact 6aata aatgttattg taaaaaatgt agctatcatt gtctagtttg ctttctgaca gcttag gcatttccta tggcaggaag aagcggagac agcgacgaag agctcctcca gtgagg atcatcaaaa tcctatatca aagcaaccct tatcccaaac ccgaggggac 24ggct cggaggaatc gaagaagaag gtggagagca agacagagac agatccgtgc 3g 3DNAArtificial sequenceLeast squares of reconstruction of clade C tat gene sequence 85atggagccag tagatcctaa cctagagccc tggaaccatc caggaagtca gcctaaaact 6aata agtgttattg taaacactgt agctatcatt gtctagtttg ctttcagaca gcttag gcatttccta tggcaggaag aagcggagac agcgacgaag cgctcctcca gtgagg atcatcaaaa tcctatatca aagcaaccct taccccaaac ccgaggggac 24ggct cggaagaatc gaagaagaag gtggagagca agacagagac agatccattc 3g 3DNAArtificial sequenceMinimum of means reconstructions of clade C tat gene sequence 86atggagccag tagatcctaa cctagagccc tggaaccatc caggaagtca gcctaaaact 6aata agtgttattg taaacactgt agctatcatt gtctagtttg ctttcagaca gcttag gcatttccta tggcaggaag aagcggagac agcgacgaag cgctcctcca gtgagg atcatcaaaa tcctatatca aagcaaccct taccccaaac ccgaggggac 24ggct cggaggaatc gaagaagaag gtggagagca agacagagac agatccattc 3g 3DNAArtificial sequenceMost recent common ancestor reconstructions of clade C vif gene sequence 87atggaaaaca gatggcaggt gctgattgtg tggcaggtag acaggatgaa gattagaaca 6agtt tagtaaaaca ccatatgtat gtttcaagga gagctaaagg atggttttat atcact atgaaagcag acatccaaaa ataagttcag aagtacacat cccattaggg ctagat tagtaataaa aacatattgg ggtttgcata caggagaaag agattggcat 24catg gagtctccat agaatggaga ctgagaagat atagcacaca agtagaccct 3ggcag accaactaat tcatatgcat tattttgatt gttttgcaga ctctgccata 36gcca tattaggaca tatagttagc cctaggtgtg actatcaagc aggacataac 42ggat ctctacaata cttggcactg acagcattaa taaaaccaaa aaagataaag 48ctgc ctagtgttaa gaaattagta gaggatagat ggaacaagcc ccagaagacc 54caca gagggagcca tacaatgaat ggacactag 57988579DNAArtificial sequenceLeast squares of reconstruction of clade C vif gene sequence 88atggaaaaca gatggcaggt gctgattgtg tggcaggtag acaggatgaa gattagaaca 6agtt tagtaaagca ccatatgtat gtttcaagga gagctaatgg atggttttac atcatt atgaaagcag acatccaaaa gtaagttcag aagtacacat cccattaggg ctagat tagtaataaa aacatattgg ggtttgcaaa caggagaaag agattggcat 24catg gagtctccat agaatggaga ttgagaagat atagcacaca agtagaccct 3ggcag accagctaat tcatatgcat tattttgatt gttttgcaga ctctgccata 36gcca tattaggaca catagttatt cctaggtgtg actatcaagc aggacataat 42ggat ctctacaata cttggcactg acagcattga taaaaccaaa aaagataaag 48ctgc ctagtgttag gaaattagta gaggatagat ggaacaagcc ccagaagacc 54cgca gagggaacca tacaatgaat ggacactag 57989579DNAArtificial sequenceMinimum of means center of tree reconstruction of clade C vif gene sequence 89atggaaaaca gatggcaggt gctgattgtg tggcaggtag acaggatgaa gattagaaca 6agtt tagtaaagca ccatatgtat gtttcaagga gagctaatgg atggttttac atcatt atgaaagcag acatccaaaa gtaagttcag aagtacacat cccattaggg ctagat tagtaataaa aacatattgg ggtttgcata caggagaaag agattggcat 24catg gagtctccat agaatggaga ttgagaagat atagcacaca agtagaccct 3ggcag accagctaat tcatatgcat tattttgatt gttttgcaga ctctgccata 36gcca tattaggaca catagttatt cctaggtgtg actatcaagc aggacataat 42ggat ctctacaata cttggcactg acagcattga taaaaccaaa aaagataaag 48ctgc ctagtgttag gaaattagta gaggatagat ggaacaagcc ccagaagacc 54cgca gagggaacca tacaatgaat ggacactag 5799Artificial sequenceMost recent common ancestor reconstructions for clade C vpr gene sequence 9caag ccccagaaga ccaggggcca cagagggagc catacaatga atggacacta 6ttag aggaacttaa gcaggaagct gtcagacatt ttcctagacc atggctccat taggac aacatatcta tgaaacctat ggggatactt gggcgggagt tgaagctata gaattc tgcaacaact actgtttatt catttcagaa ttgggtgcca acatagcaga 24atta ttcgacagag aagagcaaga aatggagcca gtagatccta a 29NAArtificial sequenceLeast squares center of tree reconstruction of clade C vpr gene sequence 9caag ccccagaaga ccaggggccg cagagggaac catacaatga atggacacta 6ttag aggaactcaa gcaggaagct gtcagacact ttcctagacc atggctccat taggac aatatatcta tgaaacctat ggggatactt ggacaggagt cgaagctcta gaatac tgcaacaact actgtttatt catttcagaa ttgggtgcca gcatagcaga 24attt tgcgacagag aagagcaaga aatggagcca gtagatccta a 29NAArtificial sequenceMinimum of means center of tree reconstruction of clade C vpr gene sequence 92atggaacaag ccccagaaga ccaggggccg cagagggaac catacaatga atggacacta 6ttag aggaactcaa gcaggaagct gtcagacact ttcctagacc atggctccat taggac aacatatcta tgaaacctat ggggatactt ggacgggagt tgaagctcta gaattc tgcaacaact actgtttatt catttcagaa ttgggtgcca gcatagcaga 24atta tgcgacagag aagagcaaga aatggagcca gtagatccta a 29NAArtificial sequenceMost recent common ancestor reconstruction of clade C vpu gene sequence 93atgttagatt taatagcaag agtagattat agattaggag taggagcatt gatagtagca 6atag caatagttgt gtggaccata gtatatatag aatataggaa attggtaaga gaaaaa tagactggtt aattaaaaga attagggaaa gagcagaaga cagtggcaat gtgatg gggatacaga ggaattgtca acactggtgg atatggggca tcttaggctt 24gtta atgatttgta a 26NAArtificial sequenceLeast squares center of tree reconstruction of clade C vpu gene sequence 94atgttagatt tactagcaag agtagattat agattaggag taggagcatt gatagtagca 6atag caatagttgt gtggaccata gtatatatag aatataggaa attgttaaga gaaaaa tagactggtt aattaaaaga attagggaaa gagcagaaga cagtggcaat gtgagg gggatactga ggaattgtca acaatggtgg atatggggca tcttaggctt 24gtta atgatttgta a 26NAArtificial sequenceMinimum of means center of tree reconstruction of clade C vpu gene sequence 95atgttagatt tactagcaag agtagattat agattaggag taggagcatt gatagtagca 6atag caatagttgt gtggaccata gtatatatag aatataggaa attgttaaga gaaaaa tagactggtt aattaaaaga attagggaaa gagcagaaga cagtggcaat gtgagg gggatactga ggaattatca acaatggtgg atatggggca tcttaggctt 24gtta atgatttgta a 26RTArtificial sequenceMost recent common ancestor reconstruction of clade C gag protein sequence 96Met Gly Ala Arg Ala Ser Ile Leu Arg Gly Gly Lys Leu Asp Thr Trpys Ile Arg Leu Arg Pro Gly Gly Lys Lys His Tyr Met Ile Lys 2R>
3u Val Trp Ala Ser Arg Glu Leu Glu Arg Phe Ala Leu Asn Pro 35 4 Leu Leu Glu Thr Ser Glu Gly Cys Lys Gln Ile Ile Lys Gln Leu 5Gln Pro Ala Leu Gln Thr Gly Thr Glu Glu Leu Lys Ser Leu Tyr Asn65 7Thr Val Ala Thr Leu Tyr Cys Val His Gln Arg Ile Glu Val Arg Asp 85 9 Lys Glu Ala Leu Asp Lys Ile Glu Glu Glu Gln Asn Lys Ser Gln Lys Thr Gln Gln Ala Glu Ala Ala Asp Gly Lys Val Ser Gln Asn Pro Ile Val Gln Asn Leu Gln Gly Gln Met Val His Gln Ala Ile Pro Arg Thr Leu Asn Ala Trp Val Lys Val Ile Glu Glu Lys Ala Phe Ser Pro Glu Val Ile Pro Met Phe Thr Ala Leu Ser Glu Gly Ala Pro Gln Asp Leu Asn Thr Met Leu Asn Thr Val Gly Gly His Gln Ala Met Gln Met Leu Lys Asp Thr Ile Asn Glu Glu Ala Ala Glu 2sp Arg Leu His Pro Val His Ala Gly Pro Val Ala Pro Gly Gln 222g Glu Pro Arg Gly Ser Asp Ile Ala Gly Thr Thr Ser Thr Leu225 234u Gln Ile Ala Trp Met Thr Ser Asn Pro Pro Ile Pro Val Gly 245 25p Ile Tyr Lys Arg Trp Ile Ile Leu Gly Leu Asn Lys Ile Val Arg 267r Ser Pro Val Ser Ile Leu Asp Ile Lys Gln Gly Pro Lys Glu 275 28o Phe Arg Asp Tyr Val Asp Arg Phe Phe Lys Thr Leu Arg Ala Glu 29la Thr Gln Asp Val Lys Asn Trp Met Thr Asp Thr Leu Leu Val33ln Asn Ala Asn Pro Asp Cys Lys Thr Ile Leu Arg Ala Leu Gly Pro 325 33y Ala Thr Leu Glu Glu Met Met Thr Ala Cys Gln Gly Val Gly Gly 345r His Lys Ala Arg Val Leu Ala Glu Ala Met Ser Gln Ala Asn 355 36n Thr Asn Ile Met Met Gln Arg Gly Asn Phe Lys Gly Pro Arg Arg 378l Lys Cys Phe Asn Cys Gly Lys Glu Gly His Ile Ala Arg Asn385 39rg Ala Pro Arg Lys Lys Gly Cys Trp Lys Cys Gly Lys Glu Gly 44ln Met Lys Asp Cys Thr Glu Arg Gln Ala Asn Phe Leu Gly Lys 423p Pro Ser His Lys Gly Arg Pro Gly Asn Phe Leu Gln Ser Arg 435 44o Glu Pro Thr Ala Pro Pro Ala Glu Ser Phe Arg Phe Glu Glu Thr 456o Ala Pro Lys Gln Glu Pro Lys Asp Arg Glu Pro Leu Thr Ser465 478s Ser Leu Phe Gly Ser Asp Pro Leu Ser Gln 485 49RTArtificial sequenceLeast squares center of tree reconstruction of clade C gag protein sequence 97Met Gly Ala Arg Ala Ser Ile Leu Arg Gly Gly Lys Leu Asp Thr Trpys Ile Arg Leu Arg Pro Gly Gly Lys Lys His Tyr Met Leu Lys 2His Leu Val Trp Ala Ser Arg Glu Leu Glu Arg Phe Ala Leu Asn Pro 35 4 Leu Leu Glu Thr Ser Glu Gly Cys Lys Gln Ile Met Lys Gln Leu 5Gln Pro Ala Leu Gln Thr Gly Thr Glu Glu Leu Arg Ser Leu Tyr Asn65 7Thr Val Ala Thr Leu Tyr Cys Val His Glu Lys Ile Glu Val Arg Asp 85 9 Lys Glu Ala Leu Asp Lys Ile Glu Glu Glu Gln Asn Lys Ser Gln Lys Thr Gln Gln Ala Glu Ala Ala Asp Gly Lys Val Ser Gln Asn Pro Ile Val Gln Asn Leu Gln Gly Gln Met Val His Gln Ala Ile Pro Arg Thr Leu Asn Ala Trp Val Lys Val Ile Glu Glu Lys Ala Phe Ser Pro Glu Val Ile Pro Met Phe Thr Ala Leu Ser Glu Gly Ala Pro Gln Asp Leu Asn Thr Met Leu Asn Thr Val Gly Gly His Gln Ala Met Gln Met Leu Lys Asp Thr Ile Asn Glu Glu Ala Ala Glu 2sp Arg Leu His Pro Val His Ala Gly Pro Val Ala Pro Gly Gln 222g Glu Pro Arg Gly Ser Asp Ile Ala Gly Thr Thr Ser Thr Leu225 234u Gln Ile Ala Trp Met Thr Ser Asn Pro Pro Val Pro Val Gly 245 25p Ile Tyr Lys Arg Trp Ile Ile Leu Gly Leu Asn Lys Ile Val Arg 267r Ser Pro Val Ser Ile Leu Asp Ile Lys Gln Gly Pro Lys Glu 275 28o Phe Arg Asp Tyr Val Asp Arg Phe Phe Lys Thr Leu Arg Ala Glu 29la Thr Gln Asp Val Lys Asn Trp Met Thr Asp Thr Leu Leu Val33ln Asn Ala Asn Pro Asp Cys Lys Thr Ile Leu Arg Ala Leu Gly Pro 325 33y Ala Thr Leu Glu Glu Met Met Thr Ala Cys Gln Gly Val Gly Gly 345y His Lys Ala Arg Val Leu Ala Glu Ala Met Ser Gln Ala Asn 355 36n Thr Asn Ile Met Met Gln Arg Ser Asn Phe Lys Gly Pro Lys Arg 378l Lys Cys Phe Asn Cys Gly Lys Glu Gly His Ile Ala Arg Asn385 39rg Ala Pro Arg Lys Lys Gly Cys Trp Lys Cys Gly Lys Glu Gly 44ln Met Lys Asp Cys Thr Glu Arg Gln Ala Asn Phe Leu Gly Lys 423p Pro Ser His Lys Gly Arg Pro Gly Asn Phe Leu Gln Ser Arg 435 44o Glu Pro Thr Ala Pro Pro Ala Glu Ser Phe Arg Phe Glu Glu Thr 456o Ala Pro Lys Gln Glu Pro Lys Asp Arg Glu Pro Leu Thr Ser465 478s Ser Leu Phe Gly Ser Asp Pro Leu Ser Gln 485 49RTArtificial sequenceMinimum of means center of tree reconstruction of clade C gag protein sequence 98Met Gly Ala Arg Ala Ser Ile Leu Arg Gly Gly Lys Leu Asp Thr Trpys Ile Arg Leu Arg Pro Gly Gly Lys Lys His Tyr Met Leu Lys 2His Leu Val Trp Ala Ser Arg Glu Leu Glu Arg Phe Ala Leu Asn Pro 35 4 Leu Leu Glu Thr Ser Glu Gly Cys Lys Gln Ile Met Lys Gln Leu 5Gln Pro Ala Leu Gln Thr Gly Thr Glu Glu Leu Arg Ser Leu Tyr Asn65 7Thr Val Ala Thr Leu Tyr Cys Val His Glu Lys Ile Glu Val Arg Asp 85 9 Lys Glu Ala Leu Asp Lys Ile Glu Glu Glu Gln Asn Lys Ser Gln Lys Thr Gln Gln Ala Glu Ala Ala Ala Asp Gly Lys Val Ser Gln Tyr Pro Ile Val Gln Asn Leu Gln Gly Gln Met Val His Gln Ala Ser Pro Arg Thr Leu Asn Ala Trp Val Lys Val Ile Glu Glu Lys Ala Phe Ser Pro Glu Val Ile Pro Met Phe Thr Ala Leu Ser Glu Gly Thr Pro Gln Asp Leu Asn Thr Met Leu Asn Thr Val Gly Gly His Ala Ala Met Gln Met Leu Lys Asp Thr Ile Asn Glu Glu Ala Ala 2rp Asp Arg Leu His Pro Val His Ala Gly Pro Val Ala Pro Gly 222t Arg Glu Pro Arg Gly Ser Asp Ile Ala Gly Thr Thr Ser Thr225 234n Glu Gln Ile Ala Trp Met Thr Ser Asn Pro Pro Val Pro Val 245 25y Asp Ile Tyr Lys Arg Trp Ile Ile Leu Gly Leu Asn Lys Ile Val 267t Tyr Ser Pro Val Ser Ile Leu Asp Ile Lys Gln Gly Pro Lys 275 28u Pro Phe Arg Asp Tyr Val Asp Arg Phe Phe Lys Thr Leu Arg Ala 29ln Ala Thr Gln Asp Val Lys Asn Trp Met Thr Asp Thr Leu Leu33al Gln Asn Ala Asn Pro Asp Cys Lys Thr Ile Leu Arg Ala Leu Gly 325 33o Gly Ala Thr Leu Glu Glu Met Met Thr Ala Cys Gln Gly Val Gly 345o Gly His Lys Ala Arg Val Leu Ala Glu Ala Met Ser Gln Ala 355 36n Asn Thr Asn Ile Met Met Gln Arg Ser Asn Phe Lys Gly Pro Lys 378e Val Lys Cys Phe Asn Cys Gly Lys Glu Gly His Ile Ala Arg385 39ys Arg Ala Pro Arg Lys Lys Gly Cys Trp Lys Cys Gly Lys Glu 44is Gln Met Lys Asp Cys Thr Glu Arg Gln Ala Asn Phe Leu Gly 423e Trp Pro Ser His Lys Gly Arg Pro Gly Asn Phe Leu Gln Ser 435 44g Pro Glu Pro Thr Ala Pro Pro Ala Glu Ser Phe Arg Phe Glu Glu 456r Pro Ala Pro Lys Gln Glu Pro Lys Asp Arg Glu Pro Leu Thr465 478u Lys Ser Leu Phe Gly Ser Asp Pro Leu Ser Gln 485 49RTArtificial sequenceMost recent common ancestor reconstruction of clade C gptein sequence 99Met Arg Val Met Gly Ile Gln Arg Asn Cys Gln Gln Trp Trp Ile Trple Leu Gly Phe Trp Met Leu Met Ile Cys Ser Val Val Gly Asn 2Leu Trp Val Thr Val Tyr Tyr Gly Val Pro Val Trp Lys Glu Ala Lys 35 4 Thr Leu Phe Cys Ala Ser Asp Ala Lys Ala Tyr Glu Arg Glu Val 5His Asn Val Trp Ala Thr His Ala Cys Val Pro Thr Asp Pro Asn Pro65 7Gln Glu Met Val Leu Glu Asn Val Thr Glu Asn Phe Asn Met Trp Lys 85 9 Asp Met Val Asp Gln Met His Glu Asp Ile Ile Ser Leu Trp Asp Ser Leu Lys Pro Cys Val Lys Leu Thr Pro Leu Cys Val Thr Leu Cys Thr Asn Val Asn Asn Thr Asn Asn Thr Asn Ser Thr Met Asn Glu Met Lys Asn Cys Ser Phe Asn Ile Thr Thr Glu Ile Arg Asp Lys Lys Lys Lys Glu Tyr Ala Leu Phe Tyr Arg Leu Asp Ile Val Pro Asn Glu Asn Asn Asn Asn Thr Ser Glu Tyr Arg Leu Ile Asn Cys Thr Ser Ala Ile Thr Gln Ala Cys Pro Lys Val Ser Phe Asp Pro 2ro Ile His Tyr Cys Ala Pro Ala Gly Tyr Ala Ile Leu Lys Cys 222n Lys Thr Phe Asn Gly Thr Gly Pro Cys Lys Asn Val Ser Thr225 234n Cys Thr His Gly Ile Lys Pro Val Val Ser Thr Gln Leu Leu 245 25u Asn Gly Ser Leu Ala Glu Glu Glu Ile Ile Ile Arg Ser Glu Asn 267r Asn Asn Ala Lys Thr Ile Ile Val Gln Leu Asn Glu Ser Val 275 28u Ile Val Cys Thr Arg Pro Asn Asn Asn Thr Arg Lys Ser Met Arg 29ly Pro Gly Gln Thr Phe Tyr Ala Thr Gly Asp Ile Ile Gly Asp33le Arg Gln Ala His Cys Asn Ile Ser Gly Arg Glu Trp Asn Asn Thr 325 33u Gln Gln Val Ala Glu Lys Leu Arg Lys His Phe Pro Asn Lys Thr 345s Phe Ala Pro Ser Ser Gly Gly Asp Leu Glu Ile Thr Thr His 355 36r Phe Asn Cys Arg Gly Glu Phe Phe Tyr Cys Asn Thr Ser Lys Leu 378n Ser Thr Tyr Asn Ser Thr Asn Ser Thr Asn Ser Thr Ile Thr385 39ro Cys Arg Ile Lys Gln Ile Ile Asn Met Trp Gln Gly Val Gly 44la Met Tyr Ala Pro Pro Ile Ala Gly Asn Ile Thr Cys Lys Ser 423e Thr Gly Leu Leu Leu Thr Arg Asp Gly Gly Lys Asn Glu Thr 435 44n Glu Thr Glu Thr Phe Arg Pro Gly Gly Gly Asp Met Arg Asp Asn 456g Ser Glu Leu Tyr Lys Tyr Lys Val Val Glu Ile Lys Pro Leu465 478l Ala Pro Thr Lys Ala Lys Arg Arg Val Val Glu Arg Glu Lys 485 49g Ala Val Gly Leu Gly Ala Val Phe Leu Gly Phe Leu Gly Ala Ala 55er Thr Met Gly Ala Ala Ser Ile Thr Leu Thr Val Gln Ala Arg 5525Gln Leu Leu Ser Gly Ile Val Gln Gln Gln Ser Asn Leu Leu Arg Ala 534u Ala Gln Gln His Met Leu Gln Leu Thr Val Trp Gly Ile Lys545 556u Gln Ala Arg Val Leu Ala Met Glu Arg Tyr Leu Lys Asp Gln 565 57n Leu Leu Gly Ile Trp Gly Cys Ser Gly Lys Leu Ile Cys Thr Thr 589l Pro Trp Asn Ser Ser Trp Ser Asn Lys Ser Gln Asp Asp Ile 595 6rp Asp Asn Met Thr Trp Met Glu Trp Asp Arg Glu Ile Asn Asn Tyr 662p Thr Ile Tyr Arg Leu Leu Glu Glu Ser Gln Asn Gln Gln Glu625 634n Glu Gln Asp Leu Leu Ala Leu Asp Ser Trp Glu Asn Leu Trp 645 65n Trp Phe Asp Ile Ser Asn Trp Leu Trp Tyr Ile Lys Ile Phe Ile 667e Val Gly Gly Leu Ile Gly Leu Arg Ile Ile Phe Ala Val Leu 675 68r Ile Val Asn Arg Val Arg Gln Gly Tyr Ser Pro Leu Ser Phe Gln 69eu Thr Pro Asn Pro Arg Gly Pro Asp Arg Leu Glu Arg Ile Glu77lu Glu Gly Gly Glu Gln Asp Arg Asp Arg Ser Ile Arg Leu Val Ser 725 73y Phe Leu Ala Leu Ala Trp Asp Asp Leu Arg Ser Leu Cys Leu Phe 745r His Arg Leu Arg Asp Phe Ile Leu Ile Ala Ala Arg Thr Val 755 76u Leu Leu Gly Arg Ser Ser Leu Arg Gly Leu Gln Arg Gly Trp Glu 778u Lys Tyr Leu Gly Ser Leu Val Gln Tyr Trp Gly Gln Glu Leu785 79ys Ser Ala Ile Ser Leu Leu Asp Thr Ile Ala Ile Ala Val Ala 88ly Thr Asp Arg Ile Ile Glu Val Val Gln Arg Ala Cys Arg Ala 823u Asn Ile Pro Arg Arg Ile Arg Gln Gly Phe Glu Ala Ala Leu 835 84nRTArtificial sequenceLeast squares of center of tree reconstructions of clade C gptein sequence Arg Val Arg Gly Ile Leu Arg Asn Cys Gln Gln Trp Trp Ile Trple Leu Gly Phe Trp Met Leu Met Ile Cys Asn Val Val Gly Asn 2Leu Trp Val Thr Val Tyr Tyr Gly Val Pro Val Trp Lys Glu Ala Lys 35 4 Thr Leu Phe Cys Ala Ser Asp Ala Lys Ala Tyr Glu Lys Glu Val 5His Asn Val Trp Ala Thr His Ala Cys Val Pro Thr Asp Pro Asn Pro65 7Gln Glu Met Val Leu Glu Asn Val Thr Glu Asn Phe Asn Met Trp Lys 85 9 Asp Met Val Asp Gln Met His Glu Asp Ile Ile Ser Leu Trp Asp Ser Leu Lys Pro Cys Val Lys Leu Thr Pro Leu Cys Val

Thr Leu Cys Ser Asn Val Asn Ala Thr Asn Thr Thr Asn Asn Thr Met Lys Glu Ile Lys Asn Cys Ser Phe Asn Ala Thr Thr Glu Ile Arg Asp Lys Lys Gln Lys Val Tyr Ala Leu Phe Tyr Arg Leu Asp Ile Val Pro Asn Glu Asn Asn Ser Asn Ser Ser Glu Tyr Arg Leu Ile Asn Cys Thr Ser Ala Ile Thr Gln Ala Cys Pro Lys Val Ser Phe Asp Pro 2ro Ile His Tyr Cys Ala Pro Ala Gly Tyr Ala Ile Leu Lys Cys 222n Lys Thr Phe Asn Gly Thr Gly Pro Cys Asn Asn Val Ser Thr225 234n Cys Thr His Gly Ile Lys Pro Val Val Ser Thr Gln Leu Leu 245 25u Asn Gly Ser Leu Ala Glu Glu Glu Ile Ile Ile Arg Ser Glu Asn 267r Asn Asn Val Lys Thr Ile Ile Val His Leu Asn Glu Ser Val 275 28u Ile Val Cys Thr Arg Pro Asn Asn Asn Thr Arg Lys Ser Ile Arg 29ly Pro Gly Gln Thr Phe Tyr Ala Thr Gly Asp Ile Ile Gly Asp33le Arg Gln Ala His Cys Asn Ile Ser Glu Glu Glu Trp Asn Lys Thr 325 33u Gln Arg Val Gly Lys Lys Leu Glu Glu His Phe Pro Asn Lys Thr 345s Phe Glu Pro Ser Ser Gly Gly Asp Leu Glu Ile Thr Thr His 355 36r Phe Asn Cys Arg Gly Glu Phe Phe Tyr Cys Asn Thr Ser Lys Leu 378n Ser Thr Tyr Asn Gly Thr Asn Ser Thr Asn Thr Thr Ile Thr385 39ro Cys Arg Ile Lys Gln Ile Ile Asn Met Trp Gln Glu Val Gly 44la Met Tyr Ala Pro Pro Ile Ala Gly Asn Ile Thr Cys Lys Ser 423e Thr Gly Leu Leu Leu Val Arg Asp Gly Gly Lys Asn Asn Thr 435 44n Asn Thr Glu Ile Phe Arg Pro Gly Gly Gly Asp Met Arg Asp Asn 456g Ser Glu Leu Tyr Lys Tyr Lys Val Val Glu Ile Lys Pro Leu465 478e Ala Pro Thr Lys Ala Lys Arg Arg Val Val Glu Arg Glu Lys 485 49g Ala Val Gly Ile Gly Ala Val Phe Leu Gly Phe Leu Gly Ala Ala 55er Thr Met Gly Ala Ala Ser Ile Thr Leu Thr Val Gln Ala Arg 5525Gln Leu Leu Ser Gly Ile Val Gln Gln Gln Ser Asn Leu Leu Arg Ala 534u Ala Gln Gln His Met Leu Gln Leu Thr Val Trp Gly Ile Lys545 556u Gln Thr Arg Val Leu Ala Ile Glu Arg Tyr Leu Lys Asp Gln 565 57n Leu Leu Gly Ile Trp Gly Cys Ser Gly Lys Leu Ile Cys Thr Thr 589l Pro Trp Asn Ser Ser Trp Ser Asn Lys Ser Gln Glu Asp Ile 595 6rp Asp Asn Met Thr Trp Met Gln Trp Asp Arg Glu Ile Ser Asn Tyr 662p Thr Ile Tyr Arg Leu Leu Glu Asp Ser Gln Asn Gln Gln Glu625 634n Glu Lys Asp Leu Leu Ala Leu Asp Ser Trp Lys Asn Leu Trp 645 65n Trp Phe Asp Ile Thr Asn Trp Leu Trp Tyr Ile Lys Ile Phe Ile 667e Val Gly Gly Leu Ile Gly Leu Arg Ile Ile Phe Ala Val Leu 675 68r Ile Val Asn Arg Val Arg Gln Gly Tyr Ser Pro Leu Ser Phe Gln 69eu Thr Pro Asn Pro Arg Gly Pro Asp Arg Leu Gly Arg Ile Glu77lu Glu Gly Gly Glu Gln Asp Arg Asp Arg Ser Ile Arg Leu Val Ser 725 73y Phe Leu Ala Leu Ala Trp Asp Asp Leu Arg Ser Leu Cys Leu Phe 745r His Arg Leu Arg Asp Phe Ile Leu Val Ala Ala Arg Ala Val 755 76u Leu Leu Gly Arg Ser Ser Leu Arg Gly Leu Gln Arg Gly Trp Glu 778u Lys Tyr Leu Gly Ser Leu Val Gln Tyr Trp Gly Leu Glu Leu785 79ys Ser Ala Ile Ser Leu Leu Asp Thr Ile Ala Ile Ala Val Ala 88ly Thr Asp Arg Ile Ile Glu Leu Ile Gln Arg Ile Cys Arg Ala 823g Asn Ile Pro Arg Arg Ile Arg Gln Gly Phe Glu Ala Ala Leu 835 84nRTArtificial sequenceMinimum of means of center of tree reconstructions for clade C gptein sequence Arg Val Arg Gly Ile Leu Arg Asn Cys Gln Gln Trp Trp Ile Trple Leu Gly Phe Trp Met Leu Met Ile Cys Asn Val Val Gly Asn 2Leu Trp Val Thr Val Tyr Tyr Gly Val Pro Val Trp Lys Glu Ala Lys 35 4 Thr Leu Phe Cys Ala Ser Asp Ala Lys Ala Tyr Glu Lys Glu Val 5His Asn Val Trp Ala Thr His Ala Cys Val Pro Thr Asp Pro Asn Pro65 7Gln Glu Met Val Leu Glu Asn Val Thr Glu Asn Phe Asn Met Trp Lys 85 9 Asp Met Val Asp Gln Met His Glu Asp Ile Ile Ser Leu Trp Asp Ser Leu Lys Pro Cys Val Lys Leu Thr Pro Leu Cys Val Thr Leu Cys Ser Asn Val Asn Thr Thr Asn Thr Thr Asn Asn Thr Met Lys Glu Ile Lys Asn Cys Ser Phe Asn Val Thr Thr Glu Leu Arg Asp Lys Lys Lys Lys Glu Tyr Ala Leu Phe Tyr Arg Leu Asp Ile Val Pro Asn Glu Asn Asn Asn Asn Ser Ser Glu Tyr Arg Leu Ile Asn Cys Thr Ser Ala Ile Thr Gln Ala Cys Pro Lys Val Ser Phe Asp Pro 2ro Ile His Tyr Cys Ala Pro Ala Gly Tyr Ala Ile Leu Lys Cys 222n Lys Thr Phe Asn Gly Thr Gly Pro Cys Asn Asn Val Ser Thr225 234n Cys Thr His Gly Ile Lys Pro Val Val Ser Thr Gln Leu Leu 245 25u Asn Gly Ser Leu Ala Glu Glu Glu Ile Ile Ile Arg Ser Glu Asn 267r Asn Asn Ala Lys Thr Ile Ile Val His Leu Asn Glu Ser Val 275 28u Ile Val Cys Thr Arg Pro Asn Asn Asn Thr Arg Lys Ser Ile Arg 29ly Pro Gly Gln Thr Phe Tyr Ala Thr Gly Asp Ile Ile Gly Asp33le Arg Gln Ala His Cys Asn Ile Ser Glu Glu Glu Trp Asn Lys Thr 325 33u Gln Arg Val Gly Lys Lys Leu Glu Glu His Phe Pro Asn Lys Thr 345s Phe Glu Pro Ser Ser Gly Gly Asp Leu Glu Ile Thr Thr His 355 36r Phe Asn Cys Arg Gly Glu Phe Phe Tyr Cys Asn Thr Ser Lys Leu 378n Ser Thr Tyr Asn Gly Thr Asn Ser Thr Asn Ser Thr Ile Thr385 39ln Cys Arg Ile Lys Gln Ile Ile Asn Met Trp Gln Glu Val Gly 44la Met Tyr Ala Pro Pro Ile Ala Gly Asn Ile Thr Cys Lys Ser 423e Thr Gly Leu Leu Leu Val Arg Asp Gly Gly Lys Asn Asp Thr 435 44n Asp Thr Glu Ile Phe Arg Pro Gly Gly Gly Asp Met Arg Asp Asn 456g Ser Glu Leu Tyr Lys Tyr Lys Val Val Glu Ile Lys Pro Leu465 478e Ala Pro Thr Lys Ala Lys Arg Arg Val Val Glu Arg Glu Lys 485 49g Ala Val Gly Ile Gly Ala Val Phe Leu Gly Phe Leu Gly Ala Ala 55er Thr Met Gly Ala Ala Ser Ile Thr Leu Thr Val Gln Ala Arg 5525Gln Leu Leu Ser Gly Ile Val Gln Gln Gln Ser Asn Leu Leu Arg Ala 534u Ala Gln Gln His Met Leu Gln Leu Thr Val Trp Gly Ile Lys545 556u Gln Thr Arg Val Leu Ala Ile Glu Arg Tyr Leu Lys Asp Gln 565 57n Leu Leu Gly Ile Trp Gly Cys Ser Gly Lys Leu Ile Cys Thr Thr 589l Pro Trp Asn Ser Ser Trp Ser Asn Lys Ser Gln Glu Asp Ile 595 6rp Asp Asn Met Thr Trp Met Gln Trp Asp Arg Glu Ile Ser Asn Tyr 662p Thr Ile Tyr Arg Leu Leu Glu Asp Ser Gln Asn Gln Gln Glu625 634n Glu Lys Asp Leu Leu Ala Leu Asp Ser Trp Lys Asn Leu Trp 645 65n Trp Phe Asp Ile Thr Asn Trp Leu Trp Tyr Ile Lys Ile Phe Ile 667e Val Gly Gly Leu Ile Gly Leu Arg Ile Ile Phe Ala Val Leu 675 68r Ile Val Asn Arg Val Arg Gln Gly Tyr Ser Pro Leu Ser Phe Gln 69eu Thr Pro Asn Pro Arg Gly Pro Asp Arg Leu Gly Arg Ile Glu77lu Glu Gly Gly Glu Gln Asp Arg Asp Arg Ser Ile Arg Leu Val Ser 725 73y Phe Leu Ala Leu Ala Trp Asp Asp Leu Arg Ser Leu Cys Leu Phe 745r His Arg Leu Arg Asp Phe Ile Leu Val Ala Ala Arg Ala Val 755 76u Leu Leu Gly Arg Ser Ser Leu Arg Gly Leu Gln Arg Gly Trp Glu 778u Lys Tyr Leu Gly Ser Leu Val Gln Tyr Trp Gly Leu Glu Leu785 79ys Ser Ala Ile Ser Leu Leu Asp Thr Ile Ala Ile Ala Val Ala 88ly Thr Asp Arg Ile Ile Glu Leu Ile Gln Arg Ile Cys Arg Ala 823g Asn Ile Pro Arg Arg Ile Arg Gln Gly Phe Glu Ala Ala Leu 835 84nRTArtificial sequenceMost recent common ancestor of reconstructions of clade C nef protein sequences Gly Gly Lys Trp Ser Lys Ser Ser Ile Val Gly Trp Pro Ala Vallu Arg Ile Arg Arg Thr Ala Pro Ala Ala Glu Gly Val Gly Ala 2Ala Ser Gln Asp Leu Asp Lys His Gly Ala Leu Thr Ser Ser Asn Thr 35 4 Ala Thr Asn Ala Asp Cys Ala Trp Leu Glu Ala Gln Glu Glu Glu 5Glu Val Gly Phe Pro Val Arg Pro Gln Val Pro Leu Arg Pro Met Thr65 7Tyr Lys Gly Ala Val Asp Leu Ser Phe Phe Leu Lys Glu Lys Gly Gly 85 9 Glu Gly Leu Ile Tyr Ser Lys Lys Arg Gln Glu Ile Leu Asp Leu Val Tyr His Thr Gln Gly Tyr Phe Pro Asp Trp Gln Asn Tyr Thr Gly Pro Gly Ile Arg Phe Pro Leu Thr Phe Gly Trp Cys Phe Lys Val Pro Val Asp Pro Arg Glu Val Glu Glu Ala Asn Glu Gly Glu Asn Asn Cys Leu Leu His Pro Met Ser Gln His Gly Met Glu Asp Glu Arg Glu Val Leu Lys Trp Lys Phe Asp Ser His Leu Ala Arg Arg Met Ala Arg Glu Leu His Pro Glu Tyr Tyr Lys Asp Cys 27PRTArtificial sequenceLeast squares and minimum of means of center of tree reconstructions of clade C nef protein sequence Gly Gly Lys Trp Ser Lys Ser Ser Ile Val Gly Trp Pro Ala Vallu Arg Ile Arg Arg Thr Glu Pro Ala Ala Glu Gly Val Gly Ala 2Ala Ser Gln Asp Leu Asp Lys His Gly Ala Leu Thr Ser Ser Asn Thr 35 4 Ala Asn Asn Ala Asp Cys Ala Trp Leu Glu Ala Gln Glu Glu Glu 5Glu Glu Val Gly Phe Pro Val Arg Pro Gln Val Pro Leu Arg Pro Met65 7Thr Tyr Lys Gly Ala Phe Asp Leu Ser Phe Phe Leu Lys Glu Lys Gly 85 9 Leu Glu Gly Leu Ile Tyr Ser Lys Lys Arg Gln Glu Ile Leu Asp Trp Val Tyr His Thr Gln Gly Tyr Phe Pro Asp Trp Gln Asn Tyr Pro Gly Pro Gly Val Arg Tyr Pro Leu Thr Phe Gly Trp Cys Phe Leu Val Pro Val Asp Pro Arg Glu Val Glu Glu Ala Asn Glu Gly Glu Asn Asn Cys Leu Leu His Pro Met Ser Gln His Gly Met Glu Asp Asp Arg Glu Val Leu Lys Trp Lys Phe Asp Ser His Leu Ala Arg His Met Ala Arg Glu Leu His Pro Glu Tyr Tyr Lys Asp Cys 29PRTArtificial sequenceMost recent common ancestor for reconstruction of clade C pol protein sequences Phe Arg Glu Asn Leu Ala Phe Pro Gln Gly Glu Ala Arg Glu Pheer Glu Gln Thr Arg Ala Asn Ser Pro Thr Ser Arg Glu Leu Gln 2Val Arg Gly Asp Asn Pro Arg Ser Glu Ala Gly Ala Glu Arg Gln Gly 35 4 Leu Asn Phe Pro Gln Ile Thr Leu Trp Gln Arg Pro Leu Val Ser 5Ile Lys Val Gly Gly Gln Ile Lys Glu Ala Leu Leu Asp Thr Gly Ala65 7Asp Asp Thr Val Leu Glu Asp Ile Asn Leu Pro Gly Lys Trp Lys Pro 85 9 Met Ile Gly Gly Ile Gly Gly Phe Ile Lys Val Arg Gln Tyr Asp Ile Leu Ile Glu Ile Cys Gly Lys Lys Ala Ile Gly Thr Val Leu Gly Pro Thr Pro Val Asn Ile Ile Gly Arg Asn Met Leu Thr Gln Gly Cys Thr Leu Asn Phe Pro Ile Ser Pro Ile Glu Thr Val Pro Val Lys Leu Lys Pro Gly Met Asp Gly Pro Lys Val Lys Gln Trp Pro Thr Glu Glu Lys Ile Lys Ala Leu Thr Ala Ile Cys Glu Glu Met Lys Glu Gly Lys Ile Thr Lys Ile Gly Pro Glu Asn Pro Tyr Asn 2ro Val Phe Ala Ile Lys Lys Lys Asp Ser Thr Lys Trp Arg Lys 222l Asp Phe Arg Glu Leu Asn Lys Arg Thr Gln Asp Phe Trp Glu225 234n Leu Gly Ile Pro His Pro Ala Gly Leu Lys Lys Lys Lys Ser 245 25l Thr Val Leu Asp Val Gly Asp Ala Tyr Phe Ser Val Pro Leu Asp 267p Phe Arg Lys Tyr Thr Ala Phe Thr Ile Pro Ser Ile Asn Asn 275 28u Thr Pro Gly Ile Arg Tyr Gln Tyr Asn Val Leu Pro Gln Gly Trp 29ly Ser Pro Ala Ile Phe Gln Ser Ser Met Thr Lys Ile Leu Glu33ro Phe Arg Ala Gln Asn Pro Glu Ile Val Ile Tyr Gln Tyr Met Asp 325 33p Leu Tyr Val Gly Ser Asp Leu Glu Ile Gly Gln His Arg Ala Lys 345u Glu Leu Arg Glu His Leu Leu Lys Trp Gly Phe Thr Thr Pro 355 36p Lys Lys His Gln Lys Glu Pro Pro Phe Leu Trp Met Gly Tyr Glu 378s Pro Asp Lys Trp Thr Val Gln Pro Ile Gln Leu Pro Glu Lys385 39er Trp Thr Val Asn Asp Ile Gln Lys Leu Val Gly Lys Leu Asn

44la Ser Gln Ile Tyr Pro Gly Ile Lys Val Arg Gln Leu Cys Lys 423u Arg Gly Ala Lys Ala Leu Thr Asp Ile Val Pro Leu Thr Glu 435 44u Ala Glu Leu Glu Leu Ala Glu Asn Arg Glu Ile Leu Lys Glu Pro 456s Gly Val Tyr Tyr Asp Pro Ser Lys Asp Leu Ile Ala Glu Ile465 478s Gln Gly His Asp Gln Trp Thr Tyr Gln Ile Tyr Gln Glu Pro 485 49e Lys Asn Leu Lys Thr Gly Lys Tyr Ala Lys Met Arg Ser Ala His 55sn Asp Val Lys Gln Leu Thr Glu Ala Val Gln Lys Ile Ala Met 5525Glu Ser Ile Val Ile Trp Gly Lys Thr Pro Lys Phe Arg Leu Pro Ile 534s Glu Thr Trp Glu Thr Trp Trp Thr Asp Tyr Trp Gln Ala Thr545 556e Pro Glu Trp Glu Phe Val Asn Thr Pro Pro Leu Val Lys Leu 565 57p Tyr Gln Leu Glu Lys Glu Pro Ile Ala Gly Ala Glu Thr Phe Tyr 589p Gly Ala Ala Asn Arg Glu Thr Lys Leu Gly Lys Ala Gly Tyr 595 6al Thr Asp Lys Gly Arg Gln Lys Val Val Ser Leu Thr Glu Thr Thr 662n Lys Thr Glu Leu Gln Ala Ile Gln Leu Ala Leu Gln Asp Ser625 634r Glu Val Asn Ile Val Thr Asp Ser Gln Tyr Ala Leu Gly Ile 645 65e Gln Ala Gln Pro Asp Lys Ser Glu Ser Glu Leu Val Asn Gln Ile 667u Gln Leu Ile Lys Lys Glu Lys Val Tyr Leu Ser Trp Val Pro 675 68a His Lys Gly Ile Gly Gly Asn Glu Gln Val Asp Lys Leu Val Ser 69ly Ile Arg Lys Val Leu Phe Leu Asp Gly Ile Asp Lys Ala Gln77lu Glu His Glu Lys Tyr His Ser Asn Trp Arg Ala Met Ala Ser Glu 725 73e Asn Leu Pro Pro Ile Val Ala Lys Glu Ile Val Ala Ser Cys Asp 745s Gln Leu Lys Gly Glu Ala Met His Gly Gln Val Asp Cys Ser 755 76o Gly Ile Trp Gln Leu Asp Cys Thr His Leu Glu Gly Lys Val Ile 778l Ala Val His Val Ala Ser Gly Tyr Ile Glu Ala Glu Val Ile785 79la Glu Thr Gly Gln Glu Thr Ala Tyr Phe Ile Leu Lys Leu Ala 88rg Trp Pro Val Lys Val Ile His Thr Asp Asn Gly Ser Asn Phe 823r Ala Ala Val Lys Ala Ala Cys Trp Trp Ala Gly Ile Gln Gln 835 84u Phe Gly Ile Pro Tyr Asn Pro Gln Ser Gln Gly Val Val Glu Ser 856n Lys Glu Leu Lys Lys Ile Ile Gly Gln Val Arg Asp Gln Ala865 878s Leu Lys Thr Ala Val Gln Met Ala Val Phe Ile His Asn Phe 885 89s Arg Lys Gly Gly Ile Gly Gly Tyr Ser Ala Gly Glu Arg Ile Ile 99le Ile Ala Thr Asp Ile Gln Thr Lys Glu Leu Gln Lys Gln Ile 9925Ile Lys Ile Gln Asn Phe Arg Val Tyr Tyr Arg Asp Ser Arg Asp Pro 934p Lys Gly Pro Ala Lys Leu Leu Trp Lys Gly Glu Gly Ala Val945 956e Gln Asp Asn Ser Asp Ile Lys Val Val Pro Arg Arg Lys Ala 965 97s Ile Ile Arg Asp Tyr Gly Lys Gln Met Ala Gly Ala Asp Cys Val 989y Arg Gln Asp Glu Asp 995RTArtificial sequenceLeast squares center of tree reconstruction of clade C pol protein sequence Phe Arg Glu Asn Leu Ala Phe Pro Gln Gly Glu Ala Arg Glu Pheer Glu Gln Thr Arg Ala Asn Ser Pro Thr Ser Arg Glu Leu Gln 2Val Arg Gly Asp Asn Pro Arg Ser Glu Ala Gly Ala Glu Arg Gln Gly 35 4 Leu Asn Phe Pro Gln Ile Thr Leu Trp Gln Arg Pro Leu Val Ser 5Ile Lys Val Gly Gly Gln Ile Lys Glu Ala Leu Leu Asp Thr Gly Ala65 7Asp Asp Thr Val Leu Glu Glu Ile Asn Leu Pro Gly Lys Trp Lys Pro 85 9 Met Ile Gly Gly Ile Gly Gly Phe Ile Lys Val Arg Gln Tyr Asp Ile Leu Ile Glu Ile Cys Gly Lys Lys Ala Ile Gly Thr Val Leu Gly Pro Thr Pro Val Asn Ile Ile Gly Arg Asn Met Leu Thr Gln Gly Cys Thr Leu Asn Phe Pro Ile Ser Pro Ile Glu Thr Val Pro Val Lys Leu Lys Pro Gly Met Asp Gly Pro Lys Val Lys Gln Trp Pro Thr Glu Glu Lys Ile Lys Ala Leu Thr Ala Ile Cys Glu Glu Met Lys Glu Gly Lys Ile Thr Lys Ile Gly Pro Glu Asn Pro Tyr Asn 2ro Val Phe Ala Ile Lys Lys Lys Asp Ser Thr Lys Trp Arg Lys 222l Asp Phe Arg Glu Leu Asn Lys Arg Thr Gln Asp Phe Trp Glu225 234n Leu Gly Ile Pro His Pro Ala Gly Leu Lys Lys Lys Lys Ser 245 25l Thr Val Leu Asp Val Gly Asp Ala Tyr Phe Ser Val Pro Leu Asp 267y Phe Arg Lys Tyr Thr Ala Phe Thr Ile Pro Ser Ile Asn Asn 275 28u Thr Pro Gly Ile Arg Tyr Gln Tyr Asn Val Leu Pro Gln Gly Trp 29ly Ser Pro Ala Ile Phe Gln Ser Ser Met Thr Lys Ile Leu Glu33ro Phe Arg Ala Gln Asn Pro Glu Ile Val Ile Tyr Gln Tyr Met Asp 325 33p Leu Tyr Val Gly Ser Asp Leu Glu Ile Gly Gln His Arg Ala Lys 345u Glu Leu Arg Glu His Leu Leu Lys Trp Gly Phe Thr Thr Pro 355 36p Lys Lys His Gln Lys Glu Pro Pro Phe Leu Trp Met Gly Tyr Glu 378s Pro Asp Lys Trp Thr Val Gln Pro Ile Gln Leu Pro Glu Lys385 39er Trp Thr Val Asn Asp Ile Gln Lys Leu Val Gly Lys Leu Asn 44la Ser Gln Ile Tyr Pro Gly Ile Lys Val Arg Gln Leu Cys Lys 423u Arg Gly Ala Lys Ala Leu Thr Asp Ile Val Pro Leu Thr Glu 435 44u Ala Glu Leu Glu Leu Ala Glu Asn Arg Glu Ile Leu Lys Glu Pro 456s Gly Val Tyr Tyr Asp Pro Ser Lys Asp Leu Ile Ala Glu Ile465 478s Gln Gly His Asp Gln Trp Thr Tyr Gln Ile Tyr Gln Glu Pro 485 49e Lys Asn Leu Lys Thr Gly Lys Tyr Ala Lys Met Arg Thr Ala His 55sn Asp Val Lys Gln Leu Thr Glu Ala Val Gln Lys Ile Ala Met 5525Glu Ser Ile Val Ile Trp Gly Lys Thr Pro Lys Phe Arg Leu Pro Ile 534s Glu Thr Trp Glu Thr Trp Trp Thr Asp Tyr Trp Gln Ala Thr545 556e Pro Glu Trp Glu Phe Val Asn Thr Pro Pro Leu Val Lys Leu 565 57p Tyr Gln Leu Glu Lys Glu Pro Ile Ala Gly Ala Glu Thr Phe Tyr 589p Gly Ala Ala Asn Arg Glu Thr Lys Ile Gly Lys Ala Gly Tyr 595 6al Thr Asp Arg Gly Arg Gln Lys Ile Val Ser Leu Thr Glu Thr Thr 662n Lys Thr Glu Leu Gln Ala Ile Gln Leu Ala Leu Gln Asp Ser625 634r Glu Val Asn Ile Val Thr Asp Ser Gln Tyr Ala Leu Gly Ile 645 65e Gln Ala Gln Pro Asp Lys Ser Glu Ser Glu Leu Val Asn Gln Ile 667u Gln Leu Ile Lys Lys Glu Arg Val Tyr Leu Ser Trp Val Pro 675 68a His Lys Gly Ile Gly Gly Asn Glu Gln Val Asp Lys Leu Val Ser 69ly Ile Arg Lys Val Leu Phe Leu Asp Gly Ile Asp Lys Ala Gln77lu Glu His Glu Lys Tyr His Ser Asn Trp Arg Ala Met Ala Ser Glu 725 73e Asn Leu Pro Pro Ile Val Ala Lys Glu Ile Val Ala Ser Cys Asp 745s Gln Leu Lys Gly Glu Ala Ile His Gly Gln Val Asp Cys Ser 755 76o Gly Ile Trp Gln Leu Asp Cys Thr His Leu Glu Gly Lys Ile Ile 778l Ala Val His Val Ala Ser Gly Tyr Ile Glu Ala Glu Val Ile785 79la Glu Thr Gly Gln Glu Thr Ala Tyr Phe Ile Leu Lys Leu Ala 88rg Trp Pro Val Lys Val Ile His Thr Asp Asn Gly Ser Asn Phe 823r Ala Ala Val Lys Ala Ala Cys Trp Trp Ala Gly Ile Gln Gln 835 84u Phe Gly Ile Pro Tyr Asn Pro Gln Ser Gln Gly Val Val Glu Ser 856n Lys Glu Leu Lys Lys Ile Ile Gly Gln Val Arg Asp Gln Ala865 878s Leu Lys Thr Ala Val Gln Met Ala Val Phe Ile His Asn Phe 885 89s Arg Lys Gly Gly Ile Gly Gly Tyr Ser Ala Gly Glu Arg Ile Ile 99le Ile Ala Thr Asp Ile Gln Thr Lys Glu Leu Gln Lys Gln Ile 9925Ile Lys Ile Gln Asn Phe Arg Val Tyr Tyr Arg Asp Ser Arg Asp Pro 934p Lys Gly Pro Ala Lys Leu Leu Trp Lys Gly Glu Gly Ala Val945 956e Gln Asp Asn Ser Asp Ile Lys Val Val Pro Arg Arg Lys Ala 965 97s Ile Ile Lys Asp Tyr Gly Lys Gln Met Ala Gly Ala Asp Cys Val 989y Arg Gln Asp Glu Asp 995RTArtificial sequenceMinimum of means center of tree reconstruction of clade C pol protein sequence Phe Arg Glu Asn Leu Ala Phe Pro Gln Gly Glu Ala Arg Glu Pheer Glu Gln Thr Arg Ala Asn Ser Pro Thr Ser Arg Glu Leu Gln 2Val Arg Gly Asp Asn Pro Leu Ser Glu Ala Gly Ala Glu Arg Gln Gly 35 4 Leu Asn Phe Pro Gln Ile Thr Leu Trp Gln Arg Pro Leu Val Thr 5Ile Lys Val Gly Gly Gln Leu Lys Glu Ala Leu Leu Asp Thr Gly Ala65 7Asp Asp Thr Val Leu Glu Glu Ile Asn Leu Pro Gly Lys Trp Lys Pro 85 9 Met Ile Gly Gly Ile Gly Gly Phe Ile Lys Val Arg Gln Tyr Asp Ile Leu Ile Glu Ile Cys Gly Lys Lys Ala Ile Gly Thr Val Leu Gly Pro Thr Pro Val Asn Ile Ile Gly Arg Asn Met Leu Thr Gln Gly Cys Thr Leu Asn Phe Pro Ile Ser Pro Ile Glu Thr Val Pro Val Lys Leu Lys Pro Gly Met Asp Gly Pro Lys Val Lys Gln Trp Pro Thr Glu Glu Lys Ile Lys Ala Leu Thr Ala Ile Cys Glu Glu Met Lys Glu Gly Lys Ile Thr Lys Ile Gly Pro Glu Asn Pro Tyr Asn 2ro Val Phe Ala Ile Lys Lys Lys Asp Ser Thr Lys Trp Arg Lys 222l Asp Phe Arg Glu Leu Asn Lys Arg Thr Gln Asp Phe Trp Glu225 234n Leu Gly Ile Pro His Pro Ala Gly Leu Lys Lys Lys Lys Ser 245 25l Thr Val Leu Asp Val Gly Asp Ala Tyr Phe Ser Val Pro Leu Asp 267y Phe Arg Lys Tyr Thr Ala Phe Thr Ile Pro Ser Ile Asn Asn 275 28u Thr Pro Gly Ile Arg Tyr Gln Tyr Asn Val Leu Pro Gln Gly Trp 29ly Ser Pro Ala Ile Phe Gln Ser Ser Met Thr Lys Ile Leu Glu33ro Phe Arg Ala Gln Asn Pro Glu Ile Val Ile Tyr Gln Tyr Met Asp 325 33p Leu Tyr Val Gly Ser Asp Leu Glu Ile Gly Gln His Arg Ala Lys 345u Glu Leu Arg Glu His Leu Leu Lys Trp Gly Phe Thr Thr Pro 355 36p Lys Lys His Gln Lys Glu Pro Pro Phe Leu Trp Met Gly Tyr Glu 378s Pro Asp Lys Trp Thr Val Gln Pro Ile Gln Leu Pro Glu Lys385 39er Trp Thr Val Asn Asp Ile Gln Lys Leu Val Gly Lys Leu Asn 44la Ser Gln Ile Tyr Pro Gly Ile Lys Val Arg Gln Leu Cys Lys 423u Arg Gly Ala Lys Ala Leu Thr Asp Ile Val Pro Leu Thr Glu 435 44u Ala Glu Leu Glu Leu Ala Glu Asn Arg Glu Ile Leu Lys Glu Pro 456s Gly Val Tyr Tyr Asp Pro Ser Lys Asp Leu Ile Ala Glu Ile465 478s Gln Gly His Asp Gln Trp Thr Tyr Gln Ile Tyr Gln Glu Pro 485 49e Lys Asn Leu Lys Thr Gly Lys Tyr Ala Lys Met Arg Thr Ala His 55sn Asp Val Lys Gln Leu Thr Glu Ala Val Gln Lys Ile Ala Met 5525Glu Ser Ile Val Ile Trp Gly Lys Thr Pro Lys Phe Arg Leu Pro Ile 534s Glu Thr Trp Glu Ala Trp Trp Thr Asp Tyr Trp Gln Ala Thr545 556e Pro Glu Trp Glu Phe Val Asn Thr Pro Pro Leu Val Lys Leu 565 57p Tyr Gln Leu Glu Lys Glu Pro Ile Ala Gly Ala Glu Thr Phe Tyr 589p Gly Ala Ala Asn Arg Glu Thr Lys Ile Gly Lys Ala Gly Tyr 595 6al Thr Asp Arg Gly Arg Gln Lys Ile Val Ser Leu Thr Glu Thr Thr 662n Lys Thr Glu Leu Gln Ala Ile Gln Leu Ala Leu Gln Asp Ser625 634r Glu Val Asn Ile Val Thr Asp Ser Gln Tyr Ala Leu Gly Ile 645 65e Gln Ala Gln Pro Asp Lys Ser Glu Ser Glu Leu Val Asn Gln Ile 667u Gln Leu Ile Lys Lys Glu Arg Val Tyr Leu Ser Trp Val Pro 675 68a His Lys Gly Ile Gly Gly Asn Glu Gln Val Asp Lys Leu Val Ser 69ly Ile Arg Lys Val Leu Phe Leu Asp Gly Ile Asp Lys Ala Gln77lu Glu His Glu Lys Tyr His Ser Asn Trp Arg Ala Met Ala Ser Glu 725 73e Asn Leu Pro Pro Ile Val Ala Lys Glu Ile Val Ala Ser Cys Asp 745s Gln Leu Lys Gly Glu Ala Ile His Gly Gln Val Asp Cys Ser 755 76o Gly Ile Trp Gln Leu Asp Cys Thr His Leu Glu Gly Lys Ile Ile 778l Ala Val His Val Ala Ser Gly Tyr Ile Glu Ala Glu Val Ile785 79la Glu Thr Gly Gln Glu Thr Ala Tyr Phe Ile Leu Lys Leu Ala 88rg Trp Pro Val Lys Val Ile His Thr Asp Asn Gly Ser Asn Phe 823r Ala Ala Val Lys Ala Ala Cys Trp Trp Ala Gly Ile Gln Gln 835 84u Phe Gly Ile Pro Tyr Asn Pro Gln Ser Gln Gly Val Val Glu Ser 85BR> 855 86n Lys Glu Leu Lys Lys Ile Ile Gly Gln Val Arg Asp Gln Ala865 878s Leu Lys Thr Ala Val Gln Met Ala Val Phe Ile His Asn Phe 885 89s Arg Lys Gly Gly Ile Gly Gly Tyr Ser Ala Gly Glu Arg Ile Ile 99le Ile Ala Thr Asp Ile Gln Thr Lys Glu Leu Gln Lys Gln Ile 9925Ile Lys Ile Gln Asn Phe Arg Val Tyr Tyr Arg Asp Ser Arg Asp Pro 934p Lys Gly Pro Ala Lys Leu Leu Trp Lys Gly Glu Gly Ala Val945 956e Gln Asp Asn Ser Asp Ile Lys Val Val Pro Arg Arg Lys Ala 965 97s Ile Ile Lys Asp Tyr Gly Lys Gln Met Ala Gly Ala Asp Cys Val 989y Arg Gln Asp Glu Asp 995RTArtificial sequenceMost common recent ancestor reconstructions of clade C rev protein sequence Ala Gly Arg Ser Gly Asp Ser Asp Glu Ala Leu Leu Gln Ala Valle Ile Lys Ile Leu Tyr Gln Ser Asn Pro Tyr Pro Lys Pro Glu 2Gly Thr Arg Gln Ala Arg Arg Asn Arg Arg Arg Arg Trp Arg Ala Arg 35 4 Arg Gln Ile His Ser Ile Ser Glu Arg Ile Leu Ser Thr Cys Leu 5Gly Arg Pro Ala Glu Pro Val Pro Leu Gln Leu Pro Pro Leu Glu Arg65 7Leu His Leu Asp Cys Ser Glu Asp Cys Gly Thr Ser Gly Thr Gln Gln 85 9 Gln Gly Thr Thr Glu Gly Val Gly Ser Pro RTArtificial sequenceLeast squares center of tree reconstructions of clade C rev protein sequence Ala Gly Arg Ser Gly Asp Ser Asp Glu Ala Leu Leu Gln Ala Valle Ile Lys Ile Leu Tyr Gln Ser Asn Pro Tyr Pro Lys Pro Glu 2Gly Thr Arg Gln Ala Arg Lys Asn Arg Arg Arg Arg Trp Arg Ala Arg 35 4 Arg Gln Ile His Ser Ile Ser Glu Arg Ile Leu Ser Thr Cys Leu 5Gly Arg Pro Ala Glu Pro Val Pro Leu Gln Leu Pro Pro Ile Glu Arg65 7Leu His Ile Gly Asp Ser Glu Ser Ser Gly Thr Ser Gly Thr Gln Gln 85 9 Gln Gly Thr Thr Glu Gly Val Gly Ser Pro RTArtificial sequenceMinimum of means center of tree reconstructions of clade C rev protein sequence Ala Gly Arg Ser Gly Asp Ser Asp Glu Ala Leu Leu Gln Ala Valle Ile Lys Ile Leu Tyr Gln Ser Asn Pro Tyr Pro Lys Pro Glu 2Gly Thr Arg Gln Ala Arg Lys Asn Arg Arg Arg Arg Trp Arg Ala Arg 35 4 Arg Gln Ile His Ser Ile Ser Glu Arg Ile Leu Ser Thr Cys Leu 5Gly Arg Pro Ala Glu Pro Val Pro Phe Gln Leu Pro Pro Ile Glu Arg65 7Leu His Ile Gly Asp Ser Glu Ser Ser Gly Thr Ser Gly Thr Gln Gln 85 9 Gln Gly Thr Thr Glu Gly Val Gly Ser Pro RTArtificial sequenceMost recent common ancestor reconstructions of clade C tat protein sequence Glu Pro Val Asp Pro Asn Leu Glu Pro Trp Asn His Pro Gly Serro Lys Thr Ala Cys Asn Lys Cys Tyr Cys Lys Lys Cys Ser Tyr 2His Cys Leu Val Cys Phe Leu Thr Lys Gly Leu Gly Ile Ser Tyr Gly 35 4 Lys Lys Arg Arg Gln Arg Arg Arg Ala Pro Pro Ser Ser Glu Asp 5His Gln Asn Pro Ile Ser Lys Gln Pro Leu Ser Gln Thr Arg Gly Asp65 7Pro Thr Gly Ser Glu Glu Ser Lys Lys Lys Val Glu Ser Lys Thr Glu 85 9 Asp Pro Cys Asp tificial sequenceLeast squares and minimum of means center of tree reconstruction of clade C tat protein sequence Glu Pro Val Asp Pro Asn Leu Glu Pro Trp Asn His Pro Gly Serro Lys Thr Pro Cys Asn Lys Cys Tyr Cys Lys His Cys Ser Tyr 2His Cys Leu Val Cys Phe Gln Thr Lys Gly Leu Gly Ile Ser Tyr Gly 35 4 Lys Lys Arg Arg Gln Arg Arg Ser Ala Pro Pro Ser Ser Glu Asp 5His Gln Asn Pro Ile Ser Lys Gln Pro Leu Pro Gln Thr Arg Gly Asp65 7Pro Thr Gly Ser Glu Glu Ser Lys Lys Lys Val Glu Ser Lys Thr Glu 85 9 Asp Pro Phe Asp 92PRTArtificial sequenceMost recent common ancestor reconstructions of clade C vif protein sequence Glu Asn Arg Trp Gln Val Leu Ile Val Trp Gln Val Asp Arg Metle Arg Thr Trp Asn Ser Leu Val Lys His His Met Tyr Val Ser 2Arg Arg Ala Lys Gly Trp Phe Tyr Arg His His Tyr Glu Ser Arg His 35 4 Lys Ile Ser Ser Glu Val His Ile Pro Leu Gly Asp Ala Arg Leu 5Val Ile Lys Thr Tyr Trp Gly Leu His Thr Gly Glu Arg Asp Trp His65 7Leu Gly His Gly Val Ser Ile Glu Trp Arg Leu Arg Arg Tyr Ser Thr 85 9 Val Asp Pro Gly Leu Ala Asp Gln Leu Ile His Met His Tyr Phe Cys Phe Ala Asp Ser Ala Ile Arg Lys Ala Ile Leu Gly His Ile Ser Pro Arg Cys Asp Tyr Gln Ala Gly His Asn Lys Val Gly Ser Gln Tyr Leu Ala Leu Thr Ala Leu Ile Lys Pro Lys Lys Ile Lys Pro Pro Leu Pro Ser Val Lys Lys Leu Val Glu Asp Arg Trp Asn Lys Gln Lys Thr Arg Gly His Arg Gly Ser His Thr Met Asn Gly His 92PRTArtificial sequenceLeast squares center of tree reconstruction of clade C vif protein sequence Glu Asn Arg Trp Gln Val Leu Ile Val Trp Gln Val Asp Arg Metle Arg Thr Trp Asn Ser Leu Val Lys His His Met Tyr Val Ser 2Arg Arg Ala Asn Gly Trp Phe Tyr Arg His His Tyr Glu Ser Arg His 35 4 Lys Val Ser Ser Glu Val His Ile Pro Leu Gly Asp Ala Arg Leu 5Val Ile Lys Thr Tyr Trp Gly Leu Gln Thr Gly Glu Arg Asp Trp His65 7Leu Gly His Gly Val Ser Ile Glu Trp Arg Leu Arg Arg Tyr Ser Thr 85 9 Val Asp Pro Gly Leu Ala Asp Gln Leu Ile His Met His Tyr Phe Cys Phe Ala Asp Ser Ala Ile Arg Lys Ala Ile Leu Gly His Ile Ile Pro Arg Cys Asp Tyr Gln Ala Gly His Asn Lys Val Gly Ser Gln Tyr Leu Ala Leu Thr Ala Leu Ile Lys Pro Lys Lys Ile Lys Pro Pro Leu Pro Ser Val Arg Lys Leu Val Glu Asp Arg Trp Asn Lys Gln Lys Thr Arg Gly Arg Arg Gly Asn His Thr Met Asn Gly His 92PRTArtificial sequenceMinimum of means center of tree reconstructions of clade C vif protein sequence Glu Asn Arg Trp Gln Val Leu Ile Val Trp Gln Val Asp Arg Metle Arg Thr Trp Asn Ser Leu Val Lys His His Met Tyr Val Ser 2Arg Arg Ala Asn Gly Trp Phe Tyr Arg His His Tyr Glu Ser Arg His 35 4 Lys Val Ser Ser Glu Val His Ile Pro Leu Gly Asp Ala Arg Leu 5Val Ile Lys Thr Tyr Trp Gly Leu His Thr Gly Glu Arg Asp Trp His65 7Leu Gly His Gly Val Ser Ile Glu Trp Arg Leu Arg Arg Tyr Ser Thr 85 9 Val Asp Pro Gly Leu Ala Asp Gln Leu Ile His Met His Tyr Phe Cys Phe Ala Asp Ser Ala Ile Arg Lys Ala Ile Leu Gly His Ile Ile Pro Arg Cys Asp Tyr Gln Ala Gly His Asn Lys Val Gly Ser Gln Tyr Leu Ala Leu Thr Ala Leu Ile Lys Pro Lys Lys Ile Lys Pro Pro Leu Pro Ser Val Arg Lys Leu Val Glu Asp Arg Trp Asn Lys Gln Lys Thr Arg Gly Arg Arg Gly Asn His Thr Met Asn Gly His 6PRTArtificial sequenceMost recent common ancestor reconstructions for clade C vpr protein sequence Glu Gln Ala Pro Glu Asp Gln Gly Pro Gln Arg Glu Pro Tyr Asnrp Thr Leu Glu Leu Leu Glu Glu Leu Lys Gln Glu Ala Val Arg 2His Phe Pro Arg Pro Trp Leu His Ser Leu Gly Gln His Ile Tyr Glu 35 4 Tyr Gly Asp Thr Trp Ala Gly Val Glu Ala Ile Ile Arg Ile Leu 5Gln Gln Leu Leu Phe Ile His Phe Arg Ile Gly Cys Gln His Ser Arg65 7Ile Gly Ile Ile Arg Gln Arg Arg Ala Arg Asn Gly Ala Ser Arg Ser 85 996PRTArtificial sequenceLeast squares center of tree reconstruction of clade C vpr protein sequence Glu Gln Ala Pro Glu Asp Gln Gly Pro Gln Arg Glu Pro Tyr Asnrp Thr Leu Glu Ile Leu Glu Glu Leu Lys Gln Glu Ala Val Arg 2His Phe Pro Arg Pro Trp Leu His Ser Leu Gly Gln Tyr Ile Tyr Glu 35 4 Tyr Gly Asp Thr Trp Thr Gly Val Glu Ala Leu Ile Arg Ile Leu 5Gln Gln Leu Leu Phe Ile His Phe Arg Ile Gly Cys Gln His Ser Arg65 7Ile Gly Ile Leu Arg Gln Arg Arg Ala Arg Asn Gly Ala Ser Arg Ser 85 996PRTArtificial sequenceMinimum of means center of tree reconstruction of clade C vpr protein sequence Glu Gln Ala Pro Glu Asp Gln Gly Pro Gln Arg Glu Pro Tyr Asnrp Thr Leu Glu Leu Leu Glu Glu Leu Lys Gln Glu Ala Val Arg 2His Phe Pro Arg Pro Trp Leu His Ser Leu Gly Gln His Ile Tyr Glu 35 4 Tyr Gly Asp Thr Trp Thr Gly Val Glu Ala Leu Ile Arg Ile Leu 5Gln Gln Leu Leu Phe Ile His Phe Arg Ile Gly Cys Gln His Ser Arg65 7Ile Gly Ile Met Arg Gln Arg Arg Ala Arg Asn Gly Ala Ser Arg Ser 85 986PRTArtificial sequenceMost recent common ancestor reconstructions for clade C vpu protein sequence Leu Asp Leu Ile Ala Arg Val Asp Tyr Arg Leu Gly Val Gly Alale Val Ala Leu Ile Ile Ala Ile Val Val Trp Thr Ile Val Tyr 2Ile Glu Tyr Arg Lys Leu Val Arg Gln Arg Lys Ile Asp Trp Leu Ile 35 4 Arg Ile Arg Glu Arg Ala Glu Asp Ser Gly Asn Glu Ser Asp Gly 5Asp Thr Glu Glu Leu Ser Thr Leu Val Asp Met Gly His Leu Arg Leu65 7Leu Asp Val Asn Asp Leu 85TArtificial sequenceLeast squares and minimum of means center of tree reconstruction of clade C vpu protein sequence Leu Asp Leu Leu Ala Arg Val Asp Tyr Arg Leu Gly Val Gly Alale Val Ala Leu Ile Ile Ala Ile Val Val Trp Thr Ile Val Tyr 2Ile Glu Tyr Arg Lys Leu Leu Arg Gln Arg Lys Ile Asp Trp Leu Ile 35 4 Arg Ile Arg Glu Arg Ala Glu Asp Ser Gly Asn Glu Ser Glu Gly 5Asp Thr Glu Glu Leu Ser Thr Met Val Asp Met Gly His Leu Arg Leu65 7Leu Asp Val Asn Asp Leu 85RTArtificial sequenceDeduced ancestor env protein sequence Lys Ser Glu Thr Asp Arg Trp Gly Leu Thr Lys Ser Ser Thr Thrhr Thr Ala Ala Pro Thr Ser Ala Pro Val Ser Glu Lys Ile Asp 2Met Val Asn Glu Thr Ser Ser Cys Ile Ala Gln Asn Asn Cys Thr Gly 35 4 Glu Gln Glu Gln Met Ile Ser Cys Lys Phe Asn Met Thr Gly Leu 5Lys Arg Asp Lys Thr Lys Glu Tyr Asn Glu Thr Trp Tyr Ser Thr Asp65 7Leu Val Cys Glu Gln Gly Asn Ser Thr Asp Asn Glu Ser Arg Cys Tyr 85 9 Asn His Cys Asn Thr Ser Val Ile Gln Glu Ser Cys Asp Lys His Trp Asp Thr Ile Arg Phe Arg Tyr Cys Ala Pro Pro Gly Tyr Ala Leu Arg Cys Asn Asp Thr Asn Tyr Ser Gly Phe Met Pro Lys Cys Lys Val Val Val Ser Ser Cys Thr Arg Met Met Glu Thr Gln Thr Ser Thr Trp Phe Gly Phe Asn Gly Thr Arg Ala Glu Asn Arg Thr Tyr Tyr Trp His Gly Arg Asp Asn Arg Thr Ile Ile Ser Leu Asn Lys Tyr Asn Leu Thr Met Lys Cys Arg Arg Pro Gly Asn Lys Thr Val 2ro Val Thr Ile Met Ser Gly Leu Val Phe His Ser Gln Pro Ile 222p Arg Pro Lys Gln Ala Trp Cys Trp Phe Gly Gly Lys Trp Lys225 234a Ile Lys Glu Val Lys Gln Thr Ile Val Lys His Pro Arg Tyr 245 25r Gly Thr Asn Asn Thr Asp Lys Ile Asn Leu Thr Ala Pro Gly Gly 267p Pro Glu Val Thr Phe Met Trp Thr Asn Cys Arg Gly Glu Phe 275 28u Tyr Cys Lys Met Asn Trp Phe Leu Asn Trp Val Glu Asp Arg Asp 29hr Thr Gln Arg Pro Lys Glu Arg His Arg Arg Asn Tyr Val Pro33ys His Ile Arg Gln Ile Ile Asn Thr Trp His Lys Val Gly Lys Asn 325 33l Tyr Leu Pro Pro Arg Glu Gly Asp Leu Thr Cys Asn Ser Thr Val 345r Leu Ile Ala Asn Ile Asp Trp Thr Asp Gly Asn Gln Thr Asn 355 36e Thr Met Ser Ala Glu Val Ala 37Artificial sequenceDeduced ancestor env protein sequence Arg Val Lys Gly Ile Arg Lys Asn Tyr Gln His Leu Trp Arg Trphr Met Leu Leu Gly Met Leu Met Ile Cys Ser Ala Ala Glu Lys 2Leu Trp Val Thr Val Tyr Tyr Gly Val Pro Val Trp Lys Glu Ala Thr 35 4 Thr Leu Phe Cys Ala Ser Asp Ala Lys Ala Tyr Asp Thr Glu Val 5His Asn Val Trp Ala Thr His Ala Cys Val Pro Thr Asp Pro Asn Pro65 7Gln Glu Val Val Leu Glu Asn Val Thr Glu Asn Phe Asn Met Trp Lys 85 9 Asn Met Val Glu Gln Met His Glu Asp Ile Ile Ser Leu Trp Asp Ser Leu Lys Pro Cys Val Lys Leu Thr Pro Leu Cys Val Thr Leu Cys Thr Asp Asp Leu Arg Thr Asn Ala Thr Asn Thr Thr Asn Ser Ala Thr Thr Asn Thr Thr Ser Ser Gly Gly Gly Thr Met Glu Gly Glu Lys Gly Glu Ile Lys Asn Cys Ser Phe Asn Val Thr Thr Ser Ile Asp Lys Met Gln Lys Glu

Tyr Ala Leu Phe Tyr Lys Leu Asp Val Pro Ile Asp Asn Asp Asn Asn Asn Thr Asn Asn Asn Thr Ser Tyr 2eu Ile Asn Cys Asn Thr Ser Val Ile Thr Gln Ala Cys Pro Lys 222r Phe Glu Pro Ile Pro Ile His Tyr Cys Thr Pro Ala Gly Phe225 234e Leu Lys Cys Asn Asp Lys Lys Phe Asn Gly Thr Gly Pro Cys 245 25r Asn Val Ser Thr Val Gln Cys Thr His Gly Ile Arg Pro Val Val 267r Gln Leu Leu Leu Asn Gly Ser Leu Ala Glu Glu Glu Val Val 275 28e Arg Ser Glu Asn Phe Thr Asp Asn Ala Lys Thr Ile Ile Val Gln 29sn Glu Ser Val Glu Ile Asn Cys Thr Arg Pro Asn Asn Asn Thr33rg Lys Ser Ile Pro Ile Gly Pro Gly Arg Ala Leu Tyr Ala Thr Gly 325 33s Ile Ile Gly Asp Ile Arg Gln Ala His Cys Asn Leu Ser Arg Ala 345p Asn Asn Thr Leu Lys Gln Ile Val Thr Lys Leu Arg Glu Gln 355 36e Gly Asn Asn Lys Thr Thr Ile Val Phe Asn Gln Ser Ser Gly Gly 378o Glu Ile Val Met His Ser Phe Asn Cys Gly Gly Glu Phe Phe385 39ys Asn Ser Thr Gln Leu Phe Asn Ser Thr Trp His Phe Asn Gly 44rp Gly Asn Asn Asn Thr Glu Arg Ser Asn Asn Ala Ala Asp Asp 423p Thr Ile Thr Leu Pro Cys Arg Ile Lys Gln Ile Ile Asn Met 435 44p Gln Glu Val Gly Lys Ala Met Tyr Ala Pro Pro Ile Ser Gly Gln 456g Cys Ser Ser Asn Ile Thr Gly Leu Leu Leu Thr Arg Asp Gly465 478n Asn Glu Asn Thr Asn Asn Thr Asp Thr Glu Ile Phe Arg Pro 485 49y Gly Gly Asp Met Arg Asp Asn Trp Arg Ser Glu Leu Tyr Lys Tyr 55al Val Lys Ile Glu Pro Leu Gly Val Ala Pro Thr Lys Ala Lys 5525Arg Arg Val Val Gln Arg Glu Lys Arg Ala Val Gly Met Leu Gly Ala 534e Leu Gly Phe Leu Gly Ala Ala Gly Ser Thr Met Gly Ala Ala545 556t Thr Leu Thr Val Gln Ala Arg Gln Leu Leu Ser Gly Ile Val 565 57n Gln Gln Asn Asn Leu Leu Arg Ala Ile Glu Ala Gln Gln His Leu 589n Leu Thr Val Trp Gly Ile Lys Gln Leu Gln Ala Arg Val Leu 595 6la Val Glu Arg Tyr Leu Lys Asp Gln Gln Leu Leu Gly Ile Trp Gly 662r Gly Lys Leu Ile Cys Thr Thr Ala Val Pro Trp Asn Ala Ser625 634r Asn Lys Ser Leu Asp Lys Ile Trp Asn Asn Met Thr Trp Met 645 65u Trp Glu Arg Glu Ile Asp Asn Tyr Thr Gly Leu Ile Tyr Thr Leu 667u Glu Ser Gln Asn Gln Gln Glu Lys Asn Glu Gln Glu Leu Leu 675 68u Leu Asp Lys Trp Ala Ser Leu Trp Asn Trp Phe Asp Ile Thr Asn 69eu Trp Tyr Ile Lys Ile Phe Ile Met Ile Val Gly Gly Leu Val77ly Leu Arg Ile Val Phe Ala Val Leu Ser Ile Val Asn Arg Val Arg 725 73n Gly Tyr Ser Pro Leu Ser Phe Gln Thr Arg Leu Pro Ala Pro Arg 745o Asp Arg Pro Glu Gly Ile Glu Glu Glu Gly Gly Glu Arg Asp 755 76g Asp Arg Ser Gly Arg Leu Val Asn Gly Phe Leu Ala Leu Ile Trp 778p Leu Arg Ser Leu Cys Leu Phe Ser Tyr His Arg Leu Arg Asp785 79eu Leu Ile Val Ala Arg Ile Val Glu Leu Leu Gly Arg Arg Gly 88lu Ala Leu Lys Tyr Trp Trp Asn Leu Leu Gln Tyr Trp Ser Gln 823u Lys Asn Ser Ala Val Ser Leu Leu Asn Ala Thr Ala Ile Ala 835 84l Ala Glu Gly Thr Asp Arg Val Ile Glu Val Val Gln Arg Ala Cys 856a Ile Leu His Ile Pro Arg Arg Ile Arg Gln Gly Leu Glu Arg865 878u LeuAArtificial sequenceFirst round primer UP-3 tgcaga tgtgaagagg tacac 25AArtificial sequenceFirst round primer PEXTM6 ctggta tgctcatagc aa 22AArtificial sequenceSecond round primers PEXTM7 ctgcag caacagcaac agctg 25AArtificial sequenceSecond round primer UP-5 agcttc tctggttggc agtg 24

* * * * *

Current U.S. Class:	536/23.1 ; 435/252.3; 435/320.1
Current International Class:	C07H 21/02 (20060101); C12N 1/20 (20060101); C12N 15/00 (20060101)