Reconstructed ancestral sequences at the root of the inferred Gag and Nef phylogenies, representing the estimated most recent common ancestor (MRCA) of the North American epidemic.

<p>A minimum of ≥50,000 reconstructions of the ancestral sequence at the root of the Gag and Nef phylogenies were performed, and the inferred MRCA was computed as the “grand consensus” of these replicate reconstructions. For each codon, reconstruction confidence (computed as the frequency of each amino acid observed across all reconstructions) is indicated on the y-axis on a scale from 0 (0%) to 1 (100%). Blue letters represent the highest-confidence residue at each position; green letters represent lower-confidence residues. All amino acids observed at >0.01 (>1%) reconstruction frequency are shown. Yellow boxes highlight positions where the highest-confidence (blue) inferred ancestral residue differs from the North American consensus B sequence (displayed in <b><a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004295#pgen.1004295.s003" target="_blank">Figure S3</a></b>).</p

Replicative implications of Gag diversification during the North American Epidemic.

<p><b>Panel A:</b> Unrooted Maximum-Likelihood phylogenies, drawn on the same distance scale, depicting the inferred ancestor (single black dot), early-historic (red, 1979–1982), mid-historic (green, 1983–1985), late-historic (blue, 1986–1989) and modern (purple, 2000+) Gag clonal sequences from unique patients that were used to construct recombinant NL4-3 viruses for functional assessment. <b>Panel B:</b> NL4-3 normalized replication capacities of recombinant viruses containing the Gag sequence of the inferred ancestral sequence (Mean±S.E.M. of 3 replicate measurements) as well as patient-derived Gag clonal sequences (one per patient, representing the mean of ≥2 replicate measurements). An RC of 1 indicates replication equal to that of NL4-3 while RC>1 and <1 indicate RC higher or lower than NL4-3 respectively. Although visually there appears a trend towards lower replication capacity among Gag clones from early historic (1979–1982) era, there no significant differences in RC between any of the groups (Kruskal-Wallis test, p = 0.6).</p

Gag residues exhibiting significant diversification over time are biased towards known HLA-associated sites.

<p><b>Panel A:</b> Differences in Shannon entropy (Δentropy) between modern and historic sequences are shown for every Gag codon. Positive y-values indicate higher entropy in modern vs. historic sequences at that codon; negative y-values indicate the opposite. Red bars indicate significant entropy differences (defined as p<0.001, q<0.01); blue colors indicate differences that do not reach this significance threshold. Grey dots designate known HIV sites under selection by HLA (as defined in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004295#pgen.1004295-Carlson1" target="_blank">[43]</a>). Green dots designate sites that display significant evidence of pervasive positive selection (dN/dS>1; posterior probability >0.9). <b>Panel B:</b> Same as panel A, but sorted by decreasing Δentropy rather than codon order. <b>Panel C:</b> Graphical depiction of a 2×2 contingency table stratifying variable (<99% conserved) Gag codons based on their status as HLA-associated (yes vs. no), and whether they exhibited significant Δentropy between modern and historic datasets (p<0.001 [red] vs. not [blue]). Ns are indicated above each bar. <b>Panel D:</b> Graphical depiction of a 2×2 contingency table stratifying variable (<99% conserved) Gag codons based on their status as HLA-associated (yes vs. no) and evidence that they are under significant pervasive positive selection (dN/dS>1; posterior probability >0.9 [green] vs. not [black]). Ns are indicated above each bar.</p

Diversity of North American Gag and Nef sequences from historic (1979–1989) and modern (2000+) eras.

<p>Unrooted Maximum likelihood phylogenetic trees, drawn on the same distance scale, are shown for historic Gag (upper left), historic Nef (lower left), modern Gag (upper right) and modern Nef (lower right). Phylogenies are star-like, with Nef exhibiting greater diversity than Gag, and modern trees exhibiting greater diversity than historic ones. Cohort sequences are colored by sampling era: red (1979–1982), green (1983–1985), blue (1986–1989) and purple (2000+); North American sequences retrieved from the Los Alamos (LANL) database are in grey. Included in each tree is the HIV subtype B reference strain HXB2, shown in black and indicated with an arrow.</p

HLA-associated polymorphisms, identified via statistical association, in historic HIV sequences.

<p><b>Panel A:</b> Gag immune escape map, indicating the locations, specific amino acid residues and HLA restrictions of HLA-associated polymorphisms identified at q≤0.05 in our historic cohort. The HIV consensus B amino acid sequence is used as a reference. Shaded vertical bars separate blocks of 10 amino acids. “Adapted” amino acids (those over-represented in the presence of the HLA allele) are red. “Nonadapted” amino acids (those under-represented in the presence of the HLA allele) are blue. UPPERCASE letters distinguish polymorphisms that survive correction for HIV codon covariation (“direct” associations), while lowercase letters distinguish polymorphisms that do not survive correction for codon covariation (“indirect” associations). The notation “_ST” following an HLA (<i>e.g.</i> B58_ST) identifies associations identified at the supertype level. The locations of optimally-defined, HLA-restricted CTL epitopes straddling or adjacent to HLA-associated polymorphisms are indicated. The well-known A*02-SL9 epitope (SLYNTVATL) epitope is also shown; no historic HLA-associated polymorphisms were identified therein at q<0.05. The single “novel” historic HLA-associated polymorphism (B*49:01-62G) is indicated with a purple asterisk. A green filled circle denotes the single Gag residue (codon 67) where the ancestral founder sequence was reconstructed with <80% confidence. Orange filled circles denote the four Gag residues (67, 76, 91 and 102) where the inferred ancestral founder sequence differs from the published North American subtype B consensus sequence. None of these sites harbor HLA associations. <b>Panel B:</b> Nef historic immune escape map. Green filled circles denote the six Nef residues where the ancestral founder sequence was reconstructed with <80% confidence (15, 21, 51, 152, 178, 205); none harbor HLA associations.</p

Functional implications of Nef diversification during the North American Epidemic.

<p><b>Panel A:</b> Unrooted Maximum-Likelihood phylogenies, drawn on the same distance scale, depicting the inferred ancestor (single black dot), early-historic (red, 1979–1982), mid-historic (green, 1983–1985), late-historic (blue, 1986–1989) and modern (purple: chronic-phase, orange: acute-phase, year 2000+) Nef clonal sequences from unique patients cloned into a GFP-expression vector for functional assessment. <b>Panel B:</b> CD4 downregulation activities of the inferred ancestral Nef sequence (mean±S.E.M. of 8 replicate measurements) and patient-derived Nef clones from various eras (one per patient, representing the mean of triplicate measurements). CD4 downregulation values are normalized to that of HIV subtype B control Nef strain SF2, such that a value of 1 indicates CD4 downregulation activity equal to that of SF2 while values>1 and <1 indicate activities higher or lower than SF2 respectively. Modern Nefs exhibited significantly higher CD4 downregulation activity compared to historic Nefs (Kruskal-Wallis p<0.0001). <b>Panel C:</b> SF2-normalized HLA class I downregulation activities of inferred ancestral (mean±S.E.M. of 8 replicate measurements) and patient-derived Nef sequences (one per patient, mean of triplicate measurements). Modern Nefs exhibited significantly higher HLA downregulation activity compared to historic Nefs (Kruskal-Wallis p<0.0001).</p