Structural localization of interface mutations.

<p><b>A</b>) Two CA chains are shown as gray/black and green ribbons. The interface residues evaluated in this study are highlighted in bold and red-orange-yellow color. Other previously studied residues <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0066065#pone.0066065-vonSchwedler1" target="_blank">[7]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0066065#pone.0066065-Bartonova1" target="_blank">[8]</a> are highlighted in cyan-blue-purple color. The fitness impact of mutations is represented by the color shade ranging from small (yellow/cyan), moderate (orange/blue) to lethal (red/purple). <b>B</b>) Prototype residues participating in inter-domain helix capping hydrogen bonds, represented by orange lines, are shown on left. The mutations that resulted in loss of hydrogen bonds are modeled and highlighted on the right.</p

Fitness Costs of Mutations at the HIV-1 Capsid Hexamerization Interface

<div><p>The recently available x-ray crystal structure of HIV-1 capsid hexamers has provided insight into the molecular interactions crucial for the virus’s mature capsid formation. Amino acid changes at these interaction points are likely to have a strong impact on capsid functionality and, hence, viral infectivity and replication fitness. To test this hypothesis, we introduced the most frequently observed single amino acid substitution at 30 sites: 12 at the capsid hexamerization interface and 18 at non-interface sites. Mutations at the interface sites were more likely to be lethal (Fisher’s exact test p = 0.027) and had greater negative impact on viral replication fitness (Wilcoxon rank sum test p = 0.040). Among the interface mutations studied, those located in the cluster of hydrophobic contacts at NTD-NTD interface and those that disrupted NTD-CTD inter-domain helix capping hydrogen bonds were the most detrimental, indicating that these interactions are particularly important for maintaining capsid structure and/or function. These functionally constrained sites provide potential targets for novel HIV drug development and vaccine immunogen design.</p></div

Relationship between sequence conservation and replication fitness.

<p>Relative fitness of all mutants evaluated as a function of database frequency of the amino acid found in the prototype COTM-CA sequence. Values shown are an average from two experiments, done in triplicate. The replication fitness of non-viable viruse is plotted as zero.</p

Viral replication fitness and growth kinetics of COTM-CA mutants in pairwise competition assays.

<p><b>A</b>) Relative fitness of the 21 viable mutants in CEMx174 cells. Values shown are an average from two experiments, with three replicates each. Error bars represent 95% confidence intervals. The dotted line represents neutral fitness. <b>B</b>) Growth kinetics of the five mutants (black lines) with substantial lower fitness compared to the COTM-CA prototype virus (gray lines). <b>C</b>) Growth kinetics of the four mutants with higher replication fitness than the prototype. Values shown are the average from one selected experiment done in triplicate. Error bar represents the standard deviation.</p

Viability and replication fitness of interface and non-interface mutants.

<p><b>A</b>) Fraction of viable and non-viable mutants in each group. <b>B</b>) Relative fitness of viable mutants at interface and non-interface sites. Values shown are an average from two experiments done in triplicate.</p

Hubness across HIV-1 Subtype C Gag.

<p>The number of co-varying partners and the Shannon Entropy are represented for each site along the Gag protein. The blue (lower) part of the bars represent the number of AA-to-AA associations and the red (upper) part of the bars represent the number of HLA-to-AA associations at each site. The secondary axis refers to the Shannon Entropy at each site in Gag (continuous black line).</p

Fitness competition assays between viruses mutated at residues in the sub-network associated with the HLA-B*81 epitope TPQDLNTML.

<p>The relative fitness of viruses presenting a mutation at site 177, 186 or at both sites is compared to that of the wt COT virus. Fitness competition assays were performed against a wt COT virus; the proportion of viral RNA from the mutant and wt viruses was measured at day zero, three and five (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0012463#s4" target="_blank">methods</a>).</p

Relationship between CTL targeting and viremia.

<p>Protein-specific protective ratios were plotted as a function of the mean entropy of each HIV-1 protein. Protective ratios were calculated as the Log₁₀ of the viral load of all the individuals who did not mount a CTL response against a protein over the viral load of all the individuals who had one or more CTL response(s) directed against that protein.</p

Amino Acid associations in HIV-1 subtype C Gag.

<p>Associations are depicted with a circular map: AA interactions among residues are represented with arcs, which are color-coded with a white to purple gradient – white corresponding to the strongest associations (i.e., lower q-values). HLA-restricted sites are identified by the HLA allele designations around the circle.</p

Relationship between viral loads and co-varying associations linking conserved sites.

<p>Shown are associations that involved an HLA-associated site (in bold) or at which a mutation had a significant impact on viral loads.</p>a<p>Number of individuals.</p>b<p>Consensus AA at both co-varying sites.</p>c<p>Rare residues at both co-varying sites.</p>d<p>Consensus AA at one site and a rare AA at the other co-varying site.</p