Non-parametric geographic subdivision tests comparing pre- ASCT viruses with those at each rebound.

<p>Non-parametric geographic subdivision tests comparing pre- ASCT viruses with those at each rebound.</p

Inferred neighbour-joining phylogenetic tree of single genome derived HIV-1 env sequences.

<p>Two APOBEC hypermutated sequences found in the third rebound have been removed. Pre melphalan HIV DNA (-27 to -38, orange open diamonds), first rebound HIV RNA (+6, blue closed diamonds), first rebound HIV DNA (+6, blue open diamonds), second rebound HIV RNA (+515, magenta closed diamonds), third rebound HIV DNA (+643 to +958, green open diamonds). Rooted on MJ4 (black square), branches with bootstrap support > 75% are indicated by black asterisk. Overall population APD is 1.5%. The scale represents 0.005 nucleotide substitutions per site, equivalent to 4.5nt. The tips in the boxed grey area representative sequences predicted to confer CXCR4 tropism by both Geno2Pheno and Phenoseq genotypic algorithms.</p

Genotypically defined co-receptor usage following HIV rebounds.

<p>Tropism testing of the HIV V3 loop was performed in both Geno2Pheno and Phenoseq online genotypic algorithms. Percentage of CXCR4 sequences is plotted on the Y-axis. Only sequences found to be X4 in both programs were designated as X4. Significance was determined using a two-tailed Fishers Exact test.</p

Diversity measured as average pairwise distance at longitudinal time points.

<p>APD measured pre- ASCT and during subsequent viral rebounds. Numbers in parentheses indicate the number of single genomes acquired at each time point. Significance was determined by two-tailed.</p

Determination of T cell responses by ELISpot.

<p>(A) Frequencies of the mutations in the TW10 epitope at different time points (days post Fiebig stage I/II). The viral sequences obtained by SGA were compared to the T/F virus and the subtype B consensus sequence (ConB). Amino acid substitutions at positions 242, 247 and 248 are highlighted in red. (B) T cell response to the wild type and mutant TW10 peptides at day 102 in subject CH77 were determined using an <i>ex vivo</i> IFN-γ ELISpot assay. Peptides containing T242N, V247I or G248A mutation alone as well as in various combinations were analyzed. ELISpot data are expressed as the mean spot forming units (SFU) per million PBMC (SFU/10⁶PBMC)±SEM. Positive T cell responses were defined as: ≥30 SFU/million and >4 times above background (indicated by the dotted line). All assays were performed in triplicate.</p

Impact of the V247I or G248A mutation alone on the fitness of their cognate T/F virus.

<p>The fitness impact of the V247I or G248A mutation alone was determined by comparing the mutant V247I (A and B) or G248A (C and D) to their cognate T/F virus in the single-passage assay (A and C) and the multiple-passage assay (B and D). The percentage of each virus in the inoculum stock and the culture supernatant was determined by PASS. Mean ± standard deviations are shown.</p

No fitness costs of the early reversion mutation I64T in Tat/Rev overlapping region.

<p>(A) Frequencies of the I64T mutation in the Tat protein at different time points (days post Fiebig stage I/II). The viral sequences obtained by SGA were compared to the T/F virus and the subtype B consensus sequence (ConB). Amino acid substitutions at the I64T mutation site were highlighted in red. Relative fitness of the I64T mutant was determined by comparing to the cognate T/F virus in the single-passage assay (B) and the multiple-passage assay (C). The percentage of each virus in the inoculum stock and the culture supernatant was determined by PASS. Mean ± standard deviations are shown.</p

Structural modeling of mutations in the TW10 epitope in p24 Gag.

<p>(A) Homology models of the p24 monomer for the sequences of the T/F virus (cyan), the T242N mutant (magenta) and the NIA mutant (green) show similar structures of the helix 6 region with modest structural differences in the neighboring N-terminal hairpin and CypA binding loop. Side chains at mutation positions are shown as stick representation. (B) Mutations at position 242 (red) and positions 247/248 (green) in helix 6 were mapped to the hexameric p24 crystal structure <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0102734#pone.0102734-Pornillos1" target="_blank">[49]</a>. The mutation positions did not occur at p24 subunit interfaces. Helix 6 is located between the N-terminal hairpin (magenta) and cyclophilin-binding loop (orange) on the surface of the hexamer. (C) Mutation at position 242 (red) and positions 247/248 (green) in helix 6 (yellow) were mapped to a hexamer of hexamers in the capsid assembly <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0102734#pone.0102734-Zhao1" target="_blank">[40]</a>. The T242N (red) and V247I/G248A (magenta) mutations face outward in the assembly and do not occur at hexamer-hexamer interfaces.</p

No fitness costs of the early CD8⁺ T cell escape mutation R355K in Env.

<p>(A) The frequency of the R355K mutation in the Env_352–360T cell epitope different time points (days post Fiebig stage I/II). The viral sequences obtained by SGA were compared to the T/F virus. Amino acid substitutions at the R355K mutation site were highlighted in red. Relative fitness of the R355K mutant was determined by comparing to the cognate T/F virus in the single-passage assay (B) and the multiple-passage assay (C) as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0102734#pone-0102734-g003" target="_blank">Figure 3</a>. The percentage of each virus in the inoculum stock and the culture supernatant was determined by PASS. Mean ± standard deviations are shown.</p

Partial restoration of the fitness loss of the T242N mutant by compensatory mutations.

<p>The relative fitness was determined between the T242N mutant and the NI (A and B) and NA (C and D) mutant as well as between the T/F virus and NI (E and F) or NA (G and H) mutant in the single-passage assay (left panels) and the multiple-passage assay (right panels). Same amount (5 ng p24) of each compared virus was mixed to infect 10⁶ of purified CD4⁺ T cells in triplicates. In the single-passage assay, the viruses were cultured for three days and the culture supernatant were harvested daily by completely replacing the medium. In the multiple-passage assay, 200 µl of cell-free virus harvested at day 3 at each passage was used to infect 10⁶ of fresh CD4⁺ T cells. The viruses were subsequently passaged three additional times. The percentage of each virus in the inoculum stock and the culture supernatant was determined by PASS. Mean ± standard deviations are shown. The relative fitness was determined by modeling the replication slope of each virus in the single- and multiple-passage assays as previously described <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0102734#pone.0102734-Song1" target="_blank">[25]</a>.</p