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 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

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

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

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

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

DataSheet_1_Defining genetic diversity of rhesus macaque Fcγ receptors with long-read RNA sequencing.pdf

Fcγ receptors (FcγRs) are membrane-bound glycoproteins that bind to the fragment crystallizable (Fc) constant regions of IgG antibodies. Interactions between IgG immune complexes and FcγRs can initiate signal transduction that mediates important components of the immune response including activation of immune cells for clearance of opsonized pathogens or infected host cells. In humans, many studies have identified associations between FcγR gene polymorphisms and risk of infection, or progression of disease, suggesting a gene-level impact on FcγR-dependent immune responses. Rhesus macaques are an important translational model for most human health interventions, yet little is known about the breadth of rhesus macaque FcγR genetic diversity. This lack of knowledge prevents evaluation of the impact of FcγR polymorphisms on outcomes of preclinical studies performed in rhesus macaques. In this study we used long-read RNA sequencing to define the genetic diversity of FcγRs in 206 Indian-origin Rhesus macaques, Macaca mulatta. We describe the frequency of single nucleotide polymorphisms, insertions, deletions, frame-shift mutations, and isoforms. We also index the identified diversity using predicted and known rhesus macaque FcγR and Fc-FcγR structures. Future studies that define the functional significance of this genetic diversity will facilitate a better understanding of the correlation between human and macaque FcγR biology that is needed for effective translation of studies with antibody-mediated outcomes performed in rhesus macaques.</p

Sequence and binding characteristics of HIV-1 Env gp41-reactive <i>IGHV</i>1-69 B-CLL mAbs.

<p>(A) The gp41-reactive <i>IGHV</i>1-69 B-CLL mAbs were unmutated and preferentially used <i>IGHD</i>3-3 and <i>IGHJ</i>6 gene segments. The aa sequences of the HCDR3 regions of 5 gp41-reactive <i>IGHV</i>1-69 B-CLL IgMs were aligned to that of CLL526 IgM. Each sequence was aligned independently to CLL526 (pairwise alignment) using ClustalW and final adjustment was made manually. Gaps are indicated as dashes. The aa conserved between the sequences of CLL526 and the other IgMs are highlighted in red. The number of aa shared with CLL526 over the total aa is reported on the right for each IgM. The CLL1296 IgM was used as a negative control. (B) Binding characteristics of the B-CLL mAbs expressed as recombinant IgG₁ with HIV-1, HCV, and intestinal commensal bacterial antigens. Serial dilutions ranging from 100 µg/ml to 0.004 µg/ml of each IgG were tested in ELISA for binding to ADA AT-2-inactivated virion, MN gp41, HIV-1 BAL gp41 immunodominant region peptide SP400 (RVLAVERYLRDQQLLGIWGCSGKLICTTAVPWNASWSNKSLNKI), and HCV E2, or in Luminex assay for aerobic and anaerobic intestinal commensal bacterial whole-cell lysates. Data are expressed in OD for ELISA or mean fluorescence intensity (MFI) for Luminex assay. The dotted lines indicate the cut-off value ≥100 MFI used to denote positivity. Data are representative of at least two separate experiments.</p

HCDR3 alignment of CLL1324 to gp41-reactive <i>IGHV</i>1-69 antibodies isolated from HIV-1-infected patients.

<p>The aa sequences of the HCDR3 regions of gp41-reactive <i>IGHV</i>1-69 antibodies isolated from HIV-1-infected patients were aligned to that of CLL1324. Each sequence was aligned independently to CLL1324 (pairwise alignment) using ClustalW and final adjustment was made manually. Gaps are indicated as dashes. The aa conserved between the sequences of CLL1324 and the other antibodies are highlighted in red. The number of aa shared with CLL1324 over the total aa is reported on the right for each antibody. Only the gp41 antibody sequences with HCDR3 % similarity ≥50% are reported. The CLL1296 IgM was used as a negative control. ¹Previously published sequence <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0090725#pone.0090725-Morris1" target="_blank">[51]</a>; ²CLL1296, HIV-1-negative control mAb; ³<i>IGHV</i>1-69 antibodies with an F₅₄ allelic variant. D RF, D gene reading frame; AA₅₄, aa in position 54.</p

B-CLL cases with anti-viral reactivity correlate with poor clinical outcomes.

<p>The Kaplan-Meier plots are shown for the time to first treatment (TFT, in months) in all samples (A) and <i>IGHV</i>1-69 samples (B). The p values for Mantel-Cox test in groups A and B are 0.011 and 0.217, respectively. The Kaplan-Meier plots are shown for overall patient survival (in months) in all samples (C) and <i>IGHV</i>1-69 samples (D). The p values for Mantel-Cox test in groups C and D are <0.0001 and 0.012, respectively. Virus+ group represents B-CLL samples with ≥10 wells out of 20 wells tested showing a specific anti-viral reactivity (<b><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0090725#pone.0090725.s001" target="_blank">Figure S1</a></b>). The results for virus-binding activity of 2 B-CLL samples (CLL821 and CLL1296) were obtained from the purified IgM paraproteins (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0090725#pone-0090725-g001" target="_blank"><b>Figure 1</b></a>).</p