Lower replication of C-Env recombinants in CD4+ T-cells is not associated with lower viral gene expression.

<p><b>A and B. Western Blot analyses of intracellular Env and Gag in CD4+ T-cells exposed to subtype B and subtype C recombinant virus pairs at day 5 post infection (A) and 40 hours post infection (B).</b> CD4+ T-cells (10⁵ cells/well) infected in triplicate wells for 5 days (A) or for 40 hours (B) were washed, pooled and lysed in 75μl of reducing Lämmli buffer. Polybrene was added at the time of infection of C-EnvEC and C-Env virions for WB detection in 40-hour lysates. Proteins were resolved by SDS-PAGE and immunoblotted with a rabbit polyclonal antibody recognizing the immature p55Gag immature precursor and the mature p17 and p24 proteins, and a goat polyclonal antibody against gp120. β-actin was immunoblotted to control for loading. The corresponding supernatant (30 μl) were immunoblotted using a mouse monoclonal antibody against p24. One representative experiment of three is shown. Samples are arranged by subtype to ease visual comparison. C and B Env samples were also run on the same gel, and similar subtype and gp41CT-related differences were observed (not shown).</p

Infectivity of C-Env virions, Env incorporation in virions produced by CD4+ T-cells and cell-to-cell transmission are lessened.

<p><b>(A and B) Infectivity of virions produced by HEK 293T cells (A) and CD4+ T-cells (B) in a single round TZM-bl assay.</b> TZM-bl cells (2x10⁴ cells/well) were infected with 2 ng/ml equivalent p24 of Env or EnvEC recombinant viruses produced from HEK 293T cells <b>(A)</b> or from CD4+ T-cells 5 days post infection <b>(B)</b>. Viral entry was monitored in cell lysates 48h post-infection. Infections were performed in triplicated wells. The means of three independent experiments with HEK293T cells and of 4 independent infections with CD4+ T cells from 4 different donors are shown. Error bars report standard deviation <b>C</b>. <b>Env incorporated in virions produced by CD4+ T-cells.</b> P24 and gp120 in day 5 supernatants of CD4+ T-cells were quantified by Western Blot. One representative experiment of two is shown. <b>D and E. Cell-to-cell transmission.</b> CD4+ T-cells were infected as above with 1 ng/ml equivalent p24. After 48 hours, cell were cocultured with TZM-bl cells in the presence of IDV to avoid any contribution of free virus. The next morning, MVC and AMD3100 were added to ensure single-round infection. Luciferase in TZM-bl cells was measured after 48 hours. <b>(D)</b> The mean and standard deviation of four independent experiments performed with CD4+ T-cells from four different donors are reported. <b>(E)</b> The same data is shown for each individual virus pair.</p

Concordance between tropism measured phenotypically and inferred genotypically using the Geno2pheno_(coreceptor) and webPSSM algorithms.

<p>(<b>A</b>) Concordance for subtype B (black bars) and non-B subtype (grey bars) strains with Geno2Pheno (G2P) at different FPR cutoffs and webPSSM. (<b>B</b>) Concordance with Geno2pheno_(coreceptor) with a FPR set at 10% (black bars) and webPSSM (grey bars) for different HIV-1 subtypes. The webPSSM X4/R5 matrix was used for all subtypes, except for subtype C, for which the subtype C SI/NSI matrix was used.</p

Characteristics of phenotypic assays developed for determination of HIV-1 coreceptor usage.

<p>Abbreviations: ESTA: Enhanced Sensitivity Trofile Assay; Env: Envelope; eGFP: enhanced Green Fluorescent protein; X4: CXCX4-using strains; R5:CCR5-using strains.</p

Viruses with subtype C gp41CT have lower replication than viruses with subtype B gp41CT in CD4+ T-cells but not in MDMs.

<p><b>A and B. Replication of subtype B and subtype C recombinants in CD4+ T-cells from two different donors.</b> 10⁵ CD4+ T-cells were infected with equivalent amounts (1 ng/ml) of Env or EnvEC recombinant viruses. Infection was monitored by measuring p24 in culture supernatants at days 2 or 3, 4 or 5, 7 and 9 or 10. Examples of replication in CD4+ T cells of two different donors are shown after infection with subtype B (left panels) and subtype C (right panels) Env and EnvEC recombinant viruses. All infections were performed in triplicate wells. <b>(C and D) P24 in supernatants of 10</b>^<b>5</b> <b>CD4+ T-cells 5 days post-infection.</b> The same results at day 5 post-infection are presented and statistically significant means (paired t-test for viral pairs, unpaired t-test for inter-subtype comparisons) are indicated (<b>C</b>). In panel (<b>D</b>), the results are shown per pair. The ratio of p24 released in supernatants of cells exposed to Env recombinant viruses divided by p24 in the supernatants of cells exposed to the corresponding EnvEC recombinant virus is reported. A positive ratio indicates that the full Env recombinant replicates better than its EnvEC counterpart containing the gp41CT of NL4.3. A negative ratio implies the EnvEC recombinant virus replicates better than the Env recombinant. Because of inter-donor variability, results are presented as individual experiments performed with CD4+ T-cells from independent donors. <b>E. Replication of subtype B and subtype C recombinants in MDMs.</b> 10⁵ MDMs were infected with equivalent amounts (3 ng/ml) of subtype B (left panel) and subtype C (right panel) Env and EnvEC recombinant viruses. Infection was monitored by measuring p24 in culture supernatants at days 3, 7, 10 and 14. All infections were performed in triplicate or quadruplicate wells. <b>(F and G) p24 in supernatants of 3x10</b>^<b>5</b> <b>MDMs 7 days post-infection.</b> All infections were performed in triplicate or quadruplicate wells. Standard deviation is indicated. In panel (<b>G</b>), the same results as in panel (F) are represented for each individual strain. The ratio of p24 released in supernatants of cells exposed to Env recombinant viruses divided by p24 in the supernatants of cells exposed to the corresponding EnvEC recombinant virus is reported. Tropism is indicated as follows: R5: blue circle; X4: red circle; R5X4: purple circle.</p

Subtype C MA decreases viral production, only partially rescues viral replication in CD4+ T-cells but does not restore infectivity.

<p><b>A and B. Production of viruses containing subtype C MA.</b> The <i>matrix</i> sequences of two subtype C strains with intermediate replication capacity (MA₁₆₇₁) and with poor replication capacity (MA₀₂₆₆) were cloned into the EnvEC and Env backbones. Recombinant viruses with subtype C MAs were generated by transfecting HEK 293T cells with these backbones and the corresponding subtype C EnvEC and Env amplicons from patient samples. p24 produced was measured by ELISA 48 hours post transfection. At least three independent productions with MA_NL4.3 and two with MA₁₆₇₁ and MA₀₂₆₆ are averaged and error bars show standard deviation. Results are shown for all viruses as groups (A) and per pairs (B). <b>C and D. Replication of C-EnvEC and C-Env recombinant viruses containing subtype C MA</b>_<b>1671</b> <b>in CD4+ T-cells.</b> CD4+ T-cells were infected with C-EnvEC and C-Env recombinants containing the MA of NL4.3 or of strain 1671, and p24 was measured in the supernatants 5 days post-infection. The average and standard deviation of four independent experiments performed with CD4+ T-cells from four different donors are shown. Results are shown for all viruses as groups (C) and per pairs (D) In (D), relative infectivity with respect to the corresponding C-EnvEC value is reported. <b>E and F. Infectivity of virions produced by CD4+ T-cells in a TZM-bl single round assay.</b> TZM-bl cells were infected with supernatants from infected CD4+ T-cells collected 5 days after infection and adjusted to equivalent p24 contents (2 ng/well). Infection was monitored 48h post-infection as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0161596#pone.0161596.g003" target="_blank">Fig 3</a>. The mean of two independent experiments performed with CD4+ T-cell supernatants from two independent donors are shown and error bars report standard deviation. <b>G and H. Cell-to-cell transmission of viruses with subtype C MA</b>_<b>1671.</b> CD4+ T-cells were infected with C-EnvEC, C-Env or C-EnvMA₁₆₇₁ viral particles for 40 hours, extensively washed, then co-cultured with TZM-bl cells for a further 48 hours in the presence of IDV. MVC and AMD3100 were added the next morning. The mean of two independent experiments is shown. Error bars represent standard deviation. Panel <b>G</b> shows the same data as panel <b>F</b> detailed for each individual pair.</p

Study design/RVA design.

<p>Viral RNA was extracted from patient plasma RT-PCR amplified. Env amplicons spanning the Env ectodomain were further amplified through an inner PCR. Five independent PCRs were pooled to minimize PCR-selection. Recombinant viruses were produced by co-transfecting HEK293T cells with Afe I-linearized, luciferase-tagged, Env-deleted, viral backbone and patient-derived PCR amplicon. Normalized amounts of recombinant viruses were used to infect U87.CD4.CCR5 or U87.CD4.CXCR4 indicator cells. Infection was monitored by quantifying luminescence in the cell lysates. Depending on the outcome of the infection, viruses were classified as either CCR5 tropic, CXCR4 tropic or dual/mixed. The same patient-derived PCR amplicon used for viral production was sequenced and tropism inferred by Geno2Pheno_[coreceptor] and webPSSM algorithms. The phenotypic and genotypic results were compared. Abbreviations: Env EC: Env ectodomain; gp41-TM-CT: gp41 Transmembrane+cytoplasmic tail.</p

Detection of minority CXCR4 and CCR5 using variants within mixed viral populations.

<p>Mixtures containing known proportions of pNLAD8 and pNL4-3 (100∶0, i.e. pure NLAD8, 99∶1, 97.5∶2.5, 95∶5, 90∶10, 80∶20, 50∶50, 20∶80, 10∶90, 5∶95, 2.5∶97.5, 1∶99, 0∶100, i.e. pure NL4-3) were PCR-amplified and used to generate recombinant viruses. U87.CD4.CCR5 and U87.CD4.CXCR4 indicator cells were infected with serial 2-fold dilutions (x-axis) of mixtures (z-axis) to determine the threshold for detection of minority variants. Infection was quantified 48 hours after infection by measuring luciferase activity in cell lysates (y-axis). Black bars report infection of U87.CD4.CXCR4 cells and grey bars report infection of U87.CD4.CCR5 cells. Panels A and B report the same data, oriented to focus on NL4-3 minority variants (A) or on NLAD8 minority variants (B).</p

The gp41CT of subtype C Envs does not tether virus to CD4+ T-cells.

<p>CD4+ T-cells (10⁵ cells/well) were infected with Env or EnvEC recombinant virus pairs for 5 days, treated with subtilisin or with buffer alone, and virus in each fraction was measured by p24 ELISA. The amount of p24 that released upon subtilisin-treatment was reported to total p24 (i.e. released in the supernatant+virus attached to the PM). Three experiments with CD4+ T-cells from different donors are shown (average). Error bars represent standard deviation.</p

Distribution of PCR amplification success stratified by viral load.

<p>The Env ectodomain was amplified from plasma viral RNA by a one-step RT-PCR followed by an inner PCR. Five independent PCR amplifications were pooled to minimize primer-related selection. 292 samples from patients infected with HIV subtypes A1, B, C, D, F, G, CRF01_AE and CRF02_AG were included. Viral load ranged from 466 to 1,350,000 RNA copies/mL.</p