FIG 3 .

Analysis of HIV-1 Env-mediated cell-to-cell viral transfer. (A) An outline of the experimental model used for the analysis of Env-mediated cell-to-cell fusion and viral transfer is shown. Briefly, 293T cells were cotransfected with env and a Δenv HIV-1 expression plasmid. After 24 h, effector cells producing HIV-1 particles were cocultured with primary CD4⁺ T cells to force synapsis formation and massive binding of budding particles to target cells. Env expression and HIV-1 binding were analyzed by flow cytometry using anti-Env and anti-p24 antibodies, respectively. (B) HIV-1 transfer to primary CD4⁺ T cells induced by the CCR5-using Env from BaL.01 or the CXCR4-using Env from NL4.3. Cocultures were performed in the presence of the following inhibitors: 1 μg/ml of the anti-CD4 antibody Leu3A or 10 μg/ml of the CCR5 antagonist TAK779 or the CXCR4 antagonist AMD3100, the fusion inhibitor peptide C34, and the reverse transcriptase inhibitor AZT. The dotted line corresponds to background levels of signal obtained in cocultures of CD4⁺ T cells and 293T cells transfected with the Δenv plasmid PSG3. Data are means ± standard deviations (SD) from two different experiments. (C) Expression of HIV-1 Env in cells untransfected (control [black line]), transfected with the Δenv plasmid (PSG3 [green line]), or cotransfected with this plasmid and the indicated env from NL4.3 (blue line), the D368R mutant (red line), or the 41.2 mutant (purple line). (D) HIV-1 transfer (bound virus) to CD4⁺ T cells assessed by flow cytometry after coculture with 293T cells expressing Env from NL4.3 (black bars), the D368R mutant (gray bars), or the 41.2 mutant (open bars) and CD4⁺ T cells. Cocultures were performed in the presence of inhibitors as in panel B; the anti-gp120 antibody that blocks the CD4 binding site IgGb12 was used at 10 μg/ml. The dotted line corresponds to background levels of signal obtained in cocultures of CD4⁺ T cells and 293T cells transfected with the Δenv plasmid PSG3. Data are means ± SD from three different experiments. (E) Analysis of the ability to induce cell-to-cell virus transfer of HIV-1 Env proteins obtained from cluster LTNP-ECs (dark gray bars) or progressors (light gray bars) or reference strains (SF162, NL4.3, BaL.01, and 89ES_061 [black bars]). Data are from three independent experiments, comparing mean values between groups. (P values compare medians between groups using a nonparametric Mann-Whitney test.)