Fate of HIV-1 virions in primary DCs and in DC-SIGN⁺ cell lines. (A) Fate of HIV-1 in DCs. Cells were exposed to HIV_NL4-3 for 2 hours, incubated at 37°C for the indicated time points, and cell-associated Gag contents were measured by ELISA. When stated, cells were treated with pronase before p24 measurement in order to remove virions present at the cell surface. Viral release in supernatants is also shown. Left panel: Viral inoculum was 200 ng p24 for 10⁶ cells. Right panel: effect of bafilomycin-A1, an inhibitor of vesicular acidification, on intracellular p24. Bafilomycin-A1 (250 nM) was added 30 minutes before viral exposure and maintained for 4 hours. Viral inoculum was 55 ng p24/10⁶ cells. Data (in ng p24/5 × 10⁵ cells) are mean ± SD of duplicates. (B) Surface DC-SIGN expression levels in primary immature DCs, and in 3 human HLA-A2⁺ B-cell–derived lines (C1RA2, T2, and T3), before (thin curves) and after (thick curves) transduction with a lentiviral vector encoding for DC-SIGN (yielding C1RA2-DCS, T2-DCS, and T3-DCS cells, respectively). Cells were stained with anti–DC-SIGN Abs and analyzed by flow cytometry. An isotypic mAb was used as a negative control (dotted line). (C) Effect of DC-SIGN on viral capture. C1RA2 and C1RA2-DCS cells were exposed to HIV_NLAD8 at 10 ng (low input) or 500 ng (high input) of p24/10⁶ cells for 2 hours at 37°C. Cell-associated p24 contents (in ng p24/2.5 × 10⁵ cells) were then measured. Data are mean ± SD of duplicates. (D) Fate of HIV-1 in C1RA2-DCS cells. Cells were exposed to HIV_NLAD8 for 2 hours, incubated at 37°C for the indicated time points, and intracellular p24 contents (in ng p24/2.5 × 10⁵ cells) were measured by ELISA. Viral inputs were 25 (black circles) or 250 (white circles) ng p24 for 10⁶ cells. Similar results were obtained with HIV_NL43. Data are representative of 3 independent experiments.