CD36 is present in the VCC and is required for efficient HIV-1 release by primary macrophages. (A) Confocal sections of macrophages infected with HIV-1 NLAD8 for 7 d and stained for the indicated markers. Merge images include DAPI staining in blue. Bars, 10 µm. (B) Quantification of the codistribution of Gag with the receptors imaged in A. For each cell, mean Pearson’s coefficient was calculated and for each group (n > 15), data are presented as geometric mean with 95% C.I. (C) Immuno-EM of HIV-1–infected macrophages. Ultrathin cryosections were double labeled for p17 Gag with PAG 10 (Protein A coupled to gold particles of 10 nm diameter) and for CD36 with PAG15. The arrow indicates a viral particle stained with both anti-CD36 and anti-p17 Gag antibodies. (D) Schematic representation of the experimental design. (E) Measurement of p24 Gag released in the supernatant of macrophages that had been infected with HIV-1 and transfected with the indicated siRNA. Washes were performed at day 3. Supernatants were collected 24 h after and analyzed by ELISA for their p24 Gag content. Data are presented as mean ± SEM of four replicates. One-way ANOVA with Tukey’s Multiple Comparison Test was used as statistic test (**, P ≤ 0.01; ***, P ≤ 0.001). (F) Cell viability determined at the end of the experiments using CellTiter-Glo is expressed in arbitrary units of luminescence and presented as mean ± SEM of four replicates. (G) Silencing efficiency was estimated by flow cytometry analysis of the various cell populations that were fixed and stained for CD36. MFI is expressed as percentage of the control (cells transfected with siRNA specific for luciferase). Images are representative of at least two independent experiments. Experiments depicted in E and F have been repeated at least three times with different donors, and twice for the one in G.