Figure 6. The anti-CD36-mediated inhibition of HIV-1 release is specific and requires bivalent binding. (A and B) Exogenous CD81 and ICAM-1–specific antibodies are transported to the VCC. Macrophages were infected with HIV NLAD8 for 3 d, washed, and incubated for 2 h with a CD81-specific mAb (A) or an ICAM-1–specific mAb (B). mAbs were used at a final concentration of 5 µg/ml. Cells were then washed, fixed, permeabilized, and stained for Gag and CD9 to identify VCCs. Bars, 10 µm. Merge images include DAPI staining in blue. (C–F) Quantification of p24 Gag released from macrophages treated overnight with the indicated antibodies. Of note, other antibodies transported to the VCC (C and D), antibodies specific for markers of the VCC (C, D and E), and antibodies specific for other LDLRs (F) did not modulate HIV-1 release from macrophages. (G) Quantification of the p24 Gag produced by HIV-1–infected primary macrophages treated (as indicated in Fig. 5 A) with the FA6-152 antibody in the presence or not of Fc-blocking antibodies. (H) Flow cytometry titration of the anti-CD36 mAb CLB-IVC7 and its Fab fragment. HeLa-CD36 (open symbols) and HeLa cells (black symbols) were stained with serial dilutions of the anti-CD36 mAb and its Fab revealed by appropriate secondary antibodies. MFIs are plotted as a function of the Ab dilutions. (I) Quantification of p24 Gag released from macrophages treated overnight with the anti-CD36 mAb, its Fab fragment, or the isotype control. Cells were exposed to the three dilutions indicated by arrows in H. Data are shown as mean ± SEM of triplicates. In C–I, representative experiments are shown. All the experiments have been reproduced at least two times with different donors. One-way ANOVA with Tukey’s multiple comparison test was used as a statistical test (*, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001).
Figure 6.

The anti-CD36-mediated inhibition of HIV-1 release is specific and requires bivalent binding. (A and B) Exogenous CD81 and ICAM-1–specific antibodies are transported to the VCC. Macrophages were infected with HIV NLAD8 for 3 d, washed, and incubated for 2 h with a CD81-specific mAb (A) or an ICAM-1–specific mAb (B). mAbs were used at a final concentration of 5 µg/ml. Cells were then washed, fixed, permeabilized, and stained for Gag and CD9 to identify VCCs. Bars, 10 µm. Merge images include DAPI staining in blue. (C–F) Quantification of p24 Gag released from macrophages treated overnight with the indicated antibodies. Of note, other antibodies transported to the VCC (C and D), antibodies specific for markers of the VCC (C, D and E), and antibodies specific for other LDLRs (F) did not modulate HIV-1 release from macrophages. (G) Quantification of the p24 Gag produced by HIV-1–infected primary macrophages treated (as indicated in Fig. 5 A) with the FA6-152 antibody in the presence or not of Fc-blocking antibodies. (H) Flow cytometry titration of the anti-CD36 mAb CLB-IVC7 and its Fab fragment. HeLa-CD36 (open symbols) and HeLa cells (black symbols) were stained with serial dilutions of the anti-CD36 mAb and its Fab revealed by appropriate secondary antibodies. MFIs are plotted as a function of the Ab dilutions. (I) Quantification of p24 Gag released from macrophages treated overnight with the anti-CD36 mAb, its Fab fragment, or the isotype control. Cells were exposed to the three dilutions indicated by arrows in H. Data are shown as mean ± SEM of triplicates. In C–I, representative experiments are shown. All the experiments have been reproduced at least two times with different donors. One-way ANOVA with Tukey’s multiple comparison test was used as a statistical test (*, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001).

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