Figure 7. Exposure to CD36-specific antibodies induces intracellular accumulation of virions in VCCs. (A) Immunoblot analysis of Gag polypeptides present in lysates of infected macrophages exposed to the CD36 mAb (36) or its isotype control (Iso). Bands corresponding to p55 Gag (p55), p24 Gag (p24), and tubulin (Tub) are indicated. The molecular weight corresponding to the closest band of the ladder is indicated. (B–D) Quantifications of the intensity of the various Gag polypeptides present in cell lysates were performed by immunoblot analysis as in A. The experiments were repeated with cells from seven donors. p55 Gag and p24 Gag signals were normalized for α-tubulin. Statistical analyses were performed using the Wilcoxon matched pairs test (*, P ≤ 0.05). (E) Examples of 3D reconstructions obtained before and after segmentation of image stacks acquired by confocal microscopy of macrophages infected with HIV-1 Gag-iGFP for 7 d, and exposed from days 5 to 7 to the anti-CD36 mAb or its isotype control. Bars, 7 µm. Segmented reconstructions, as seen in E, were quantified using Imaris software. Graphs illustrating the variation of the total Gag intensity per cell (F) and the total volume of VCC per cell (G) are presented. Effect of the anti-CD36 on p24 Gag release was estimated in parallel by Elisa in the three donors used for these experiments. The mean inhibition of p24 Gag release was 90%. Data are presented with the median in red. Each square corresponds to a cell. Statistical analyses were performed using the Mann-Whitney test (***, P ≤ 0.001). (H) HIV-1 NLAD8–infected macrophages at 6 d p.i. were washed and treated with anti-CD36 mAbs or with its isotype control for 48 h. Cells were then fixed, embedded in epon, and processed for EM. Bar, 200 nm. (I) HIV-1–infected macrophages treated as in H were prepared for immuno-EM. mAbs present in internal compartments were detected with appropriate secondary antibodies revealed by PAG10. Bars, 200 nm. Representative images are shown in H and I. Data were reproduced with three different donors.
Figure 7.

Exposure to CD36-specific antibodies induces intracellular accumulation of virions in VCCs. (A) Immunoblot analysis of Gag polypeptides present in lysates of infected macrophages exposed to the CD36 mAb (36) or its isotype control (Iso). Bands corresponding to p55 Gag (p55), p24 Gag (p24), and tubulin (Tub) are indicated. The molecular weight corresponding to the closest band of the ladder is indicated. (B–D) Quantifications of the intensity of the various Gag polypeptides present in cell lysates were performed by immunoblot analysis as in A. The experiments were repeated with cells from seven donors. p55 Gag and p24 Gag signals were normalized for α-tubulin. Statistical analyses were performed using the Wilcoxon matched pairs test (*, P ≤ 0.05). (E) Examples of 3D reconstructions obtained before and after segmentation of image stacks acquired by confocal microscopy of macrophages infected with HIV-1 Gag-iGFP for 7 d, and exposed from days 5 to 7 to the anti-CD36 mAb or its isotype control. Bars, 7 µm. Segmented reconstructions, as seen in E, were quantified using Imaris software. Graphs illustrating the variation of the total Gag intensity per cell (F) and the total volume of VCC per cell (G) are presented. Effect of the anti-CD36 on p24 Gag release was estimated in parallel by Elisa in the three donors used for these experiments. The mean inhibition of p24 Gag release was 90%. Data are presented with the median in red. Each square corresponds to a cell. Statistical analyses were performed using the Mann-Whitney test (***, P ≤ 0.001). (H) HIV-1 NLAD8–infected macrophages at 6 d p.i. were washed and treated with anti-CD36 mAbs or with its isotype control for 48 h. Cells were then fixed, embedded in epon, and processed for EM. Bar, 200 nm. (I) HIV-1–infected macrophages treated as in H were prepared for immuno-EM. mAbs present in internal compartments were detected with appropriate secondary antibodies revealed by PAG10. Bars, 200 nm. Representative images are shown in H and I. Data were reproduced with three different donors.

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