HIV-1 perturbs and hijacks the EHD3/MICAL-L1 sorting machinery. (A) Primary human macrophages were infected with HIV-1ADA WT or mock infected for 8 d. The cells were incubated for different times with IgG-SRBCs at 37°C. Macrophages were treated as in Fig. 2 F except that staining was with anti-EEA1 followed by Cy3-labeled anti–mouse IgG. The number of internal phagosomes positive for EEA1 was counted. Results are expressed as a percentage of total internal phagosome number ± SEM (>200 phagosomes per condition, repeated in n = 3 independent experiments on different donors). (B) Primary human macrophages were noninfected (left) or infected with HIV-1YU-2WT (right) for 8 d. The cells were then fixed and stained with an anti–p24 antibody, followed by Alexa Fluor 488–coupled anti–goat IgG (top panels) and an anti–MICAL-L1 (B) or anti-EHD3 (C) antibody, followed by Cy3-labeled anti–rabbit IgG and Cy3-labeled anti–mouse IgG, respectively. Stacks of images were acquired and a maximum-intensity projection is shown in B. Bar, 5 µm; magnification in insets is 2×. (C) Stacks were deconvoluted and single optical sections are shown. 3D reconstitution was performed with Imaris. N, nucleus. Bar, 5 µm. (D and E) Macrophages differentiated for 5 d were treated with control siRNA or siRNA against MICAL-L1 (D) or siRNA against EHD3 (E) for 72 h. They were then allowed to phagocytose IgG-opsonized SRBCs for 1h, fixed, and stained to detect SRBCs with Alexa Fluor 647–coupled anti–rabbit IgG and LAMP1 with anti-LAMP1 followed by Cy3-labeled anti–mouse IgG (not depicted). LAMP1 acquisition was quantified as in Fig. 2. Results are expressed as a percentage of control cells. The means ± SEM of three independent experiments (donors) are plotted. *, P < 0.05.
