Vpr expression or EB1 depletion leads to defective phagosome maturation. (A) MDMs were nucleofected at day 5 of differentiation to express HA-Vpr or with the HA plasmid as a control, incubated for 60 min with IgG-SRBCs at 37°C, and then fixed and permeabilized. They were labeled and analyzed as described in Fig. 2 (E and F). Z-stacks of wide-field fluorescent images were acquired and deconvoluted and a Z projection (ImageJ) is shown. Bar, 10 µm. (B) The number of LAMP1-positive phagosomes was counted as in Fig. 2 and expressed as a percentage of control HA-negative cells. The means ± SEM of three independent experiments are plotted (P < 0.05). (C) MDMs differentiated for 5 d were treated with control siRNA or siRNA against EB1 for 72 h. They were then allowed to phagocytose IgG-opsonized SRBCs for 1 h, fixed, and stained to detect SRBCs with Alexa Fluor–coupled anti–rabbit IgG (not depicted, red in the merge images, right panels) and LAMP1 with anti-LAMP1 followed by Cy3-labeled anti–mouse IgG (middle panels). SRBCs are also detected with phase contrast (left panels). Z-stacks of wide-field fluorescent images were acquired and deconvoluted and a Z projection (ImageJ) is shown. Bar, 10 µm. (D) LAMP1 acquisition was quantified as in Fig. 2. Results are expressed as a percentage of control cells. The means ± SEM of three independent experiments are plotted (P < 0.05). (E) Model of Vpr-induced microtubule-dependent trafficking defects. Phagosome formation is partially inhibited by HIV Nef, perturbing the focal membrane remodeling. Phagosomes that do form in HIV-1–infected macrophages do not move efficiently onto the microtubules to reach the cell center and to undergo maturation. They show altered sorting events that are a consequence of the hijacking of the EHD3 and MICAL-L1 components of the sorting machinery. This is caused by impaired plus end loading of p150^Glued by EB1, which is mislocalized by Vpr. Vpr expression or EB1 depletion is sufficient to recapitulate these events. *, P < 0.05; **, P < 0.005.