HIV-1 infection perturbs membrane delivery of VAMP3- and TNFα-positive compartments but not VAMP7-positive compartments during phagocytosis. (A) Primary human macrophages were incubated for 5 minutes with IgG-RBCs, fixed, and stained with AMCA-anti–mouse IgG to reveal external RBCs. The cells were then permeabilized and labeled with anti-VAMP3, followed by Cy3-anti–rabbit IgG (ii). Merged images show VAMP3 in green and RBCs in red (i). Arrowheads indicate VAMP3 enrichment in phagocytic cup in formation. Cells were analyzed by wide-field fluorescence microscopy and a phase-contrast image is shown (iii). Bar represents 10 μm. (C) Macrophages were treated as in panel A, except that they were labeled with anti-TNFα, followed by Cy3-anti–mouse IgG (ii). Merged images show TNFα in green and RBCs in red (i). Arrowheads indicate TNFα enrichment in phagocytic cup in formation. Bar represents 10 μm. (E) Macrophages were treated as in panel A, except that they were labeled with anti-VAMP7, followed by Cy3-anti–mouse IgG (ii). Merged images show VAMP7 in green and RBCs in red (i). Arrowheads indicate VAMP7 enrichment in phagocytic cup in formation. Bar represents 10 μm. (B,D,F) The VAMP3 (B), TNFα (D), or VAMP7 (F) fluorescence intensities measured in the phagocytic cups were background subtracted, and divided by the respective fluorescence intensities measured in the cell body after background substraction. The ratio defines the index of recruitment. The means ± SEM of 3 independent experiments with 6 noninfected and 6 infected cells are plotted. HIV-1_ADAWT infection significantly inhibited VAMP3 and TNFα, but not VAMP7, recruitment at the phagocytic cup compared with control cells.