Figure 4.
Figure 4. Established HIV-1 infection and the viral factor Vpr are important for the phagosome maturation defect. (A) Primary human macrophages were noninfected (black bars) or infected with HIV-1ADA WT (red bars) for 2, 3, 6, or 8 d. At each time point, the cells were incubated for 1 h with IgG-SRBCs at 37°C, fixed, and permeabilized. Then, they were labeled with Cy5-labeled anti–rabbit IgG to detect the total SRBCs, an anti-p24 antibody followed by Cy2-labeled anti–goat IgG to detect the infected cells, and an anti–LAMP1, followed by Cy3-labeled anti–mouse IgG. Z-stacks of fluorescence images were acquired and analyzed with ImageJ. The number of phagosomes positive for LAMP1 was calculated for >200 phagosomes per condition at each time point. Results are expressed as a percentage of total phagosomes. The means ± SEM from two independent experiments are plotted. (B) Primary human macrophages were noninfected (black bars), infected with HIV-1ADA WT alone (red bars), or in the presence of raltegravir (violet bars) at 10 mM for 8 d. Data were analyzed as in A. (C) Primary human macrophages were noninfected or infected with HIV-1ADAWT or HIV-1ADAΔNef for 2, 3, 6, or 8 d. At each time point, the cells were incubated for 1 h with IgG-SRBCs at 37°C and fixed. External and internal SRBCs were counted and the efficiency of phagocytosis was calculated for noninfected cells (black bars), HIV-1ADAWT–infected cells (red bars), and HIV-1ADAΔNef–infected cells (green bars). Results are expressed as a percentage of control noninfected cells. The means ± SEM of three independent experiments are plotted. (D and F) Primary human macrophages were noninfected (black bars) or infected with HIV-1ADAWT (red bars) or HIV-1ADAΔNef (green bars) for 8 d. Cells were treated and results analyzed as in A and C, respectively. The means ± SEM from five independent experiments are plotted. (E and G) Primary human macrophages were noninfected (black bars) or infected with HIV-1YU-2WT (red bars) or HIV-1YU-2ΔVpr (blue bars) for 8 d. Cells were treated and results analyzed as in A and C, respectively. The means ± SEM from five independent experiments are plotted. *, P < 0.05; **, P < 0.005.

Established HIV-1 infection and the viral factor Vpr are important for the phagosome maturation defect. (A) Primary human macrophages were noninfected (black bars) or infected with HIV-1ADA WT (red bars) for 2, 3, 6, or 8 d. At each time point, the cells were incubated for 1 h with IgG-SRBCs at 37°C, fixed, and permeabilized. Then, they were labeled with Cy5-labeled anti–rabbit IgG to detect the total SRBCs, an anti-p24 antibody followed by Cy2-labeled anti–goat IgG to detect the infected cells, and an anti–LAMP1, followed by Cy3-labeled anti–mouse IgG. Z-stacks of fluorescence images were acquired and analyzed with ImageJ. The number of phagosomes positive for LAMP1 was calculated for >200 phagosomes per condition at each time point. Results are expressed as a percentage of total phagosomes. The means ± SEM from two independent experiments are plotted. (B) Primary human macrophages were noninfected (black bars), infected with HIV-1ADA WT alone (red bars), or in the presence of raltegravir (violet bars) at 10 mM for 8 d. Data were analyzed as in A. (C) Primary human macrophages were noninfected or infected with HIV-1ADAWT or HIV-1ADAΔNef for 2, 3, 6, or 8 d. At each time point, the cells were incubated for 1 h with IgG-SRBCs at 37°C and fixed. External and internal SRBCs were counted and the efficiency of phagocytosis was calculated for noninfected cells (black bars), HIV-1ADAWT–infected cells (red bars), and HIV-1ADAΔNef–infected cells (green bars). Results are expressed as a percentage of control noninfected cells. The means ± SEM of three independent experiments are plotted. (D and F) Primary human macrophages were noninfected (black bars) or infected with HIV-1ADAWT (red bars) or HIV-1ADAΔNef (green bars) for 8 d. Cells were treated and results analyzed as in A and C, respectively. The means ± SEM from five independent experiments are plotted. (E and G) Primary human macrophages were noninfected (black bars) or infected with HIV-1YU-2WT (red bars) or HIV-1YU-2ΔVpr (blue bars) for 8 d. Cells were treated and results analyzed as in A and C, respectively. The means ± SEM from five independent experiments are plotted. *, P < 0.05; **, P < 0.005.

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