Article Figures & Data
Figures
- Fig. 1.
Vpr expressed from the retroviral vector HR′Vpr is sufficient to induce apoptosis. (A) Representative diagram of Annexin V staining of mock-, HR′Thy-, and HR′Thy (Vpr+)-infected HeLa cells. Flourescence intensity of Annexin-V-FITC is represented on thex axis, and flourescence intensity of 7-AAD is represented on the y axis. The population in the lower left quadrant represents the live cells, the cells in the upper right quadrant represent the dead cell population, and the lower right quadrant represents the apototic cell population. (B) Bar graph represents five independent experiments showing the percentages of mock- (open bars), HIV-1NL4-3Thyenv(−)VprX/VSV-G- (hatched bars), and HIV-1NL4-3Thyenv(−)/VSV-G-infected cells (filled bars) staining Annexin V positive 48 h postinfection. Values obtained with HIV-1NL4-3Thyenv(−)/VSV-G were significantly different from those obtained with HIV-1NL4-3Thyenv(−)VprX/VSV-G, which in turn were higher than those obtained from media alone (P < 0.0001 by Page's test for ordered alternatives in a two-way layout) (22). (C) Bar graph represents three independent experiments showing the percentages of mock- (open bars), HR′Thy(Vpr−)- (hatched bars), and HR′Vpr-infected cells (filled bars) staining Annexin V positive 72 h postinfection. Values obtained with HR′Vpr were significantly higher than those obtained with HR′Thy(Vpr−), which in turn were higher than those obtained with media alone (P < 0.0001 by Skillings-Wolf nonparametric test for ordered alternatives in a two-way unbalanced layout) (22).
- Fig. 2.
CrmA and p35 reduce Vpr-induced apoptosis. Transfection efficiencies were analyzed by flow cytometry at 48 h posttransfection. The data shown is representative of three independent experiments. (A) Bar graph representing Annexin V staining of cells transfected with the indicated viral anti-apoptosis gene and infected with HIV-1NL4-3Thyenv(−)/VSV-G. The percentage of Annexin V staining is represented on the y axis. The data indicated are results from 72 h posttransfection and 48 h postinfection. (B) Bar graph representing Annexin V staining of cells transfected with the indicated viral anti-apoptosis gene and infected with HIV-1NL4-3Thyenv(−)VprX/VSV-G. The percentage of Annexin V staining is represented on the y axis. The data indicated are results from 72 h posttransfection and 48 h postinfection.
- Fig. 3.
Synthetic peptide inhibitor of apoptosis has no significant effect on cell cycle arrest but does reduce the amount of Vpr-induced apoptosis. HeLa cells (105) were mock infected or infected with HR′Vpr. Following infection, cells were washed and 200 μM z-VAD-fmk was added (filled bars). In addition, one set of infected cultures (open bars) received DMSO alone, which was used to dissolve the inhibitors. Fresh inhibitor was added every 24 h throughout the experiment. Infected cells were analyzed 72 h postinfection. Data shown is representative of three independent experiments.
- Fig. 4.
Synthetic caspase inhibitor protects human T cells from Vpr-induced apoptosis. SupT1 cells (4 × 105) were mock infected or infected with HR′Vpr for 4 h in the presence of 10 μg of polybrene per ml. Following infection, cells were washed and DMSO or 100 μM z-VAD-fmk, dissolved in DMSO, was added. Fresh DMSO or z-VAD-fmk was added every 24 h for the duration of the experiment. The experiment was carried out in triplicate, and percentage of apoptosis is plotted against time for HR′Vpr-infected cells treated with either DMSO or z-VAD-fmk as indicated. The difference between estimated mean response at day 3 is significantly lower for z-VAD treatment than for treatment with DMSO (P < 0.005 by testing contrasts for fixed effects in a mixed linear model for repeated measurements) (30).
- Fig. 5.
Synthetic peptide inhibitor of apoptosis reduces the levels of apoptosis while increasing the amount of p24 produced during an HIV-1 infection. HeLa cells (105) were infected with HIV-1NL4-3Thyenv(−)/VSV-G for 4 h in the presence of 10 μg of polybrene per ml. Following infection, the cells were washed twice with PBS and fresh media containing DMSO or 100 μM z-VAD-fmk, dissolved in DMSO, was added. Every 12 h, fresh DMSO or z-VAD-fmk was added to the media. The data shown is representative of four independent experiments. (A) Bar graph representing the percentage of Annexin V-staining cells within the cultures infected with HIV-1NL4-3Thyenv(−)/VSV-G 72 h postinfection. Thy 1.2 analysis revealed that 47.1% of the cells were infected at 48 h postinfection. (B) Bar graph representing p24 (ng/ml) at 84 h postinfection. In each of four independent experiments, we observed a similar negative association: DMSO-treated cultures had higher levels of Annexin V-positive cells and lower levels of p24 than z-VAD-fmk-treated cultures (P = 0.06 by testing the signs of independent Spearman rank correlations for evidence of no association versus negative association) (22).
