Fig. 3

NNRTI-induced killing is rapid and occurs via caspase-dependent and caspase-independent pathways. a Resting CD4 T cells were infected with single round HSA reporter HIV-1 virus and incubated with IL-7. At 5 dpi, cells were treated with 1 μM of IDV and/or RPV. Cells were harvested at the indicated times and kept on ice until all the time points were harvested. Cells were then stained for HSA, labeled with Annexin V and analyzed by flow cytometry. Uninfected control cells were treated with staurosporine. (*p = 0.0281: p-value was calculated at 30′, between RPV HSA+ and RPV HSA− populations, with an unpaired two-tailed t-test). Data are averages and SD of 3 cell donors and are representative of 3 independent experiments. b At 5 dpi, HSA virus infected cells were treated or not with a panel of caspase inhibitors (as indicated, see “Methods”) for 1 h and then treated with EFV (1 μM) for 2 h. Cells were then analyzed for Annexin V labeling and HSA expression. Histogram indicates the percentage of Annexin V positive cells within the HSA+ fraction. Data are averages and SD of 3 donors and are representative of 3 independent experiments. INI = initiator caspases 8, 9, 10; EXE = executioner caspases 3 and 6; INF = inflammatory caspases 1 and 13. PAN = pan-caspase inhibitor Z-VAD-FMK. All the inhibition were statistically different from EFV alone (p < 0.05), indicated by “*”. Inhibition with INI cocktail was statistically different from the effect of the individual caspase inhibitors 8, 9 and 10 (p < 0.05). (p-values were calculated with an unpaired two-tailed t-test). c HSA reporter virus infected cells were treated at 5 dpi with RPV (1 μM) for 2 and 6 h in the presence of Z-VAD-FMK (PAN) or not and stained for HSA expression and with Annexin V and 7-AAD. Dot plots show Annexin V and 7-AAD labeling within pre-gated HSA+ cells. Data are from 1 donor and are representative of 2 independent experiments. d HSA reporter virus infected cells were treated at 5 dpi with RPV (1 μM) for 2 h and stained for HSA expression and with DioC6. Data are 1 donor and representative of 2 independent experiments