Fig. 10

Inhibition of caspase activity and apoptosis-triggered HIV-1 activation in ACH-2 cells. ACH-2 cells were pre-treated with increasing concentration (0–200 μM) of pan-caspase inhibitor, ZVAD-FMK for 2 h prior induction of apoptosis by (a) etoposide, (b) doxorubicin and (c) vincristine. Cells were harvested 36 h post treatment and stained with Annexin V and 7-AAD to assay for apoptosis (solid line) by flow cytometry. HIV-1 unspliced viral RNA was quantified using qRT-PCR and HIV-1 activation was represented as fold change compared to control treatment (dashed line). ACH-2 cells were treated with TNF-α to induce viral replication through the conventional pathway as positive control. Apoptosis-mediated release of HIV-1 virions in culture supernatants was determined by ELISA for HIV-1 p24 antigen (dotted line). (d and e) The levels of apoptosis (white bars), HIV-1 activation (black bars) and (e) virus production followed by TNF-α treatment was determined as in panel A–C. Cells were also pretreated with Z-VAD-FMK (100 μM) for 2 h, followed by TNF-α treatment to determine whether caspase inhibition reduces HIV-1 activation triggered by conventional pathway. Results are pooled from three independent experiments ± SD