Vpr-Induced Caspase Activation and Smac Release from the Mitochondria Are Temporally Delayed in Relation to G₂ Arrest

<div><p>(A) pHR-VPR-G and pHR-VPR-R are bicistronic lentiviral vectors that encode HIV-1_NL4–3<i>vpr,</i> an internal ribosome entry site, and either the gene for GFP or that for mRFP, respectively; pHR-VPR(R80A) was derived from pHR-VPR (both the mRFP and the GFP versions) by site-directed mutagenesis; DHIV3 is an envelope-truncated (see gray box) version of HIV-1_NL4–3; DHIV3-ΔVPR was derived from DHIV-3 by introducing a frameshift mutation in <i>vpr</i>.</p><p>(B) SupT1 T lymphocytes were transduced by spin-infection in the presence of 10 μg/ml Polybrene with indicated vectors. Mock-infected cells were subjected to spin-infection in the presence of 10 μg/ml Polybrene without virus. Cells were collected at specified time points post-transduction, stained with propidium iodide, and analyzed for DNA content by flow cytometry to determine cell cycle profiles. The percentage of cells transduced with pHR vectors and DHIV3 viruses ranged between 70% to 80% and between 65% to 70%, respectively, as determined by mRFP or GFP expression (with pHR vectors) or intracellular p24 staining (with DHIV3 vectors).</p><p>(C) Caspase activation was measured as an indication of apoptosis. SupT1 cells were infected with indicated vectors, harvested, and incubated with FITC-VAD-FMK. The percentage of caspase-active cells at each time point was measured by flow cytometry.</p><p>(D) Infected SupT1 cells were lysed, and lysates were fractionated into mitochondrial (m) and cytoplasmic (c) fractions and then assayed by Western blot. Western blots were probed with antibodies specific to Smac to measure release from mitochondria and with anti-VDAC antibodies to measure mitochondrial contamination in the cytoplasmic fractions. As a positive control for apoptosis, SupT1 cells were treated with 0.8 μg/ml doxorubicin (dox) for 48 h.</p><p>(E) Examples of flow cytometric analysis of caspase activation, corresponding to the 72-h time points from (C).</p>