Intracellular PR activation and NNRTI induced killing of MT-4 cells persistently infected with HIV. (A) NNRTI induced enhancement of intracellular Gag processing in chronically infected MT-4 cells . MT-4-IIIB cells were cultured in the presence of DMSO (lane 1), 1 μM GW-678248 (lane 2), 200 nM DRV (lane 3), 1 μM GW-678248 + 200 nM DRV (lane 4), 1 μM VRX-480773 (lane 5), or 1 μM VRX-480773 + 200 nM DRV (lane 6), respectively. Cell lysates were harvested and analyzed by immunoblot using antiserum raised against HIV-1 CA. Positions of Gag and Gag-Pol processing products are marked at the right, molecular mass standards are indicated to the left (in kDa). Lysates shown here were harvested at day 2 post addition of compounds; longer incubation periods (6 days) resulted in a more pronounced accumulation of unprocessed Gag in the DRV treated samples, but the pattern in the NNRTI treated samples became difficult to detect due to cell death. (B) NNRTI induced killing of chronically infected MT-4 cells. The MT4-LTR-EGFP parental cell line or its persistently HIV-1 infected derivative MT4-LTR-EGFP-IIIB, respectively, were seeded at a density of 1.5 × 105 cells/ml and incubated for 6 days in the presence of 0.1% DMSO (white bars), 200 nm DRV (gray bars), 1 μM VRX-480773 (black bars) or 1 μM VRX-480773 + 200 nM DRV (hatched bars), respectively. Live cells were counted after trypan blue staining. Data represent mean values and standard deviations from three parallel cultures. P-values were calculated with GraphPad Prism using an unpaired two-tailed t-test. n.s., non significant. (C) MT4-CMV-EGFP (circles) or MT4-LTR-EGFP-IIIB (triangles) cells were seeded in 96-well plates at a density of 105 cells/ml and incubated for 4 days in the presence of various concentrations of the indicated NNRTI, either with (open symbols) or without (filled symbols) the addition of 100 nM DRV. EGFP intensity per well was quantitated at the end of the incubation period by measuring total fluorescence intensity per well based on analysis of microscopic images as described in Methods. The graphs show exemplary data for three NNRTIs. Mean values and standard deviations from three independent wells of one representative experiment are shown. Lines represent fits of the data to a standard dose response equation (4 parameters), yielding CC50 values on virus producing cells in the absence of DRV (filled triangles) of 0.35 μM for GW-678248 and 2.44 μM for EFV, respectively. Data from several independent experiments for these compounds as well as for the other NNRTIs were used to calculate the CC50 values summarized in Table 1.