5 fold relative to the DMSO control. The most efficient com pounds IDX 12899, GW 678248 and VRX 480773 selleck chem inhibitor showed strong b Gal activity enhancement at 250 nM, while 1 uM of ETV or EFV was required to achieve the maximal effect. At high NNRTI concentrations microscopically detectable impairment of cell growth, accompanied by a decrease in b Gal activity and high signal variability between replicates indicative of cyto toxic effects was observed, and concentrations above 2. 5 uM NNRTI were therefore excluded from the analysis shown here. this Inhibitors,Modulators,Libraries effect was most pronounced for TMC 120, ETV and VRX 480773. The cytotoxicity observed for TMC 120 under the conditions used, which was con firmed by CC50 determination using a T cell line, likely presents an Inhibitors,Modulators,Libraries explanation for Inhibitors,Modulators,Libraries a discrepancy between our findings and those of Figueiredo et al.
who had reported a stimulation of Gag processing upon shorter incubation of cells with 5 uM TMC 120. Under our experimental conditions we could Inhibitors,Modulators,Libraries not measure repro ducible b Gal activities at this concentration due to cell death. we can also not exclude that cytotoxicity might have obscured stimulatory effects of TMC 120 at lower concentrations. The ranking in the efficacy of compounds was confirmed by immunoblot analysis of lysates from cells incubated with 0. 5 uM of the respective inhibitors, which showed clear differences between the Inhibitors,Modulators,Libraries compounds with respect to the enhancement of Gag pro cessing directly paralleling the results obtained in the alpha complementation assay.
Selective PR dependent killing of HIV expressing Gemcitabine buy T cells by NNRTIs The described drug induced PR activation might be exploited to selectively kill HIV infected cells. In order to test this hypothesis, we established the persistently infected T cell lines MT4 IIIB and MT4 LTR EGFP IIIB, where the expression of HIV encoded proteins in 99% of cells could be detected by intracellular p24 staining. In MT4 LTR EGFP IIIB cells, HIV expres sion could additionally be detected through long terminal repeat driven expression of the gfp marker gene. As a control we used uninfected MT 4 cells or MT4 CMV EGFP cells, constitutively expressing EGFP from a CMV promoter, respectively. The use of persistently infected cells enabled us to study the effects of NNRTIs on virus producing cells regardless of their effect on reverse transcription, since the proportion of virus pro ducing cells in this system does not depend on infection of new host cells. Immunoblot analysis of cell lysates after treatment with two of the more potent NNRTIs, VRX 480773 and GW 678248, confirmed that NNRTI mediated enhancement of Gag processing also occurred in virus producing cells, as apparent from the decreased ratio of Gag to intermediate and fully mature processing products.