Fig. 2

Effect of RT mutations on infectivity, reverse transcription and uncoating. a, b, c 293T cells were infected with either WT (black) GFP-HIV VLP or VLP carrying mutations in RT at A114V (purple), E478Q (green) or H539F (blue). a The percentage of GFP positive cells was measured by flow cytometry 72 h.p.i. with increasing amounts of VLP, and plotted relative to the number of cells infected by 100 μl WT VLP. b The levels of early and late cDNA were detected by qPCR 6 h.p.i. c Fate-of-CA assay. HeLa cells were infected with WT or RT mutant GFP-HIV VLP. Cells were lysed 2 or 20 h.p.i. and lysate separated into soluble and pellet fractions by centrifugation through a sucrose cushion. CA was detected by immunoblotting, quantified and the percentage of total CA in the pellet fraction is plotted. d In situ uncoating assay. HeLa cells were infected with dual-labeled WT or RT mutant HIV VLP in the presence (orange) or absence of 10 µM NVP and then fixed and stained for CA at the indicated times post-infection. Baf A (red cirle) was added to one sample as a negative control for fusion. The percentage of fused particles that still stain for CA was calculated 1, 2, or 4 h post-infection. e Saturation assay. Vero cells were infected with 2 fold serial dilutions of freshly harvested 293T cell supernatants containing WT or RT mutant LacZ-HIV VLPs. After 4 h, cultures were challenged with a fixed titre of WT GFP-HIV VLP. The percentage of GFP positive cells 72 h later is plotted relative to the infection seen following pre-infection with undiluted WT LacZ-HIV VLP. Each panel shows the mean and SEM of >3 independent experiments