Fig. 4

Effect of FST mutations on infectivity, reverse transcription and uncoating. a Schematic representation of FST mutants. b, c 293T cells were infected with either WT (black) GFP-HIV VLP, VLP carrying a chimeric (blue) or partially deleted (green) 3′LTR. b 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. c The levels of early, intermediate (GFP gene) and late cDNA were detected by qPCR 6 h.p.i. d Fate-of-CA assay. HeLa cells were infected with either WT (black) GFP-HIV VLP or VLP carrying the RT mutation A114V (purple), a chimeric 3′LTR (blue) or partially deleted 3′LTR (green). Cells were lysed 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 plotted. e In situ uncoating assay. HeLa cells were infected with dual-labeled WT or mutant HIV VLP and then fixed and stained for CA at the indicated times post-infection. Baf A (red circle) 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.p.i. f Saturation assay. Vero cells were infected with 2 fold serial dilutions of freshly harvested 293T cell supernatants containing WT, mutant GFP-HIV VLPs or P38A capsid mutant GFP-HIV VLP (orange). After 4 h, cultures were challenged with a fixed titre of WT LacZ-HIV VLP. The level of β-galactosidase activity in cell lysates was measured after 72 h and is plotted relative to the level of infection seen following pre-infection with undiluted WT GFP-HIV VLP. Each panel shows the mean and SEM of at least 3 independent experiments