FIG 2.

Effects of the expression of the different IFITM3 mutants on the production and infectivity of HIV-1 virion particles. (A) Experimental scheme used to further characterize the antiviral effects of IFITM3 mutants during the negative imprinting of virion particle infectivity. Briefly, HIV-1 virion particles were produced by transient DNA transfection of HEK293T cells with DNAs coding for the different IFITM3 mutants along with the NL4-3 proviral clone and an HIV-1-based miniviral genome bearing a self-inactivating LTR and an expression cassette for GFP. Two days afterwards, cells were lysed, and virion particles were purified by ultracentrifugation through a 25% sucrose cushion, prior to normalization by exo-RT activity and further analyses. (B) Western blot panels presenting typical results obtained for both cell and viral lysates. Mutant 19-24 was the sole mutant whose detection required hybridization with a distinct anti-IFITM3 antibody compared to the remaining mutants. (C) Quantification of the amounts of virion particles produced in the presence of the different IFITM3 mutants by exo-RT. The graph presents data obtained from 6 experiments. *, P < 0.05 as determined by a Student t test upon comparison to WT IFITM3 (two tailed, unpaired). (D) Normalized amounts of virions were used to challenge target HeLaP4 cells, and the extent of infection was measured 2 to 3 days afterwards by flow cytometry. The graph presents data obtained with 4 to 8 independent experiments. *, P < 0.05 as determined by a Student t test for comparisons between the indicated mutant and WT IFITM3 (two tailed, unpaired). All mutants displayed statistically significant differences over the control according to the same test (not displayed on the graph for simplicity).