Fig. 6

VSV-G pseudotyping leads to stronger restriction by IFNα and IFITM2/3. a, b THP1 wild-type or overexpressing IFITM3-HA were treated for 24 h with 0 or 1000 U/mL IFNα for 24 h, and then infected with the indicated amounts of either HIV-1 WT (a, left panel) or HIV-1Δenv(VSV-G) (a, right panel) packaging the Vpr-β-lactamase fusion protein for 3 h. Cells were then incubated with the fluorescent CCF2-AM substrate for 2 h, fixed, and acquired by flow cytometry immediately. a Representative dose response experiment. b Combined data from four independent experiments, using a viral dose within linear range. Each symbol representing data from one experiment. *p < 0.05; **p < 0.01 (t test). c, d THP1 cells transduced with a Non-Targeting Control (NTC) sgRNA or with an sgRNA targeting both IFITM2 and IFITM3 (IFITM2/3) were infected, in the absence of IFNα, with HIV WT (c, left panel) or HIV-1Δenv(VSV-G) (c, right panel) packaging Vpr BlaM for 3 h. Viral entry was measured as in panel a. c One representative dose response experiment. d Combined data from four independent experiments, using a viral dose within linear range. Each symbol represents one experiment. *p < 0.05 (t test). e, f THP1 cells transduced with a non-targeting sgRNA (NTC) or with a sgRNA against IFITM2 and IFITM3 (IFITM2/3) were infected, in absence of IFNα, with HIV WT (e, left panel) or HIV-1Δenv(VSV-G) (e, right panel). After 24 h, viral input was washed, and cells were cultured in presence of 1 μM of the entry inhibitor T-20, to prevent subsequence rounds of infection. The percentage of Gag positive cells were measured by flow cytometry at 48 h p.i. E: One representative dose response experiment. f Combined data from four independent experiments, using a viral dose within linear range. Each symbol representing data from one experiment. Each symbol represents one experiment. *p < 0.05, **p < 0.01 (t test)