Figure 2

IFITMs are HIV-1 virion associated proteins. A) HEK293T cells were transfected with DNAs coding IFITMs and HIV-1 and two days after, both cell lysates and supernatant purified by ultracentrifugation through a 25% sucrose cushion were harvested and analyzed by WB. The panels present typical results obtained out of 10 independent experiments. B and C) HEK293T cells were transfected as above by maintaining a fixed amount of Gag-Pol/Env and by varying the amount of DNAs coding the different IFITMs. Virion particles were purified by ultracentrifugation, normalized by exo-RT and either analyzed by WB to determine the amount of IFITMs incorporated onto the virion particles (B), or used to challenge HEK293T cells to determine their infectivity (C). D) Virions produced in the presence of IFITMs were first concentrated and purified through sucrose as described above then layered onto a linear OptiprepTM velocity gradient (5 to 20% w/v) for an ultracentrifugation step of 45 minutes at 28,000 rpm. Aliquots were harvested from the top of the gradient, precipitated with TCA and analyzed by WB. The proportion of CA and IFITMs present in each fraction with respect to the total CA and IFITMs in all fractions was determined by densitometry and is presented here solely for IFITM3 due to space constraints. The density of each fraction was determined prior to TCA precipitation with a bench densitometer and is presented here as a dotted grey line (g/mL). The graph and associated WB panels are representative of 3 independent experiments. E) Virion particles produced from SupT1 cells stably expressing Flag-IFITMs were subjected or not to CD45 depletion, prior to WB analysis. The Western blot panels are representative of 3 independent experiments.