Figure 5.

Co-immunoprecipitation of IFITM proteins by CoV S proteins

(A) HEK293T cells were cotransfected with empty control vector or constructs expressing V5- or Flag-tagged 229E, NL63, OC43, MERS, CoV-1 and CoV-2 S proteins and IFITM1, IFITM2, or IFITM3, as indicated. 48h post-transfection, cell lysates were harvested for Co-IP assays. Similar results were obtained in two independent experiments.

(B) Calu-3 cells were transfected with either non-targeting siRNA or siRNA targeting IFITM1, IFITM2 or IFITM3 and treated with IFN-β (500U/ml) or left untreated. Cell were incubated on ice or at 37°C for 1 h, exposed to SARS-CoV-2 (MOI 2.5), and further incubated on ice or at 37°C for 2 h. Subsequently, cells were washed once by PBS and lysed for western blot analysis using the indicated antibodies. Shown is one representative example of three western blots performed.

(C) HEK293T cells were cotransfected with construct expressing V5-tagged CoV-2 S proteins and Flag-tagged mouse IFITMs. Whole cell lysates were harvested at 48h post-transfection for Co-IP analysis. Similar results were obtained in two independent experiments.

(D) Huh7 cells overexpressing the indicated mouse IFITMs were infected with VSVpp carrying the SARS-CoV-2 S protein. GFP+ cells were determined 24 h post-infection. Bars represent the mean of three independent experiments (±SEM). ∗, p < 0.05; ∗∗, p < 0.01; ∗∗∗, p > 0.001.