Fig. 2.

WNV infection is inhibited in VCP knockdown cells. (A) HeLa cells were treated with either siRNA against VCP [siVCP (1), (2) and (3)] or control siRNA (siCont). The siRNA-treated cells were inoculated with WNV (MOI = 1) at 48 h.p.i. The inoculated cells were harvested at 24 h.p.i. The expression of endogenous VCP protein and WNV envelope protein after treatment with the indicated siRNA were examined by immunoblotting with mouse anti-VCP antibody and mouse anti-WNV/Kunjin envelope protein. The expression of actin was examined after reprobing as an endogenous control. (B) WNV-infected cells from (A), after 24 h incubation with WNV, the cells were harvested and examined by immunofluorescence assay. WNV-infected cells were stained with anti-JEV antibody (green) and cell nuclei were counterstained with DAPI (blue). (C) Positivity of WNV-infected cells from (B). Mean ± SD from three independent experiments is shown; ** p < 0.01 (one-way ANOVA). (D) The culture supernatants from (A) were collected at 24 h.p.i. and the viral titers of the harvested supernatants were determined using plaque assay. Mean ± SD from three independent experiments is shown; ** p < 0.01 (one-way ANOVA).