Figure 4

Effects of autophagic blockade on ChikV infection. (A) HEK.293 cells were pretreated with the inhibitor of autophagy 3-MA (10 mM) for 2 hours or left untreated (Ct). Then, the cells were infected by ChikV at a MOI = 1, as described in Materials and Methods. ChikV infected cells were visualized by immufluorescence, using a monoclonal antibody directed against E1 glycoprotein. Magnification = 200× (B) Quantitative analysis of ChikV expression in cells treated or not with 3-MA. (C) Consequences of 3-MA treatment on viral particle release. Viral RNA level was determined from cell supernatant. Values are expressed as fold change relative to levels detected from untreated cells. (D) Intracellular expression of ChikV structural proteins in HEK.293 maintained in medium alone or supplemented by 10 mM of 3-MA was detected by Western blot. (E) HEK.293 cells were infected by ChikV with a MOI = 1 after transfection with Beclin-1 or unrelated siRNA. Expression of Beclin-1 was monitored by immunoblotting using a specific mAb directed against Beclin-1. Quantitative analysis of the viral RNA in cell culture supernatant was determined 24 h post infection. Results are represented as mean values from three independent experiments with standard errors. * P < 0.05; ** P < 0.01; *** P < 0.001.