Mutant SFV Replicase triggers IFN-β in a RIG-I- and MDA-5-dependent manner.
(A) COP-5 cells were transfected with siRNAs against MDA-5, RIG-I, and LGP2 or combinations thereof. After 48 hr, cells were transfected with poly(I:C) dsRNA, and after 24 hr, the amount of secreted IFN-β was measured by ELISA (upper panel). The efficiency of RLR protein knockdown was assessed by immunoblot assay (lower panel). Cells lysates were separated by SDS-PAGE and immunoblotted with different antibodies. Neg. ctrl., negative control non-targeting siRNA; mock, transfection without siRNA. (B) COP-5 cells were transfected with siRNAs as described in (A). After 48 hr, cells were transfected with pRep-RDR plasmid DNA, and after 48 hr, the amount of secreted IFN-β was measured as described in (A) (upper panel). The RLR knockdown efficiency (lower panel) was assessed as described in (A). Expression of SFV replicase was analyzed using antibodies against nsP4 and nsP2; ACTIN was used as loading control. (C and D) MEFs were transfected with siRNAs as described in (A). After 48 hr, cells were infected with SFV4-Rluc-RDR at different MOIs. After an additional 12 hr, the amount of IFN-β was measured (C) and cell lysates were prepared for the Renilla luciferase reporter assay (D). Immunoblots (A and B) and panels (C and D) are representative examples of two independent experiments. Error bars (A and B) represent the standard deviation of three experiments. ***p<0.001 (t-test).