Figure 2. Induction of the interferon response by 5′pppRNA is dependent on functional RIG-I signaling.

(A) WT and RIG-I−/− MEFs were co-transfected with IFNα4 or IFNβ promoter reporter plasmid (200 ng) along with 5′pppRNA (500 ng/ml) or expression plasmids encoding a constitutively active form of RIG-I (ΔRIG-I) (100 ng). IRF-7 expression plasmid (100 ng) was added for transactivation of the IFNα4 promoter. Luciferase activity was analyzed 24 h post-transfection by the Dual-Luciferase Reporter assay. Relative luciferase activity was measured as fold induction relative to the basal level of reporter gene. Error bars represent SEM from nine replicates performed in three independent experiments. (B) Mda5−/−, TLR3−/−, TLR7−/− and RIG-I−/− MEFs were co-transfected with IFNβ promoter reporter plasmid (200 ng) along with 5′pppRNA (500 ng/ml). Luciferase activity was analyzed 24 h post-transfection by the Dual-Luciferase Reporter assay. Relative luciferase activity was measured as fold induction relative to the basal level of reporter gene. Promoter activity in the knockout MEFs was then normalized against the activity in their respective wt MEFs to obtain the percentage of fold activation. Error bars represent SEM from nine replicates performed in three independent experiments. (C) A549 cells were either left untreated or transfected with a control siRNA or RIG-I siRNA. After 48 h, 5′pppRNA (10 ng/mL) was transfected and at 8 h after treatment, WCEs were analyzed by SDS-PAGE and immunoblotted for pIRF3 Ser-396, IRF3, pSTAT1 Tyr 701, STAT1, IFIT1, RIG-I, and β-Actin. Results are from a representative experiment; all immunoblots are from the same samples.