Fig. 4.

RIG-I SV inhibits RIG-I-mediated antiviral signaling. (A) RIG-I SV inhibits the SeV-induced IFN-β promoter activation. HEK293T were transfected with vector or increasing amounts of RIG-I T₅₅I or RIG-I SV together with IFN-β luciferase and pGK-β-gal. At 24 h after transfection, cells were mock-treated or infected with SeV, and luciferase activity was determined as described in Fig. 1D. Data represent the mean ± SD (n = 3). RIG-I SV inhibits virus-induced IRF3 phosphorylation (B) and dimerization (C). (B) At 24 h after infection with Myc-RIG-I (10 μg), Flag-RIG-I SV (2 μg) or Flag-RIG-I T₅₅I (2 μg), cells were mock-treated or infected with SeV (50 HA units/ml) for 16 h. WCLs were subjected to IB with α-Phospho-IRF3 (Ser₃₉₆), α-IRF3, α-Myc, α-Flag, or α-actin. (C) At 24 h after transfection with Flag-IRF3, Flag-RIG-I WT, RIG-I SV, or RIG-I T₅₅I, cells were mock-treated or infected with SeV (50 HA units/ml) for 18 h. WCLs were used for native PAGE and subjected to IB with α-IRF3. (D) RIG-I SV inhibits the virus-induced nuclear translocation of IRF3. At 24 h after transfection with IRF3-eGFP, HEK293T stably expressing vector, Flag-RIG-I, Flag-RIG-I SV, or Flag-RIG-I T₅₅I were mock-treated or infected with SeV and stained with α-RIG-I (red) and Hoechst 33256 (nucleus). (E) RIG-I SV expression increases VSV-eGFP replication. HEK293T stably expressing vector, RIG-I, RIG-I SV, or RIG-I T₅₅I were infected with VSV-eGFP at MOI 0.5. At 24 h after infection, virus titer and replication were determined by plaque assay and GFP expression, respectively. (F) RIG-I SV suppresses virus-induced IFN-β production. HEK293T stably expressing vector, RIG-I, RIG-I SV, or RIG-I T₅₅I were infected with SeV (50 HA units/ml) for 20 h. IFN-β production was measured by ELISA. Data represent the mean ± SD (n = 3).