(A), (B), (C) RING domain deletion mutant REULΔRING suppresses SV-induced activation of ISRE (A), NF-κB (B) and IFN-β (C) promoter in a dose-dependent manner. HEK293T cells (1×105) were transfected with the indicated reporter plasmid (50 ng), pRL-TK Renilla luciferase plasmid (50 ng) and the indicated amounts of an expression plasmid for REULΔRING or empty plasmids. 24 h after transfection, cells were left uninfected or infected with SV for 8 h before luciferase assays were performed. (D) REULΔRING has no effect on polyI:C-induced IFN-β promoter. The transfections were done as in (A), (B) and (C). 16 h after transfection, cells were further transfected with polyI∶C (4 µg) or untransfected for 12 h before luciferase assays were performed. (E) Effects of REUL RNAi plasmids on the expression of transfected REUL. HEK293T cells (2×105) were transfected with expression plasmids for HA-REUL and HA-WDR34 as control (0.5 µg each), and the indicated RNAi plasmids (1 µg). At 48 h after transfection, cell lysates were analyzed by Western blot with anti-HA and anti-GAPDH antibody. (F), (G), (H) REUL RNAi plasmids suppress SV-induced activation of ISRE (F), NF-κB (G) and IFN-β (H) promoter. HEK293T cells (1×105) were transfected with the indicated reporter plasmid (50 ng), pRL-TK Renilla luciferase plasmid (50 ng), and the indicated REUL RNAi (1 µg). At 40 h after transfection, cells were left uninfected or infected with SV for 8 h before luciferase assays were performed. (I) REUL RNAi suppresses RIG-I-, but not MDA5-mediated gene expression. HEK293T cells (2×105) were transfected with indicated plasmids. At 40 h after transfection, cells were left uninfected or infected with SV for 8 h before total RNA was isolated and RT-PCR was performed using indicated primers. (J) REUL suppresses NDV-eGFP replication. HEK293T cells (1×105) were transfected with the indicated plasmids. At 20 h after transfection, cells were infected with NDV-eGFP at MOI 0.001. At 40 h after infection, virus titer and replication were determined by plaque assay or GFP expression visualized by fluorescence microscopy. Pfu, plaque-forming unit. (K) REUL accelerates RIG-I-mediated anti-VSV response. HEK293T cells (2×105) were transfected with indicated plasmids (0.5 µg each). At 30 h after transfection, cells were infected with VSV (MOI = 0.001) and supernatants were harvested at 12, 18, 24 and 30 h post infection. Supernatants were analyzed for VSV production using standard plaque assays. Plaques were counted and titers calculated as plaque-forming units (pfu/ml). (L) Knockdown of REUL inhibits RIG-I-mediated anti-VSV response. The experiments were carried out as in (K).