Figure 7. NS1 inhibits the Riplet-dependent RIG-I ubiquitination and IFN induction in murine cells.

(A) Mouse Hepa 1.6 cells were transfected with vector or Flag-tagged mRiplet with or without NS1-PR8. WCLs were subjected to IP with RIG-I antibody, followed by IB with anti-Ub or anti-RIG-I antibody. Expression of mRiplet, NS1, and ubiquitin (Ub) was determined in the WCLs by IB with anti-Flag, anti-NS1 or anti-Ub antibody. (B) Mouse Hepa1.6 cells were transfected with IFN-β luciferase reporter plasmid together with empty vector or mRIG-I together with or without mRiplet and NS1-PR8. At 24 h posttransfection, cells were lysed and subjected to luciferase assay. Data shown is representative of 3 independent experiments and depicted is the mean ± SD (n = 3). (C) Influenza NS1 protein specifically inhibits the Riplet-dependent ubiquitination of mouse RIG-I. Hepa1.6 cells were transiently transfected with non-silencing control siRNA (si.C), or with a siRNA specific for mouse Riplet (si.Riplet) together with empty vector or NS1-PR8. At 24 h posttransfection, cells were infected with SeV (50 HA units/ml) for 22 h. WCLs were used for IP with anti-RIG-I antibody, followed by IB with anti-Ub or anti-RIG-I antibody. (D–F) Knockdown of endogenous Riplet in mouse embryonic fibroblasts enhances influenza A virus replication. WT or TRIM25 −/− MEFs were transfected with non-silencing control siRNA (si.C), or with a siRNA specific for mouse Riplet (si.Riplet). At 30 h posttransfection, cells were infected with recombinant A/PR/8/34 WT virus (MOI 0.1). Knockdown of endogenous Riplet was confirmed by RT-PCR (D). Supernatants were assayed for progeny virus yields 24 h postinfection in standard plaque titrations (E). Virus yields are depicted in Pfu/ml. The results of three independent experiments are shown. Furthermore, viral NS1 protein expression was determined in the WCLs of infected cells (F).