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. 2012 Nov 29;8(11):e1003059. doi: 10.1371/journal.ppat.1003059

Figure 5. NS1-mediated suppression of RIG-I ubiquitination and IFN induction in mouse is TRIM25-independent.

Figure 5

(A) Ubiquitination of the RIG-I CARDs in WT and TRIM25 −/− MEF cells. WT and TRIM25 −/− MEFs were transfected with human GST-RIG-I 2CARD (GST-h2CARD) or mouse GST-RIG-I 2CARD (GST-m2CARD). At 40 h posttransfection, WCLs were subjected to GST-pulldown (GST-PD), followed by IB with anti-GST antibody. Arrow heads indicate ubiquitinated bands. Arrow indicates non-ubiquitinated 2CARD. (B) Ubiquitination of the RIG-I CARDs in TRIM25 −/− cells reconstituted with mouse or human TRIM25. At 40 h posttransfection with human or mouse GST-RIG-I 2CARD together with empty vector, V5-tagged hTRIM25 or mTRIM25, WCLs of TRIM25 −/− MEFs were subjected to GST-PD, followed by IB with anti-GST or anti-ubiquitin (Ub) antibody. Expression of TRIM25 proteins was determined by IB with anti-V5 antibody. Arrow heads indicate ubiquitinated bands. Arrow indicates non-ubiquitinated 2CARD. (C) NS1 inhibits the RIG-I CARD ubiquitination induced by human TRIM25 but not by mouse TRIM25. TRIM25 −/− MEFs were transfected with GST, human GST-RIG-I 2CARD (GST-h2CARD) or mouse GST-RIG-I 2CARD (GST-m2CARD) together with vector, hTRIM25-V5 or mTRIM25-V5 with or without NS1. At 40 h posttransfection, WCLs were subjected to GST-PD, followed by IB with anti-GST for detecting RIG-I CARD ubiquitination, anti-V5 for detecting TRIM25 binding, or anti-NS1 antibody for detecting NS1 binding. Arrow heads indicate ubiquitinated bands. Arrow indicates non-ubiquitinated 2CARD. (D) NS1 interacts with mouse RIG-I in a TRIM25-independent manner. TRIM25 −/− MEFs were transfected with empty vector, V5-hTRIM25, or V5-mTRIM25 together with NS1-PR8. WCLs were subjected to IP with anti-RIG-I antibody, followed by IB with anti-NS1, anti-V5, or anti-RIG-I antibody. Expression of NS1 and TRIM25 proteins in the WCLs was determined by anti-NS1 and anti-V5 antibody, respectively. (E) NS1 inhibits endogenous mouse RIG-I ubiquitination in a TRIM25-independent manner. TRIM25 −/− MEFs were transfected with increasing amounts of NS1-PR8. At 32 h posttransfection, cells were infected with SeV (50 HA units/ml) for 10 h. WCLs were subjected to IP with anti-RIG-I antibody, followed by IB with anti-Ub or anti-RIG-I antibody. (F) NS1 inhibits IFN-β induction in murine cells in a TRIM25-independent manner. WT and TRIM25 −/− MEFs were transfected with an IFN-β luciferase construct together with empty vector or NS1. At 24 h posttransfection, cells were either mock-treated, or infected with SeV (10 HA units/ml) for 16 h. Samples were then subjected to a dual luciferase assay. Data represent the mean ± SD (n = 3).