Riplet promotes TRIM25-mediated full-length RIG-I activation.

<p>(A, B) Wild-type and Riplet KO MEFs were infected with SeV at MOI = 0.2. The mRNA expressions of IFN-β (A) and –α2 (B) at the indicated times after viral infection were determined by RT-qPCR. <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003533#s2" target="_blank">Results</a> are presented as mean ± SD (n = 3). (C, D) The activation of IFN-β promoter was examined by reporter gene assay using a p125luc IFN-β reporter. RIG-I CARDs (C) or full-length RIG-I (D) expression vectors were transfected into HEK293 cells together with a Riplet and/or TRIM25 expression vector as indicated. 24 hours after the transfection, the reporter activities were determined. (E, F) The activation of IFN-β promoter was examined by reporter gene assay using a p125luc IFN-β reporter. Full-length RIG-I expression vectors were transfected into HEK293 cells together with a Riplet and/or TRIM25 expression vector as indicated. 24 hours after the transfection, the cells were stimulated with 50 ng of HCV 3′ UTR dsRNA by transfection or infected with VSV at MOI = 1 for 24 hours, and then reporter activation was determined. Data are presented as mean ± SD (n = 3). *p<0.05. (G) HEK293FT cells were stimulated with 0.8 µg of HCV double-stranded RNA (HCV RNA) using lipofectamine 2000 in 6-well plate. Cell lysates were prepared at the indicated times, followed by immunoprecipitation with an anti-RIG-I mAb (Alme-1).