Figure 5.

TRIP negatively regulates cellular antiviral response. (A) HEK293 cells transfected with the indicated plasmids were infected with SeV for 12 h. IFN-β, RANTES, and TRIP mRNA expressions were analyzed by RT-PCR. (B) HEK293 cells were transfected with IFN-β or IRF3 reporter together with TRIP or control plasmid, and luciferase activity was analyzed after infection with SeV for 12 h. (C) HEK293 cells were transfected with the indicated plasmids. 24 h after transfection, cells were infected with SeV or left uninfected for 6 h. Cell lysates were separated by native (top) or SDS (bottom two) PAGE and analyzed by immunoblotting with the indicated antibodies. (D) Mouse peritoneal macrophages were transfected with control siRNA or TRIP siRNA for 36 h. Quantitative RT-PCR analysis of IFN-β and RANTES mRNA in peritoneal macrophages infected with SeV for indicated time periods. (E) Western blot analysis of the expression of TBK1, IRF3, TRAF3, and STAT1 in HEK293 cells transfected with control or Flag-TRIP plasmid and then infected with SeV for indicated time periods. (F) In vitro kinase assays of TBK1 in the lysates of peritoneal macrophages transfected with control siRNA or TRIP siRNA 1 and then infected with SeV for 2 h, assessed with the substrates MBP (for TBK1). (G) HEK293 cells were transfected with the indicated plasmids. 24 h later, cells were further transfected with 0.1 µg poly(I:C). 18 h later, cells were infected with VSV (MOI, 0.1), and the supernatants were harvested at 12 h after infection. Supernatants were analyzed for VSV titers. Intracellular VSV RNA replicates were measured by quantitative RT-PCR. (H) Mouse peritoneal macrophages were transfected with control siRNA (Ctrl) or TRIP siRNA (siRNA). VSV titers and intracellular VSV RNA replicates were measured as in G. **, P < 0.01. Data are representative of three experiments (mean and SD of triplicate samples in B, D, F, G, and H). Similar results were obtained in three independent experiments in A, C, and E.