FIG. 7.

Involvement of RIG-I in SeV- and influenza A virus-induced IFN-β production. (A and B) RIG-IC inhibits activation of IFN-β promoter by viral infection. 293T cells were transfected with 0.5 μg of pIFNβ-CAT, 0.5 μg of pCAGGS-Luc, and 0.5 μg of pCAGGS, pCAGGS-NS1, pCAGGS-Flag-RIG-IC, or pCAGGS-Flag-RIG-I, as indicated, using CaPO₄. The reporter assays were conducted in triplicate. At 24 h posttransfection, cells were mock infected or infected with SeV, influenza A/PR/8/34 virus, or delNS1 virus at an MOI of 1. Cells were collected at 24 h postinfection and subjected to CAT assays. Relative CAT activity was normalized against relative luciferase activity. The y axis indicates activation (n-fold) of the IFN-β promoter by setting the reporter activities of empty pCAGGS-transfected and mock-infected cells at 1. (C and D) Suppression of virus-induced IFN-β production by RIG-IC and NS1. Before cells were collected, culture media were harvested and subjected to a bioassay for determination of IFN levels/production based on RFP expression in NDV-mRFP-infected Vero cells. The y axis indicates the red fluorescence produced by NDV-mRFP. (E) siRNA for RIG-I inhibits activation of IFN-β promoter by viral infection. To knock down the expression of RIG-I, RNA interference was performed with siRNA (see Materials and Methods). Expression of RIG-I was downregulated in Stealth_1199-transfected cells but not in Stealth_control_1199-transfected cells (data not shown), and that of β-actin was not affected by the transfection of both Stealth_1199 and Stealth_control_1199 (data not shown). After incubation, cells were mock infected or infected with SeV or delNS1. After the infection, cells were incubated for 24 h and then cells were collected and subjected to CAT assay. The y axis indicates activation (n-fold) of IFN-β promoter by setting the reporter activity of Stealth_control_1199 and the reporter plasmid-transfected and mock-infected cells at 1.