HCV NS2 and NS3/4A inhibit RIG-I and Cardif-induced IFN promoter activity. HEK293 cells were transfected with ΔRIG-I (constitutively active form of RIG-I) (A) or Cardif (B) expression plasmids alone or together with NS3/4A, NS3/4A-S139A or NS2 expression constructs (0.03 μg or 0.3 μg). IFN-β promoter activities were measured in cell lysates as described in the legend for Figure 1. Relative IFN-β promoter activities standardized with Renilla expression. (C) IFN-β promoter was induced by transfecting with increasing (0.03–0.3 μg) amounts of ΔRIG-I, Cardif or IKKε expression constructs either alone or together with NS3/4A (0.1 μg) expression construct. The effect of NS3/4A on IFN-β promoter activities were measured in HEK293 cell lysates as described in the legend for Figure 1. (D) Cells were transfected with Cardif and increasing amounts (0.1–1.0 μg) of NS2, NS3/4A and NS3/4A-S139A expression constructs. Total cell lysates were prepared and Cardif and viral protein expression was visualized by western blotting.