FIG 6 .

ASCC3 functions through an IRF-3- and IRF-7-dependent pathway. (A) Primary Ikkβ^−/− MEFs were transfected with pCAGGS-GFP or pCAGGS-ASCC3-HA and then infected with WNV at an MOI of 0.05. Viral titers were monitored through a focus-forming assay at indicated time points. The results are the averages of three independent experiments performed in duplicate, and asterisks indicate differences that are statistically significant (***, P < 0.001; **, P < 0.01). (B) Primary wild-type (WT) and Irf3^−/− × Irf7^−/− DKO MEFs were transduced with scrambled shRNA or shRNA against murine Ascc3. Cells were either untreated or treated with 10 IU/ml murine IFN-β for 6 h. Total RNA was harvested, and expression of Ascc3 was determined by qRT-PCR. (C) Multistep growth analysis of WNV infection was performed on the corresponding cells after IFN-β treatment. (D to F) Expression of three ISGs was assayed in shRNA-transduced WT and Irf3^−/− × Irf7^−/− DKO MEFs at 72 h post-WNV infection. Relative expression levels of Ifi44 (D), Irf1 (E), and Rsad2 (F) were normalized to scrambled shRNA-transduced cells. The results are the averages of three independent experiments performed in triplicate, and asterisks indicate differences from the scrambled shRNA control that are statistically significant (***, P < 0.001; **, P < 0.01; *, P < 0.05; n.s., not significant).