FIG 6.

N protein inhibits TBK1-mediated IRF3 phosphorylation and sequesters the interaction between IRF3 and TBK1. (A) HEK-293T cells were cotransfected with an empty vector or expression plasmids encoding HA-tagged N protein and Flag-tagged TBK1. Twenty-four hours after transfection, the cells were mock infected or infected with SEV for 8 h, followed by immunoblotting assays with antibodies directed against phosphorylated IRF3 (Ser396), IRF3, HA, Flag, or β-actin. (B) HEK-293T cells were transfected with increasing amounts (0, 1.5, 3, or 6 μg) of a plasmid expressing HA-tagged N protein. Twenty-four hours after transfection, the cells were mock infected or infected with SEV for 8 h. The cell lysates were immunoprecipitated with rabbit anti-TBK1 or rabbit normal IgG antibody (lane 2). They were then analyzed by immunoblotting (IB) with antibodies directed against TBK1, IRF3, or HA. The whole-cell lysates (WCL) were used to analyze the expression of TBK1, IRF3, and N protein by immunoblotting using anti-TBK1, anti-IRF3, and anti-HA antibodies, respectively.