FIG 3.

N protein blocks the phosphorylation and nuclear translocation of endogenous IRF3. (A) HEK-293T cells were mock transfected or transfected with an expression plasmid encoding HA-tagged N protein 24 h before being infected with SEV or not for 8 h. Cell lysates were collected for immunoblot analysis with antibodies directed against phosphorylated IRF3 (Ser396), IRF3, HA, or β-actin. (B) HEK-293T cells were transfected with plasmid pCAGGS-HA-N or an empty vector. At 24 h after transfection, the cells were infected with SEV for 8 h. After the fixation and permeation of the cells, an immunofluorescence analysis was performed to detect endogenous IRF3 (red) and N protein (green) with rabbit anti-IRF3 and mouse anti-HA antibodies, respectively. DAPI staining (blue) indicates the locations of the cell nuclei. Fluorescent images were acquired with a confocal laser scanning microscope (Fluoview ver. 3.1; Olympus, Japan). Cells transfected with an empty vector or mock infected with SEV were used as the negative controls.