Figure 7.

BFRF1 blocks the SeV-induced phosphorylation, dimerization, and nuclear translocation of IRF3. (A, B) HEK293T cells were transfected with vector or Flag-tagged BFRF1 expression plasmid. At 24 h post-transfection, cells were mock-infected or infected with 100 HAU/ml SeV for 8 or 16 h. Then, whole-cell extracts were prepared and subjected to IRF3 phosphorylation analysis (A) for phosphorylated IRF3 (Ser396), total IRF3, β-actin, BFRF1-Flag, and native PAGE analysis (B) for IRF3 dimerization, using related Abs as indicated. (C, D) Hone1-EBV cells were co-transfected with BZLF1-HA expression plasmid and pSuper-shBFRF1-retro-puro or pSuper-shRandom-retro-puro expression plasmid. At 24 h post-transfection, cells were mock-infected or infected with 100 HAU/ml SeV for 16 h. Then, whole-cell extracts were prepared and subjected to IRF3 phosphorylation analysis (C) and native PAGE analysis (D), as indicated in (A, B), respectively. (E) HeLa cells were transfected with vector or Myc-tagged BFRF1 expression plasmid, at 24 h post-transfection, cells were mock-infected or infected with 100 HAU/ml SeV for 16 h. Cells were then probed with primary Abs mouse anti-Myc mAb and rabbit anti-IRF3 pAb, and secondary Abs FITC-conjugated goat anti-mouse IgG (green) and Cy5-conjugated goat anti-rabbit IgG (red), respectively. Cells were counterstained with Hoechst to visualize the nuclear DNA (blue). Images were obtained by confocal microscopy using a 63× lens objective. All scale bars indicate 10 μm. Statistical analysis of the subcellular localization of IRF3 in the absence or presence of BFRF1 was shown in Table 1.