FIG 6.

PPRV N protein disrupts TBK1-IRF3 complex formation. (A) HEK-293T cells were transfected with empty vector or increasing amounts of Flag-N-expressing plasmids (1, 2, or 5 μg) for 24 h and then infected with SeV for 16 h. The cells were lysed and immunoprecipitated with IRF3. The WCLs and IP complexes were analyzed by Western blotting using anti-IRF3, anti-TBK1, anti-Flag, and anti-β-actin antibodies. (B) HEK-293T cells were transfected with empty vector or increasing amounts of Flag-N-expressing plasmids (1, 2, or 5 μg) for 24 h and then transfected with poly(I·C) for 24 h. The cells were lysed and immunoprecipitated with IRF3. The WCLs and IP complexes were analyzed by Western blotting using anti-IRF3, anti-TBK1, anti-Flag, and anti-β-actin antibodies. (C) HEK-293T cells were cotransfected with equal amounts of empty vector- or Flag-N-expressing plasmids and HA-IRF3 or HA-tagged IRF3 truncated mutants expressing plasmids (HA-IRF3-Δ-1-140, HA-IRF3-Δ-140-400, or HA-IRF3-Δ-400-427) for 48 h. The cells were then lysed and immunoprecipitated with an anti-Flag antibody. The WCLs and IP complexes were analyzed by Western blotting using an anti-HA, anti-Flag, or anti-β-actin antibody.