FIG 3.
HAV 3Cpro disrupts NEMO-mediated IFN-β induction. (A) HEK293T cells cultured in 60-mm dishes were transfected with Flag-tagged NEMO-K277A expression plasmid (2 μg) along with plasmid encoding HA-tagged 3Cpro (1 μg). Cell lysates were prepared 30 h posttransfection and analyzed by Western blotting. (B) HEK293T cells cultured in 60-mm dishes were transfected with Flag-tagged NEMO expression plasmid (2 μg), along with the indicated 3Cpro expression plasmids (1 μg). Cell lysates were prepared 30 h posttransfection and analyzed by Western blotting. (C) HEK293T cells cultured in 24-well plates were transfected with the indicated reporter plasmid (0.1 μg), pRL-TK (0.02 μg), and Flag-tagged NEMO-K277A expression plasmid (0.5 μg), along with increasing quantities (0, 0.0625, 0.125, 0.25, or 0.5 μg) of plasmid encoding 3Cpro. Luciferase assays were performed at 36 h after the transfection. (D) HEK293T cells cultured in 24-well plates were cotransfected with the indicated reporter plasmid, pRL-TK plasmid, and Flag-tagged NEMO-K277A expression plasmid (0.5 μg) along with the designated 3Cpro expression plasmids (0.5 μg). An empty vector (pCMV-HA) was used as a control. Cell extracts were collected 36 h after transfection and analyzed for firefly and Renilla luciferase expression.