FIG. 4.

Effect of VP35 and dsRNA-binding mutants on virus- and RIG-I-mediated activation of IRF-3. HEK293 cells were transfected with 4 μg of plasmids that express wild-type VP35 (VP35), R312A, and K309A, with or without 40 ng of RIG-I plasmid as indicated. Twenty-four h posttransfection, cells were either mock SeV infected or infected with Sendai virus (MOI of 8), as indicated. Eight h postinfection, cells were lysed and proteins were separated in a continuous 7.5% native gel that was prerun with and without 0.2% sodium deoxycholate in the cathode and anode chambers, respectively. Endogenous IRF-3 was detected by Western blotting with a primary rabbit anti-hIRF-3 (1:500) antibody and a secondary goat anti-rabbit immunoglobulin G-horseradish peroxidase (1:5,000) antibody (top panel). Expression of P56 and the different forms of VP35 in the same cell lysates was analyzed by Western blotting following separation of proteins by 12.5% sodium dodecyl sulfate-PAGE. A mouse anti-human β-tubulin antibody was used as a loading control.