FIG. 2.

Effects of mutations in the IRF-3 inhibitory domain of VP35 on viral transcription/replication. The 3E-5E CAT minigenome transcription/replication system was used to determine the effects of VP35 mutations on viral transcription. 293T cells were transfected in six-well plates with the 3E-5E minigenome (1 μg) along with expression plasmids for pT7-pol (1 μg), pCEZ-NP (1 μg), pCEZ-VP35 (0.5 μg), pCEZ-VP30 (0.3 μg), and pCEZ-L (1 μg). Forty-eight hours posttransfection, the cells were lysed, and CAT levels were measured by an ELISA. Results shown are averages of wells transfected in duplicate and are representative of three independent experiments. Lysates were subjected to SDS-PAGE and Western blotting to determine VP35 and actin expression levels, and expression ratios were calculated as described in the legend to Fig. 1. Neg Control, negative control.