FIG. 6.

Functional characterization of P-DDSS and P-AASS and their corresponding X mutants. (A) Mammalian two-hybrid analysis with the indicated pCA-Gal4 and pCA-VP16 constructs was carried out as described in the legend of Fig. 2. Expression of Renilla luciferase was used to normalize for transfection efficiency. The normalized firefly luciferase expression observed with P-wt was set to 100%. (B) Cell extracts were prepared for coimmunoprecipitation studies 24 h posttransfection and subjected to immunoprecipitation (IP) using anti-HA-agarose (αHA). Precipitated material was separated by 15% SDS-PAGE and analyzed by Western blotting (WB) for the presence of X and P using an X/P-specific antibody. (C) Cofactor activity of the P mutants in the presence or absence of X mutant proteins was analyzed with the BDV minireplicon assay as described in the legend of Fig. 1C. Increasing levels of X-expressing plasmids correspond to 20, 40, and 80 ng. Mean values of at least three independent assays are shown. Complete reaction mixtures without P served as a negative control reaction (no P). (D) Double immunofluorescence analysis using polyclonal rabbit anti-P-antibodies (αP) and mouse monoclonal anti-X-antibodies (αX) of Vero cells persistently infected with the indicated recombinant viruses.