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. 2007 Mar 21;81(11):5497–5507. doi: 10.1128/JVI.02233-06

FIG. 7.

FIG. 7.

Impact of single amino acid substitutions at amino acid position 70 or 86 of BDV-P. (A) Cofactor activity of P mutants using the BDV minireplicon assay as described in the legend of Fig. 1C. Mean values of at least three independent assays are shown. Complete reaction mixtures without P served as a negative control reaction (no P). (B) Table showing the ability to rescue BDV mutants. (C) Mammalian two-hybrid analysis. Expression of Renilla luciferase was used to normalize for transfection efficiency. The normalized firefly luciferase expression observed with P-wt was set to 100%. (D) Coimmunoprecipitation studies. Cell extracts were prepared 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 the indicated HA-tagged and X proteins using an X/P-specific antibody (αX/P). (E) Cofactor activity of the P mutants in the presence or absence of X wt or mutant proteins using the BDV minireplicon system. Mean values of at least three independent assays are shown. Complete reaction mixtures without P served as a negative control reaction (no P). Increasing levels of X-expressing plasmids correspond to 20, 40, and 80 ng.