Fig 6.
Inhibition of TBSV replication by recombinant Pkc1p in vitro. (A) Scheme of the CFE-based TBSV replication assay. Purified recombinant p33 and p92pol replication proteins of TBSV and in vitro-transcribed TBSV DI-72 (+)repRNA were added to the whole-cell extract prepared from the wt yeast strain. The purified recombinant yeast Pkc1p was added before (exp. C) or during (exp. B) the CFE-based TBSV replication assay. (B) Denaturing PAGE analysis of the 32P-labeled TBSV repRNA products obtained in the in vitro CFE-based TBSV replication assay in the presence of recombinant Pkc1p (1× = 200 ng). Each experiment was repeated three times. (C) CFE-based assay similar to that in panel B, except that Pkc1p was preincubated with p33/p92 in the reaction buffer for 30 min at 25°C. (D) Western blot analysis of purified recombinant GST-Pkc1p with anti-GST antibody.