Fig 4. Suppression of TBSV recRNA accumulation by Ded1p in in vitro replication assays.
(A) The membrane-enriched fraction (MEF) was isolated from wt or ded1–199ts yeast expressing the tombusvirus p33 and p92 replication proteins in combination with the DI-AU-FP repRNA, followed by in vitro tombusvirus replication assay. The denaturing PAGE analysis shows the emergence of recRNAs and degRNA as indicated. (B–C) Northern blot analysis of the polarity of the TBSV RNAs synthesized in the MEF assay (see panel A, except the assay was done with nonlabeled ribonucleotides) using 32P-labeled probes to detect (-)-strands and (+)-strands, respectively. The ratios between various TBSV RNAs were calculated as in Fig. 2. (D) Scheme of the yeast cell-free (CFE) assay for TBSV replication. Note that the assembly of the TBSV replicase takes place in the CFE using recombinant viral components as shown. The TBSV p33 and p92 replication proteins and Ded1p and its mutants (D1 is inactive, D11 has enhanced ATPase activity) were purified from E. coli. (E) Reduced replication of the full-length DI-72 repRNA in TET::DED1 yeast CFEs with high or depleted levels of Ded1p (see Fig. 1). (F-G) Denaturing PAGE analysis of the effect of Ded1p and its mutants on the replication of the full-length recombinogenic DI-AU-FP and the 5’-truncated DI-RIIΔ70 degRNA, respectively, in the CFE assay. The experiments were repeated twice.