Cell-free TBSV replication assay supports a role for Ded1p helicase in plus-strand synthesis.

<p>(A) Scheme of the CFE-based TBSV replication assay. Purified recombinant p33 and p92^pol replication proteins of TBSV and <i>in vitro</i> transcribed TBSV DI-72 (+)repRNA were added to the whole cell extract prepared from Ded1p-depleted yeast strain. The assay also contained purified recombinant Ded1p (0.3 µg) or MBP (the same molar amount as Ded1p) as a control. (B) Left panel: Detection of single- and double-stranded RNA products produced in the cell-free TBSV replication assay. The dsRNA products are also shown on the right subpanel with high contrast for better visualization. Odd numbered lanes represent replicase products, which were not heat treated (thus both ssRNA and dsRNA products are present), while the even numbered lanes show the heat-treated replicase products (only ssRNA is present). The % of dsRNA and ssRNA in the samples are shown. Note that, in the nondenatured samples, the dsRNA product represents the annealed (−)RNA and the (+)RNA, while the ssRNA products represents the newly made (+)RNA products. Right panel: A higher contrast image of portion of the left panel to aid the visualization of the dsRNA products. Each experiment was repeated three times. (C) Denaturing PAGE analysis of the ³²P-labeled TBSV repRNA products obtained in the CFE assay in the presence of ATPase inactive D1 mutant of Ded1p (0.3 µg), wt Ded1p (0.3 µg) or MBP. Each experiment was repeated three times.