FIG. 3.
Hsp70 affects the in vitro activity of the tombusvirus replicase and binds to the functional replicase. (A) In vitro replicase assay using the membrane-enriched fraction carrying the copurified repRNA. Gels show in vitro synthesis of the repRNA products by the tombusvirus replicase preparations obtained from WT (lanes 1 to 3) and ssa1 ssa2 (lanes 4 to 6) yeast. Yeast cells were harvested 16 h after the induction of TBSV replication. Note that p33 and p92 were expressed from the galactose-inducible GAL1 promoter, resulting in similar amounts of p33 and p92 proteins. (B) Western blot analysis of p33 and p92pol levels in the replicase preparations. Both proteins were expressed from the GAL1 promoter. (C) Copurification of the functional tombusvirus replicase with FLAG-tagged Ssa1p. The membrane-enriched fraction of yeast coexpressing His6-tagged p33, p92pol, and DI-72 (+)repRNA with FLAG-tagged Ssa1p was solubilized with a detergent, followed by FLAG affinity purification. The FLAG-Ssa1p preparations obtained were tested by a standard replicase assay with an added minus-stranded repRNA template for the presence of copurified tombusvirus replicase. The control preparations were also prepared using FLAG affinity purification, but from yeast expressing His6-tagged Ssa1p.