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. 2009 Dec 24;5(12):e1000705. doi: 10.1371/journal.ppat.1000705

Figure 3. Reduced activity of the tombusvirus replicase assembled in yeast with deletion of selected ESCRT genes.

Figure 3

(A) Denaturing PAGE analysis of in vitro replicase activity in the membrane-enriched fraction from wt and vps23Δ yeast using the co-purified repRNA. Note that this image shows the repRNAs made by the replicase in vitro. Asterisk marks a recombinant RNA species formed. Bottom panel shows a Western blot of p33 in the replicase preparations as seen in the top panel. (B) Decreased replication of TBSV repRNA in yeast extracts prepared from wt, vps4Δ, snf7Δ or vps24Δ yeast strains expressing p33 and p92pol. The yeast extracts were programmed with DI-72(+)repRNA and the radiolabeled in vitro repRNA products were detected via denaturing PAGE analysis. (C) Increased RNase sensitivity of TBSV minus-strand (−)repRNA during replication in a membrane-enriched replicase preparation obtained from WT, vps23Δ or vps24Δ yeast. At the end of the assays, the replicase preparations were treated with RNase A for 5 min, followed by inactivation with phenol-chloroform. The (−)repRNA protected in the replicase complex from the ribonuclease was detected using a 32P-UTP labeled probe. The untreated preparation was chosen as 100%. Note that the (−)repRNA is protected from RNase degradation by the membrane-associated viral replicase complex.