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. 2010 Nov 4;6(11):e1001175. doi: 10.1371/journal.ppat.1001175

Figure 2. Cell-free TBSV replicase assay supports a role for eEF1A in minus-strand synthesis.

Figure 2

(A) Purified recombinant p33 and p92pol replication proteins of TBSV in combination with DI-72 (+)repRNA were added to the whole cell extract prepared from eEF1A mutant or WT yeast strains as shown (lanes 1–5). Top panel: The denaturing PAGE analysis of the 32P-labeled repRNA products obtained is shown. The full-length repRNA is pointed at by an arrow. Panels below show Western blot analysis of the whole cell extracts for the indicated yeast proteins based on specific antibodies. Bottom panel shows the coomassie-blue stained SDS-PAGE gel to visualize total protein levels in the whole cell extracts. (B) Detection of single- and double-stranded RNA products produced in the cell-free TBSV replicase assay. 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 (mostly ssRNA is present). The amount of dsRNA and the ratio of ssRNA/dsRNA 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. (C) Denaturing PAGE analysis of the TBSV replicase products obtained in the cell-free replicase assay after S1 nuclease treatment, which cleaves the ssRNA, but not the dsRNA product. (D) The denaturing PAGE analysis of the 32P-labeled repRNA products obtained in the in vitro reconstitution assay is shown. The membrane fraction of the whole cell extracts prepared from eEF1A mutant strains were mixed with the supernatant fraction of CFE prepared from WT eEF1A (lanes 6–10) or the supernatant fraction of CFE from the mutant strains were added to the membrane fraction from the wt strain (lanes 11–15). The reconstituted extracts were programmed with purified recombinant TBSV p33/p92pol and (+)repRNA. Bottom panel: Western blot analysis shows the amount of endogenous eEF1A in various fractions (see above) prepared from yeast expressing various mutants of eEF1A.