To test if eEF1A stimulates the rate of initiation of (2)RNA synthesis, we analyzed the amount of abortive RNA products, which are generated during de novo initiation of RNA synthesis by the TCV RdRp [57] . We found that the amount of the 5-11 nt long abortive RNA products increased by 3.5-fold in the presence of purified eEF1A in the TCV RdRp assay (Fig. 3B , lanes 3-4 versus 1-2). We also tested the RdRp activity in the presence of Top panel: Replication of the TBSV repRNA was measured by Northern blotting 24 h after initiation of TBSV replication. The accumulation level of repRNA was normalized based on the rRNA (middle panel, the 18S ribosomal RNA levels were estimated by Northern blotting). Bottom two panels: Accumulation of p33/p92 pol and eEF1A was estimated by Western blotting using anti-His and anti-eEF1A antibody, respectively. Note that * marks an SDS-resistant p33 homodimer band. (B) An in vitro replicase assay to test the relative activity of the tombusvirus replicase obtained from yeast expressing various mutants of eEF1A. Top panel: We tested the in vitro replicase activity using comparable amounts of affinity-purified replicase with added DI-72 RI(2) RNA template. Bottom panels: Western blot analysis showing p33 viral replication protein and the co-purified eEF1A in the above purified replicase preparations. (C) Critical eEF1A residues for tombusvirus replication. Three novel mutants of eEF1A were identified, which exhibited increased tombusvirus replication (V301D, L374V/N377K, and F413L; yellow balls) while the new A76V and the previously identified T22S exhibited decreased tombusvirus replication (green balls). The structure of eEF1A was generated using Jmol with PDB coordinates 1IJE. doi:10.1371/journal.ppat.1001175.g001 eEF1A using a (+)RNA template with a mutation opening the closed structure in the promoter region that leads to increased template activity [58] . The mutated template also showed 2-fold increased abortive RNA products in the RdRp assay with eEF1A ( Fig. 3B , lanes 5 versus 6). These data strongly support the model that eEF1A stimulates the de novo initiation step in the RdRp assay. The denaturing PAGE analysis of the 32 P-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 (2)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 32 P-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] [12] [13] [14] [15] . The reconstituted extracts were programmed with purified recombinant TBSV p33/p92 pol 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. doi:10.1371/journal.ppat.1001175.g002