To test if the stimulating activity of eEF1Bc on the in vitro RdRp activity was due to binding of eEF1Bc to the (+)RNA template and/or to the TCV RdRp protein, we performed assays, in which the recombinant eEF1Bc was pre-incubated with the TCV RdRp or the (+)RNA template prior to the RdRp assay. These experiments revealed that pre-incubation of the purified eEF1Bc with the TBSV-derived SL3-2-1(+) RNA template prior to the RdRp assay led to a ,4.5-fold increase in (-)RNA products ( Figure 2B, lanes 1-2) . In contrast, pre-incubation of the TCV Author Summary RNA viruses recruit numerous host proteins to facilitate their replication and spread. Among the identified host proteins are RNA-binding proteins (RBPs), such as ribosomal proteins, translation factors and RNA-modifying enzymes. In this paper, the authors show that deletion of eukaryotic translation elongation factor 1Bgamma (eEF1Bc) reduces Tomato bushy stunt virus (TBSV) replication in a yeast model host. Knock down of eEF1Bc level in plant host also decreases TBSV accumulation. Moreover, the authors demonstrate that eEF1Bc binds to the viral RNA and is present in the tombusvirus replicase complex. Functional studies revealed that eEF1Bc promotes minusstrand synthesis by serving as an RNA chaperone. The authors also show that eEF1Bc and eukaryotic translation elongation factor 1A, another host factor, function together to promote tombusvirus replication. Cell-free TBSV replicase assay supports a role for eEF1Bc in minus-strand synthesis. Purified recombinant TBSV p33 (12 pmol) and p92 pol (1 pmol) replication proteins in combination with DI-72 (+)repRNA (4 pmol)were added to the whole cell extract prepared from tef4D (lanes 1-2) or WT yeast strains. Left panel: The nondenaturing PAGE analysis of the 32 P-labeled repRNA products obtained is shown. The full-length single-stranded repRNA is pointed at by an arrow. 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 heattreated replicase products (ssRNA is present). The amount of ssRNA 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 input (+)RNA, while the ssRNA products represents the newly made (+)RNA products. Right panel shows the coomassie-blue stained SDS-PAGE gel to visualize total protein levels in the whole cell extracts. (D) eEF1Bc stimulates TBSV repRNA synthesis in whole cell extract prepared from tef4D. Increasing amounts of purified recombinant eEF1Bc (lanes 3-4, 26 pmol; lanes 5-6, 13 pmol) were added to tef4D CFE and the in vitro synthesized 32 P-labeled TBSV repRNA was measured on denaturing PAGE. See further details in panel C. Note that the recombinant eEF1Bc added to the tef4D CFE is about 10-fold less than the total eEF1Bc present in the WT CFE. doi:10.1371/journal.ppat.1002438.g001