In vitro initiation of (-)-strand synthesis by the viral RdRp requires the gPR promoter consisting of a short 39-terminal singlestranded tail and a stem-loop (SL1) sequence [39, 50] . However, Figure 6 . Synergistic effect of eEF1Bc and eEF1A on stimulation of minus-strand synthesis by the closely-related TCV RdRp. Purified eEF1Bc (20 pmol) and eEF1A (20 pmol) were added to the TCV RdRp (2 pmol) assay as shown. The RdRp assay had two steps: first, the shown components on the top and bottom were incubated in separate tubes at room temperature to facilitate their interaction, followed 5 min later by mixing the components from the two tubes and addition of the ribonucleotides to start RNA synthesis. The RdRp activity in samples containing the template RNA and the RdRp were chosen as 100% (lanes 1-2 and 9-10). The gel image shows the results of RNA synthesis in the presence of equal amounts of purified eEF1Bc and eEF1A as shown in a TCV RdRp assay. doi:10.1371/journal.ppat.1002438.g006 The FLAG/His 6 -tagged HF-p33 was purified from yeast extracts using a FLAG-affinity column. The purified HF-p33 and the co-purified His 6 -tagged eEF1Bc were detected with anti-His antibody. Bottom panel: Western blot of HF-p33 and the His 6 -tagged eEF1Bc in the total yeast extract using anti-His antibody. (B) RT-PCR analysis to detect the co-purified TBSV (+)RNA in the affinity-purified His 6 -tagged eEF1Bc preparation from yeast replicating TBSV repRNA. Both the membrane and soluble yeast fractions were used for eEF1Bc purification and subsequent RT-PCR analysis to detect (+)repRNA. ''+'' and ''-'' mean that His 6 -tagged eEF1Bc was expressed from a plasmid or not in yeast. Samples were used for RT-PCR (lanes 3-4 and 7-8) or for PCR (without RT reaction, lanes 1-2 and 5-6). doi:10.1371/journal.ppat.1002438.g005 the gPR region is present in a 'closed' structure in the TBSV (+)RNA due to base-pairing of a portion of the gPR with the RSE present in SL3 as shown in Figure 9 . This interaction makes the TBSV (+)RNA poor template in the in vitro assay due to the difficulty for the viral RdRp to recognize and/or open the 'closed' structure [39] . Our current work with eEF1Bc, however, suggests that eEF1Bc can bind to the tetraloop region of SL1 (and to an Arich sequence in SL2) that leads to melting of the base-paired structure and opening the stem of SL1 and the RSE-gPR basepairing as shown schematically in Figure 9B . We propose that the open structure can be recognized efficiently by the viral replicase leading to efficient initiation of (-)-strand synthesis ( Figure 9B ). This model is supported by several pieces of evidence presented in this paper, including (i) stimulation of (-)-strand synthesis by eEF1Bc when the wt SL1 is present in the template; (ii) lack of stimulation of(-)-strand synthesis by eEF1Bc when a mutated SL1 (tetraloop mutant), which does not bind efficiently to eEF1Bc, was used as a template in the in vitro assay; (iii) stimulation of (-)-strand synthesis when eEF1Bc was pre-incubated with the (+)-strand template, but not when eEF1Bc was pre-incubated with the viral RdRp ( Figure 2) ; and (iv) the lack of stimulation of (+)-strand synthesis on a (-)-strand template by eEF1Bc (Figure 2 ). In addition, eEF1Bc stimulated (-)-strand synthesis by the viral RdRp when a partially complementary RNA oligo was hybridized with the SL1 region ( Figure 4B ). However, eEF1Bc could not efficiently bind to the 39-end of the TBSV RNA when it formed a hybrid (duplex) with a perfectly complementary DNA oligo ( Figure 3A) , suggesting that eEF1Bc can melt only the local secondary structure, but cannot unwind more extended duplex regions. An alternative possibility is that eEF1Bc protein stabilizes the unpaired structure (when the SL1 structure is kinetically pairing/unpairing), rather than implying that it actively "opens" the structure.