To test if eEF1A stimulates the rate of RNA synthesis in the absence of de novo initiation, we analyzed the amount of 39terminal extension (39-TEX) RNA products, which are generated from an internal primer by the TCV RdRp (Fig. 3D ) [55] . Addition of purified eEF1A did not increase the amount of 39TEX products (lanes 2, 4, 6, Fig. 3D ), suggesting that the elongation step The TBSV (+)RNA template was the short 39 end region (SL1/SL2/SL3), which contain the promoter region (SL1) for initiation and the replication silencer element (within SL3) that down-regulates initiation. The second template was SL1m with a point mutation within the promoter sequence, which is being used more efficiently by the TCV RdRp in vitro. Note that eEF1A has been shown to bind to the replication silencer element. The RdRp assay had two steps: first, the shown components were incubated at room temperature to facilitate their interaction, followed 5 min latter the addition of the shown component and the ribonucleotides to start RNA synthesis. The RdRp activity in samples containing the template RNA and the RdRp were chosen as 100% (lanes 3-4). (B) Detection of abortive RdRp products in the in vitro assay. 15% PAGE/UREA gel was used to resolve the 4-10 nt long products produced during initiation followed by rapid termination. Note that abortive RNA products are characteristic products for RNA polymerases that initiate de novo (in the absence of a traditional primer). (C) Lack of stimulation of 39-terminal extension by eEF1A in vitro. The template RNAs (shown schematically) contain a common artificial hairpin structure at the 39 end that facilitates 39-TEX by the TCV RdRp. The black bar represents 3 different sequences in the three constructs, derived from RIV(+)(includes SL1/SL2/SL3 sequences), RIII(2) and RIII(+) of DI-72 RNA, respectively. The gel image shows the results of 39-TEX in the presence of 0 or 1 mg eEF1A as shown in a TCV RdRp assay. doi:10.1371/journal.ppat.1001175.g003 during complementary RNA synthesis is not affected by eEF1A. Altogether, the obtained in vitro TCV RdRp data suggest that eEF1A can mostly stimulate the initiation step during de novo viral (2)RNA synthesis.