We confirmed a direct role for eEF1A in RNA synthesis in vitro by using a highly purified eEF1A and the recombinant TCV RdRp, which is closely homologous with the TBSV p92 pol . Interestingly, it seems that eEF1A stimulates the RdRp activity directly, since pre-incubation of eEF1A and the RdRp prior to the RdRp assay led to the highest level of stimulation of (2)RNA synthesis (Fig. 3A) . On the other hand, pre-incubation of eEF1A with the TBSV-derived template RNA led only to ,2-fold increase in RNA synthesis in vitro (Fig. 3A) . Analyzing the amount of short abortive RdRp products, which are produced through initiation followed quickly by abortive termination [57] , in the in vitro assays revealed that eEF1A strongly enhanced the initiation of minus-strand synthesis (Fig. 3B) . Although the actual mechanism of stimulation of RdRp activity by eEF1A is currently unknown, we propose that eEF1A might facilitate the proper and efficient binding of the RdRp to the 39 terminal sequence of the viral RNA prior to initiation of (2)-strand synthesis (Fig. 5) . Accordingly, eEF1A was shown to bind to the so-called replication silencer sequence (RSE) in the 39-UTR, which is required for the assembly of the viral replicase complex [22, 58] . The binding of eEF1A-RdRp complex to the RSE might assist in placing the RdRp over the 39-terminal promoter sequence, thus facilitating the initiation of (2)RNA synthesis starting from the 39-terminal cytosine. Similar function of eEF1A in stimulation of (2)RNA synthesis has been proposed for WNV, based on mutations in the viral RNA within the eEF1A binding sequence that reduced the binding affinity of RNA to eEF1A and inhibited (2)RNA synthesis in infected cells [46] . eEF1A stimulates the assembly of the viral replicase complex during TBSV replication Recent intensive work revealed that the assembly of the viral replicase complex is a regulated process involving viral-and host factors, cellular membranes and the viral (+)RNA [8, 10, 19, 63, 64, 65] . The assembly of the viral replicase also depends on steps occurring prior to the actual assembly process, such as selection of the viral template RNA and the recruitment of (+)RNA/protein factors to the sites of assembly. Although our current understanding is rather poor about the factors involved and their functions during replicase assembly, rapid progress is being made in this area due to the development of a new cell-free assay based on yeast CFE [30, 31] . The yeast CFE is capable of assembling the tombusvirus replicase complex in vitro in 40-60 min in the presence of recombinant p33/p92 pol and the viral (+)repRNA [31] , allowing for studies on direct roles of various factors. We find that inhibition of eEF1A activity by either DB or GM also inhibited the assembly of the tombusviral replicase complex based on time-course experiments (Fig. 4B ) as well as a direct replicase assembly assay (Fig. 4C) . On the contrary, the replicase activity was not inhibited by these compounds after the assembly took place (Fig. 4B-C) . It is possible that after the formation of the eEF1A-RdRp-repRNA complex DB or GM are not effective in inhibiting the stimulatory effect of eEF1A on the RNA synthesis by the viral RdRp. Additional in vitro experiments with purified tombusvirus replicase preparations confirmed the lack of inhibition of RNA synthesis by DB or GM (Fig. S4A ) on pre-assembled virus replicases.