Pre-incubation of the purified wt eEF1A with the TCV RdRp prior to the RdRp assay led to a ,5-fold increase in in vitro (2)RNA synthesis (Fig. 3A , lanes 9-10), while pre-incubation of the purified eEF1A with the TBSV (+)RNA template prior to the RdRp assay led only to a ,2-fold increase in (2)RNA products (lanes 7-8). Also, preincubation of the TCV RdRp with the (+)RNA template prior to the RdRp assay containing purified eEF1A led only to a ,2-fold increase in (2)RNA synthesis (lanes 5-6), suggesting that eEF1A can stimulate (2)RNA synthesis less efficiently after the formation of the (+)RNA-RdRp complex. Overall, data shown in Fig. 3 imply that eEF1A stimulates (2)RNA synthesis most efficiently when it forms a complex with the viral RdRp prior to binding of the template RNA to the eEF1A-RdRp complex.