Since both eEF1Bc and eEF1A bind to the 39-terminal region of the TBSV (+)RNA ( Figure 3 ) and ref: [23, 24] , it is possible that they could affect each other's functions during replication. To test the mutual effect of eEF1Bc and eEF1A on the (-)-strand RNA production of the viral RdRp, we performed in vitro RdRp assays with purified eEF1A and recombinant eEF1Bc as shown in Figure 6 . Based on previous experiments, eEF1Bc was known to stimulate (-)-strand synthesis the most when pre-incubated with the template (+)RNA ( Figure 2B ). In contrast, pre-incubation of eEF1A with the viral RdRp was more effective than preincubation of eEF1A with the template RNA [23] . Therefore, we performed the pre-incubation experiments prior to the RdRp assay as shown in Figure 6 . We found the largest stimulation of (-)-strand synthesis by the viral RdRp in a dual pre-incubation assay, when eEF1Bc was pre-incubated with the viral RNA template, while eEF1A was separately pre-incubated with the viral RdRp ( Figure 6, lanes 3-4) . Pre-incubation of eEF1Bc with the viral RNA template (lanes 5-6) or pre-incubation of eEF1A with the viral RdRp (lanes 7-8) were about half as efficient in stimulation of (-)-strand synthesis than the dual pre-incubation assay (lanes [3] [4] . Therefore, these data support the model that eEF1Bc and eEF1A both promote (-)-strand synthesis and their effect is synergistic, likely involving separate mechanisms (see Discussion). in vitro binding assay with purified eEF1Bc using an ssDNA oligo/ssRNA template duplex. The annealed ssDNA (purple)/ssRNA (black) duplexes representing the 39 end of the TBSV RNA are shown schematically. The assay contained the annealed ssDNA/ssRNA plus 0.6 and 0.4 pmol purified recombinant eEF1Bc, respectively. The 32 P-labeled free ssDNA and ssDNA/ssRNA duplex were separated on nondenaturing 5% acrylamide gels. Quantification of the ssDNA/ssRNA duplex was done with ImageQuant. (B) RNA gel shift analysis shows the role of the SL1 tetraloop in binding to eEF1Bc. The RNA templates representing the 39 end of the TBSV RNA and the mutations (circled nucleotides) are shown schematically. The eEF1Bc -32 P-labeled ssRNA complex was visualized on nondenaturing 5% acrylamide gels. The RNA transcript (0.2 pmol), and eEF1Bc (0.4, 0.5 and 0.6 pmol) were used in these assays. doi:10.1371/journal.ppat.1002438.g003