Since the TBSV (+)RNA, including the minimal SL3-2-1 sequence, forms a secondary structure where the replication silencer sequence (RSE) in SL3 base-pairs with the 39-terminal 5 nts within the genomic promoter (gPR) (both sequences are highlighted with gray boxes in Figure 4A ), it is possible that eEF1Bc helps (-)-strand synthesis by opening up the gPR. The single-stranded gPR sequence would be more accessible for (-)strand synthesis as shown based on RNA mutagenesis [39] . To test this model, we obtained a complementary RNA that formed a duplex with SL1 and neighboring sequences, but leaving SL1 including the 'GAAA' loop-sequence nonbase-paired to facilitate binding to eEF1Bc ( Figure 4B ). Interestingly, eEF1Bc was able to stimulate (-)-strand synthesis by 70%, suggesting that eEF1Bc might indeed facilitate opening up the 39-terminal structure when it is part of a duplex. eEF1Bc co-purifies with the viral replicase complex and it binds to TBSV repRNA in yeast To test if eEF1Bc is a component of the tombusvirus replicase, we purified the His 6 -Flag-tagged p33 (HF-p33) replication protein via Flag-affinity purification from the detergent-solubilized membrane fraction of yeast [10] . We detected both p33 and eEF1Bc in the purified preparation ( Figure 5A , lane 1), suggesting that eEF1Bc is likely part of the replicase complex [21] . Importantly, eEF1Bc was not found in the control samples containing the His 6 -tagged p33 (H-p33) that were also purified via the Flag-affinity procedure ( Figure 5A , lane 2). Since eEF1Bc does not seem to bind to p33 or p92 replication proteins (data not shown), it is likely that eEF1Bc was co-purified with p33 via the viral RNA template in the viral replicase complex.