In this paper, we characterize the function of eEF1Bc in TBSV replication. Our approaches based on yeast and in vitro replication assays reveal that eEF1Bc is a component of the tombusvirus replicase and binds to the 39-end of the viral RNA. Using a cellfree replication assay, we define that eEF1Bc plays a role by enhancing minus-strand synthesis by the viral replicase. The obtained data support the model that eEF1Bc opens up a 'closed' structure at the 39-end of the TBSV (+)RNA, rendering the RNA compatible for initiation of (-)-strand synthesis. Moreover, we find that eEF1Bc and eEF1A play nonoverlapping functions to enhance (-)-strand synthesis. Altogether, the two translation factors regulate TBSV replication synergistically by interacting with different portions of the viral (+)RNA and the replication proteins.