In this paper, a yeast-based cell-free TBSV replication assay and recombinant Ded1p was used to test the role of Ded1p in TBSV replication. We define that Ded1p plays a role in enhancing plusstrand (+)RNA synthesis by the viral replicase. We also find that Ded1p is a component of the tombusvirus VRC and binds to the 39-end of the viral minus-stranded (2)RNA. The obtained data support the model that Ded1p unwinds the 39-end of the TBSV (2)RNA, rendering the RNA compatible for initiation of (+)strand synthesis. Interestingly, we find that Ded1p and GAPDH play non-overlapping functions to enhance (+)-strand synthesis. Altogether, the two host factors enhance TBSV replication synergistically by interacting with the viral (2)RNA and the replication proteins.