Recombinant Ded1p DEAD-box helicase enhances TBSV RNA replication in yeast cell-free extract To address if Ded1p could play a direct role in TBSV replication, we added purified recombinant Ded1p to yeast CFE with reduced level of Ded1p (Figure 2A ). We observed a ,2-fold increased level of TBSV RNA replication (based on total singlestranded repRNA level) in the presence of recombinant Ded1p when compared with the MBP control ( Figure 2B , lane 2 versus 4). Interestingly, the amount of double-stranded RNA, which correlates with (2)-strand levels [23] , did not increase in the presence of Ded1p in the CFE assay ( Figure 2B, lanes 1 versus 3) . This finding suggests that (2)-strand RNA did not contribute to the 2-fold increase in total TBSV RNA levels when recombinant Ded1p was added to the CFE-based replication assay. Therefore, we conclude that the added recombinant Ded1p selectively increased TBSV (+)-strand RNA synthesis in vitro.