Reduced level of TBSV RNA replication in yeast cell-free extract depleted for Ded1p DEAD-box RNA helicase Since Ded1p is an essential translation factor for all yeast mRNAs [38, 39] , it is difficult to separate its direct versus indirect effect on TBSV replication in yeast model host. To circumvent this problem, we used whole cell extracts (CFE) prepared from yeast containing reduced level of Ded1p ( Figure S1 ) to support cell-free TBSV replication. As we have shown previously, TBSV (+)RNA can perform one complete cycle of replication in the CFE-based replication assay when purified recombinant p33 and p92 pol replication proteins are provided [23, 40] . The CFE-based replication assay showed that both (+) and (2)RNA synthesis decreased by over 4-fold when Ded1p was down-regulated as compared with the control CFE prepared from yeast with high level of Ded1p expression ( Figure S1 , lanes 4-6 versus 1-3). Thus, these data confirm that Ded1p is important for TBSV replication. However, the observed decrease in the CFE-based TBSV replication could be due to either reduced replication when Ded1p is limiting (direct effect) or lesser amounts of other critical host factors needed for TBSV replication in the CFE with reduced level of Ded1p (indirect effect due to Ded1p's role in host translation). To address these points, we first studied if Ded1p is present within the tombusvirus replicase and then, what is the mechanistic role of Ded1p during TBSV RNA synthesis.