Addition of purified Ded1p to the CFE during the first step did not increase TBSV replication ( Figure 3B , lane 4 versus 5). This suggests that Ded1p did not facilitate the assembly of the replicase complex, unlike other host factors, such as Hsp70 and eEF1A [23, 27] . Moreover, Ded1p was likely lost during the centrifugation/washing step in this assay due to its low association with the membrane. However, addition of Ded1p exclusively during the second step of the CFE assay resulted in ,2-fold increase in TBSV RNA replication ( Figure 3B , lane 14 versus 15), similar to the stimulatory effect of Ded1p during standard CFE replication assay ( Figure 2 ). Interestingly, a Ded1p mutant (D1) deficient in ATPase activity could not stimulate TBSV RNA synthesis in this assay ( Figure 3B , lane 13), while a mutant (D11, Figure S3C ) with increased ATPase activity [41] promoted TBSV RNA synthesis by ,2-fold ( Figure 3B , lane 11). Altogether, these findings indicate that Ded1p has a direct stimulatory function during TBSV RNA synthesis, while Ded1p is unlikely to affect viral RNA recruitment for replication or VRC assembly.