The ATPase inactive mutant D1 ( Figure 4B , lanes 5-6 and S3D, lane 4), D5 and D10 ( Figure S3D , lanes 6 and 8) could not promote (+)-strand synthesis, suggesting that the ATPase activity of Ded1p is required for the above stimulatory effect on TBSV RNA synthesis. Also, two active ATPase mutants, D3 and D11 ( Figure S3C ), did facilitate (+)-strand synthesis ( Figure S3D , lanes 5 and 9), confirming that the ATPase activity of Ded1p is required during TBSV (+)-strand RNA synthesis. Interestingly, Ded1p mutant D11, albeit has increased ATPase activity, has wt-like strand displacement activity in vitro [41] . We found that D11 behaved similarly in TBSV replicase assay ( Figure S3D ) to wt Ded1p, suggesting that the unwinding activity of Ded1p is important during TBSV replication.