The in vitro data, based on the CFE assay containing the membrane-bound VRC as well as the solubilized/purified tombusvirus replicase, showed the Ded1p can mainly stimulate TBSV (+)-strand synthesis, while its effect on (2)RNA synthesis is less pronounced. The ATPase activity of Ded1p is required for this stimulatory effect, suggesting that the helicase function of Ded1p is likely important for unwinding the secondary structure of (2)RNA template with the purified tombusvirus replicase or destabilizing the replication intermediate with the membrane-bound VRC, which might contain dsRNA structure. This function of Ded1p can explain why yeast with down-regulated Ded1p level produced small amount of (+)-stranded RNA progeny, albeit it has also shown that depleted Ded1p reduced the level of p33/p92 in yeast [20] . However, the decreased p33/p92 levels are expected to reduce both (+) and (2)RNA levels [55] ( Figure S1 ). Since the recombinant Ded1p enhanced (+)-strand synthesis by the purified recombinant tombusvirus replicase, we propose that Ded1p directly affect TBSV RNA synthesis via affecting the structure of the RNA templates. However, we cannot exclude that Ded1p could also affect the activity of the VRC due to its interaction with p33, albeit the assembly of the VRC was not affected by Ded1p in the CFE-based assay ( Figure 3) . Overall, the recruitment of a host DEAD-box helicase for replication of a small RNA virus is remarkable, since RNA viruses with less than 6 kB genomes are usually do not code for their own helicases [51] . These viruses are thought to replicate without needing a helicase by using RNA chaperones or possibly recruiting host helicases. The emerging picture with TBSV is that this virus utilizes both the viral-coded p33 RNA chaperone [48] and the host Ded1p helicase for replication by promoting (+)-strand synthesis. Both p33 and Ded1p have been shown to open up short DNA/RNA duplexes, although the activity of Ded1p was more robust than that of p33 in vitro [48] . Why would TBSV utilize both an RNA chaperone and an RNA helicase? It is possible that both proteins are needed for robust (+)RNA synthesis to make excess amount of progeny RNA. Also, Ded1p helicase could be involved in remodeling the viral RNA bound by the viral RdRp or host proteins prior or during RNA synthesis. Since Ded1p can work in both 59-to-39 and 39-to-59 directions [56] , it could be used for multiple purposes during RNA synthesis.