Ded1p facilitates initiation by the tombusvirus replicase on RNA/DNA duplex To gain further insights into the mechanism of Ded1p-driven stimulation of TBSV and FHV RNA synthesis, we exploited template structures, such as an RNA/DNA duplex, that are known to hinder RdRp-driven RNA synthesis [48, 49] . Since Ded1p is an RNA helicase [39, 41] and the ATPase/helicase function of Ded1p is needed for stimulation of (+)RNA synthesis by the tombusvirus and FHV replicases, we wanted to examine if Ded1p might facilitate RNA synthesis on a partial DNA/RNA duplex. We chose partial duplex for this assay, since Ded1p and other DEAD-box helicases are not processive enzymes and can only unwind short duplexes [34] . Also, we have shown previously that short DNA oligos hybridized to the promoter region of (2)RNA can inhibit (+)RNA synthesis by the tombusvirus replicase in vitro [42, 47, 48] . Interestingly, addition of purified Ded1p to the tombusvirus replicase assay containing the short RNA/DNA duplex ( Figure 7A Non-overlapping functions of Ded1p and GAPDH in promoting initiation by the tombusvirus replicase GAPDH (Tdh2p in yeast) RNA binding protein is also a host factor stimulating (+)RNA synthesis by the tombusvirus replicase [25, 50] . To test if Ded1p and GAPDH could play a complementary role during (+)RNA synthesis, we added the purified recombinant Ded1p and Tdh2p to the in vitro tombusvirus replicase assay based on the purified preparation ( Figure 8A ). While Ded1p mostly stimulated de novo (+)RNA synthesis initiated from the 39 end of the (2)repRNA up to ,3-fold ( Figure 8B , lane 6), Tdh2p enhanced both de novo (+)RNA synthesis and 39TEX by ,2-and ,3-fold, respectively ( Figure 8B, lane 7) . Interestingly, the two host proteins together had the largest (4.5-fold) effect on (+)RNA synthesis, while their effect on 39TEX was only ,2-fold. Based on these data, we suggest that Ded1p and Tdh2p have a We also performed similar experiments with a short partial RNA/DNA duplex as a template. Both Ded1p and Tdh2p alone could enhance (+)RNA synthesis on the short RNA/DNA duplex ( Figure 8C , lanes 2-3 versus 1) but the largest stimulatory effect (i.e., close to 5-fold increase) was seen when both host factors were included in the assay ( Figure 8C, lane 4) . Altogether, these data further support that Ded1p and GAPDH play synergistic roles in (+)RNA synthesis by the tombusvirus replicase. [9] . Since the viral RNA plays multiple roles during infection, it is likely that remodeling of the viral RNAs and RNP complexes during the switch from one step to another requires RNA helicases or RNA chaperones. Accordingly, the larger RNA viruses all code for RNA helicases [51, 52] . However, RNA viruses with shorter than 6 kB genomes usually do not code for RNA helicases. They could still use RNA helicases during infections if they can subvert selected host helicases for viral purposes. Indeed, we show here that Ded1p helicase is recruited for TBSV replication to aid (+)strand RNA synthesis.