To test if Flock house virus (FHV) replicase is affected by Ded1p, first we have developed an in vitro FHV replication assay based on affinity-purified FHV replicase preparation that can be programmed with exogenously added RNAs ( Figure 4C , lanes 1 versus 10). The in vitro FHV replicase assay revealed that the purified recombinant Ded1p increased (+)-strand RNA synthesis on the (2)-stranded FHV template by ,3-fold ( Figure 4D Figure  S4D , lane 2 versus 1). It is likely that the observed stimulation of FHV replicase activity by Ded1p is direct, since we found that Ded1p bound to the FHV repRNA ( Figure S8A ) and protein A RdRp protein in vitro ( Figure S8B ). Altogether, these data show that, similar to the tombusvirus replicase, the activity of FHV replicase is stimulated by Ded1p only on the (2)RNA templates, but not when using the (+)RNA templates.