We found that addition of purified recombinant Ded1p to the purified tombusvirus replicase programmed with the (2)repRNA stimulated (+)-strand synthesis by ,2.5-3-fold ( Figure 4B , lanes 3-4 versus 1-2; and Figure S3D , lane 3 versus 2). Interestingly, Ded1p stimulated the production of the full-length (+)-strand RNA product, while the amount of 39-terminal extension product (39TEX; due to self-priming by the 39 end of the template [42] [43] [44] , Figure S3B ) decreased in the presence of added recombinant Ded1p. Therefore, we suggest that Ded1p facilitates the de novo initiation on the (2)RNA template by the tombusvirus replicase.