To further test the direct effect of Ded1p on RNA synthesis, we utilized detergent-solubilized and affinity-purified tombusvirus -stranded RNA products produced in the cell-free TBSV replication assay. The dsRNA products are also shown on the right subpanel with high contrast for better visualization. Odd numbered lanes represent replicase products, which were not heat treated (thus both ssRNA and dsRNA products are present), while the even numbered lanes show the heat-treated replicase products (only ssRNA is present). The % of dsRNA and ssRNA in the samples are shown. Note that, in the nondenatured samples, the dsRNA product represents the annealed (2)RNA and the (+)RNA, while the ssRNA products represents the newly made (+)RNA products. Right replicase from Ded1p-depleted yeast ( Figure 4A ). This purified replicase can only synthesize complementary RNA products on added TBSV templates, but, unlike the above membrane-bound replicase in the CFE-based assay, it cannot perform a complete cycle of RNA synthesis [14, 15] .