To further test what step(s) Ded1p promotes during TBSV replication, we performed a two-step replication assay based on yeast CFE. In this assay, the first step includes the assembly of the replicase complex on the endogenous membranes present in CFE in the presence of the viral (+)repRNA, the p33/p92 replication proteins and ATP/GTP [23] ( Figure 3A , step 1). Under these conditions, the viral replication proteins recruit the repRNA to the membrane and the replicase becomes partially RNase and protease insensitive, but it cannot initiate minus-strand synthesis yet, due to the absence of CTP/UTP [23] . Then, centrifugation and washing the membranes will remove all the proteins and molecules not bound to the membrane. This is followed by addition of ATP/CTP/GTP/UTP to initiate RNA synthesis during the second step.