Further testing of Ded1p binding to (+)repRNA regions revealed that RIV(+), representing the 39 noncoding region in TBSV RNA, was bound more efficiently by Ded1p ( Figure S5 , Step-wise cell-free TBSV replication assay does not support a role for Ded1p helicase in the assembly of the TBSV VRC. (A) Scheme of the CFE-based TBSV replicase assembly and replication assays. Purified recombinant p33 and p92 pol replication proteins of TBSV and in vitro transcribed TBSV DI-72 (+)repRNA were added to the whole cell extract prepared from Ded1p-depleted yeast strain in step 1. The assay either contained or lacked the purified recombinant Ded1p (0.3 mg) or MBP during step 1. Note that the assay was performed in the presence of ATP/GTP to facilitate TBSV VRC assembly, but prevent RNA synthesis in step 1. After step 1, centrifugation was used to collect the membrane fraction of the CFE, and after washing the membranes, step 2 was performed in the presence of ATP/CTP/GTP and 32 P-UTP to allow TBSV RNA replication. In the samples presented in right panel, Ded1p or MBP were added at the beginning of step 2. (B) Denaturing PAGE analysis of the 32 P-labeled TBSV repRNA products obtained in the CFE assays in the presence of wt and various Ded1p mutants (0.3 mg each) ( Figure S3C ) or MBP. See further details in Figure 2 . Note that the various preps of D3 mutant of Ded1p showed high variation in in vitro activity (for reasons currently unknown)-see Figure 4B . Each experiment was repeated at least three times and the data were used to calculate standard deviation. To test if Ded1p can interact with the TBSV p33 replication protein, we used the membrane-based split-ubiquitin assay [31] . We found that Ded1p interacted with p33 protein in yeast ( Figure  S6A ). The pull-down experiments with MBP-p33 and MBP-p92 also showed that Ded1p bound to the tombusvirus replication proteins in vitro ( Figure S6B ). This was further supported by the reverse pull-down experiments with GST-tagged Ded1p, which resulted in the co-purification of p33 and p92 ( Figure S6C ).