Saccharomyces cerevisiae strain BY4741 (MATa his3D1 leu2D0 met15D0 ura3D0) and TET::DED1 yeast strain (yTHC library, MATa his3D1 leu2D0 met15D0 URA3::CMV-tTA) was obtained from Open Biosystems (Huntsville, AL, USA). The plasmid pESC-HIS-Gal-His33/Gal-DI-72 expressing Cucumber necrosis virus (CNV) 66His-tagged p33 and the TBSV DI-72 repRNA was described earlier [30] . The CNV p33 protein has very high sequence identity with the closely related TBSV p33, but the CNV p33 is expressed better and more active in yeast than TBSV p33. Recombinant yeast Tdh2p protein was produced in E. coli as GST fusion using plasmid pGEX-TDH2, described earlier [50] . Recombinant Ded1p helicase proteins were produced in E. coli as maltose binding protein (MBP) fusions [48] . The expression plasmid pMal-DED1 was prepared by PCR using primers #3956 (CCAGCTG-CAGTCACCACCAAGAAGAGTTG)/ #3957(CCAGGAATT-TGGCTGAACTGAGCGAACAAG) and the yeast genomic DNA as a template. The plasmids pMal-Dx, expressing different Ded1p mutants, were prepared by PCR using #3956/ #3957 primers and plasmids containing mutated DED1 sequences [41, 53] . The PCR products obtained from DED1 wt and mutant sequences were digested with EcoRI and PstI and inserted between EcoRI and PstI sites in pMalc-26 (New England Biolab).