Yeast strains (BY4741 and TET::DED1) were transformed with plasmids pGBK-HisFlagp33 (or pGBKHisp33 as a control), pGAD-HisFlagp92 (or pGAD-Hisp92 as a control) and pYC-DI72. The 66His-Flag double-tagged HF-p33 and p92 were expressed from the ADH1 promoter and DI-72 repRNA was under the Gal1 promoter. Transformed yeast were pre-grown on SC-ULH 2 media containing 2% glucose at 29uC. After centrifugation at 2,000 rpm for 3 min and washing pellet with selective media containing 2% galactose and 1 mg/ml Doxycycline, yeast were grown for 24 hours in SC-ULH 2 media containing 2% galactose at 23uC. The replicase purification was done according to a previously described procedure [24] with the following modification. Briefly, 200 mg of yeast cells were resuspended and homogenized in TG buffer [50 mM Tris-HCl [pH 7.5], 10% glycerol, 15 mM MgCl 2 , 10 mM KCl, 0.5 M NaCl, , and 1% [V/V] yeast protease inhibitor cocktail (Ypic)] by glass beads using FastPrep Homogenizer (MP Biomedicals). The membrane fraction containing the viral replicase complex was solubilized with 1 ml TG buffer containing 1% Triton X-100, 1% [V/V] Ypic as described [14, 15] . After affinity purification of HF-p33 on anti-FLAG M2-agarose affinity resin (Sigma), the resinbound replicase complex was eluted in 100 ml elution buffer , 10% glycerol, 15 mM MgCl 2 , 10 mM KCl, 50 mM NaCl, 1% Triton X-100, and 0.15 mg/ml Flag peptide (sigma)].