To test if eEF1Bc is present in the viral replicase, yeast tef4D strain was transformed with pGBK-CUP1-HisFLAGp33/GAL1-DI-72, pGAD-CUP1-Hisp92 and pYES-GAL1-HisTef4. In the control experiment, 6xHisp33was expressed from pGBK-CUP1-Hisp33/GAL1-DI-72. Yeast cultures were grown in SC-ULHmedia containing 1% raffinose and 1% galactose with 5 mM copper-sulfate for 4 days with increasing the volume of the culture from 2 ml to 100 ml to a final OD 600 of, 1.0. After harvesting of cells, co-purification of 6xHis-tagged Tef4p with HF-p33 (part of the viral replicase) was conducted by using anti-FLAG M2-agarose affinity resin as described above (in the section: FLAG-affinity purification of eEF1Bc-TBSV repRNA complex), with the exception that only solubilized ME fraction was loaded on the column. Proteins bound to affinity resin were eluted by incubation with 150 ml buffer containing FLAG peptide and precipitated with Trichloroacetic acid (TCA) [64] . Samples were analyzed by SDS-PAGE and Western blotting.