For the co-purification of TBSV DI-72 repRNA and eEF1Bc protein, the yeast tef4D strain was co-transformed with pGBK-ADH-Hisp33/GAL1-DI72, pGAD-CUP1-Hisp92 and pESC-CUP1-HisFLAG-Tef4. The pESC-CUP1-FLAGHis-Tef4 plasmid was replaced with the pESC plasmid in the control experiment. Yeast was pre-grown overnight at 29uC in 2 ml SC ULHmedium containing 2% glucose and 5 mM copper sulfate. The volume of the media was increased to 20 ml after 16 h for an additional 10 h (OD 600 of ,0.8), then the cultures were transferred to 20 ml SC ULHmedium containing 2% galactose to induce TBSV DI-72 RNA transcription at 23uC. The transcription of DI-72 RNA was stopped by changing to the media containing 2% glucose after 8 h. The cultures were diluted to 200 ml and copper sulfate was added to a final concentration of 50 mM to induce the expression of Flagtagged Tef4 protein. After incubation at 23uC for 24 h, the samples were centrifuged at 3000 rpm for 4 min. Cells (,1 g) were re-suspended in 2 ml TG Buffer (50 mM Tris-HCl [pH 7.5], 10% glycerol, 15 mM MgCl 2 , and 10 mM KCl) supplemented with 0.5 M NaCl and 1% [V/V] YPIC yeast protease inhibitor cocktail (Sigma) and RNase inhibitor (Ambion). Yeast cells were broken by glass beads in a FastPrep cell disruptor (MP Biomedicals) 4 times for 20 sec each at speed 5.5. Samples were removed and incubated 1 min in an ice-water bath after each treatment. The samples were centrifuged at 500 6g for 5 min at 4uC to remove glass beads, unbroken cells and debris then supernatant was moved into fresh pre-chilled tubes. After being centrifuged again at 500 6g for 5 min at 4uC supernatant transferred into fresh pre-chilled tubes and soluble (SU) and membrane (ME) fractions containing the viral replicase complex were separated with centrifugation at 35,000 6g for 15 min at 4uC. The SU fraction was applied on 0.1 ml anti-FLAG M2agarose affinity resin (Sigma) and Tef4 protein tagged with 6xHisand FLAG affinity tags was purified. Before applying ME fraction on the anti-FLAG M2 resin, solubilization of the membranebound replicase was performed in 1 ml TG buffer with 0.5 M NaCl, 1% [V/V] YPIC yeast protease inhibitor cocktail (Sigma), and 2% Triton X-100 via rotation for 2 hours at 4 uC. The solubilized membrane fraction was centrifuged at 35,000 6g at 4uC for 15 min and the supernatant was added to the resin preequilibrated with TG buffer supplemented with 0.5 M NaCl and 0.5% Triton X-100, followed by gentle rotation for 2 h at 4uC. The unbound proteins were removed by gravity flow, and the resin was washed two times with 1 ml TG buffer supplemented with 0.5 M NaCl, 0.5% Triton X-100 and once with 1 ml TG buffer, 0.5% Triton without NaCl. The bound proteins were eluted with 150 ml TG buffer without NaCl, 0.5% Triton X-100, supplemented with 150 mg/ml flag peptide and 1% yeast protease inhibitor cocktail via gentle tapping the column occasionally for 2 h at 4uC. After centrifugation at 600 6g 2 min at 4uC, semiquantitative RT-PCR was performed to detect TBSV repRNA copurified with eEF1Bc using primers, #359 (GTAATACGACT-CACTATAGGAAATTCTCCAGGATTTC) and #1190, amplifying full length (+)repRNA.