Whole cell yeast extract capable of supporting TBSV replication in vitro was prepared as described [31] . The in vitro TBSV replication assays were performed in 20-ml total volume containing 2 ml of whole cell extract, 0.5 mg DI-72 (+)repRNA transcript, 400 ng purified MBP-p33, 100 ng purified MBP-p92 pol (both recombinant proteins were purified from E. coli), 30 mM HEPES-KOH, pH 7.4, 150 mM potassium acetate, 5 mM magnesium acetate, 0.13 M sorbitol, 0.4 ml actinomycin D (5 mg/ml), 2 ml of 150 mM creatine phosphate, 0.2 ml of 10 mg/ml creatine kinase, 0.2 ml of RNase inhibitor, 0.2 ml of 1 M dithiothreitol (DTT), 2 ml of 10 mM ATP, CTP, and GTP and 0.25 mM UTP and 0.1 ml of [ 32 P]UTP [31] . The reaction mixture was incubated at 25uC for 3 h. The reaction was terminated by adding 100 ml stop buffer (1% sodium dodecyl sulfate [SDS] and 0.05 M EDTA, pH 8.0), followed by phenol-chloroform extraction, isopropanol-ammonium acetate precipitation, and a washing step with 70% ethanol as described [52] . The newly synthesized 32 P-labeled RNA products were separated by electrophoresis in a 5% polyacrylamide gel (PAGE) containing 0.56Tris-borate-EDTA (TBE) buffer with 8 M urea. To detect the double-stranded RNA (dsRNA) in the cell-free replication assay, the 32 P-labeled RNA samples were divided into two aliquotes: one half was loaded onto the gel without heat treatment in the presence of 25% formamide, while the other half was heat denatured at 85uC for 5 min in the presence of 50% formamide [31] . S1 nuclease digestion to remove single-stranded 32 P-labeled RNA was performed at 37uC for 30 min in a buffer containing 5 mM sodium acetate (pH 4.5 at 25uC), 0.28 M NaCl, 4.5mM ZnSO4 and 40 U S1 nuclease (Boehringer).