Expression and purification of the recombinant TBSV p33 and p92 and TCV p88C replication proteins from E. coli were carried out as described earlier with modifications [54] . Briefly, the expression plasmids were transformed separately into E. coli strain BL21 Rosetta (DE3). Protein expression was induced using isopropyl b-D-thiogalactopyranoside (IPTG) for 8 h at 16uC, then the cells were collected by centrifugation (5,000 rpm for 5 min). The recombinant TCV p88C protein was purified on an amylose resin column (NEB), as described [54] . The cells were suspended and sonicated in MBP column buffer containing 20 mM Tris-Cl pH 8.0, 150 mM NaCl, 1 mM EDTA, 10 mM b-mercaptoethanol and 1 mM phenylmethylsulfonyl fluoride (PMSF). The sonicated extract was then centrifuged at 27,000 g for 10 min, followed by incubation with amylose resin (NEB) for 1 h at 4uC. After washing the resin 3 times with the column buffer and once with a low salt column buffer (25 mM NaCl), the proteins were eluted with a low salt column buffer containing 0.18% (V/W) maltose and 6% (V/V) glycerol and stored at 280uC. MBP-p33 and MBP-p92 pol were purified as above, except 30 mM HEPES-KOH pH 7.4 was used instead of 20 mM Tris-Cl pH 8.0. eEF1A was purified from yeast as described [76] and stored in aliquots at the vapor temperature of liquid nitrogen. Protein fractions used for the replication assays were 95% pure, as determined by SDS-PAGE.