Since eEF1A is part of the tombusvirus replicase complex [25] , it is possible that C42, C53 and C62 eEF1A mutants might affect the assembly/activity of the tombusvirus replicase. To test this idea, we prepared cell-free extracts (CFE) from yeast strains expressing selected eEF1A mutants in the absence of the wt copy of eEF1A. These yeast extracts contained comparable amount of total proteins as well as the amounts of eEF1A, ALP, PGK and Hsp70 (Ssa) yeast proteins were comparable ( Fig. 2A) . The advantage of the CFE extracts is that they can then be programmed with the TBSV (+)repRNA in the presence of purified recombinant p33 and p92 pol obtained from E. coli that leads to the in vitro assembly of the viral replicase, followed by a single cycle of complete TBSV replication, resulting in both (2)stranded repRNA and (+)-stranded progeny [31, 52] . Therefore, this assay can uncouple the translation of the viral proteins from viral replication, which are interdependent during (+)RNA virus infections.