Based on the previous successful strategy of analyzing eEF1A mutants, here we generated ,6,000 random mutants covering the entire eEF1A sequence and found four mutants, which greatly affected TBSV repRNA accumulation in yeast (Fig. 1A) . Among these mutants, C42, C53 and C62 increased TBSV repRNA replication. Importantly, this effect by the eEF1A mutants was not due to changing the translation efficiency of p33/p92 pol , but likely via directly altering viral replication and affecting the activity of the viral replicase. On the other hand, N21 mutant of eEF1A resulted in decreased TBSV RNA accumulation and also led to reduction in the level of p33 replication protein. This is reminiscent of the previously characterized GDP-binding mutant T 22 S [25], which supported greatly reduced level of viral RNA replication and p33 accumulation due to shortened half-life of p33. Overall, N21 mutant further supports that one of the functions of eEF1A in TBSV replication is to stabilize the p33 replication protein in yeast.