To determine the functions of eEF1A during tombusvirus replication, we generated ,6,000 yeast strains expressing eEF1A with random mutations (see Fig. S1A ) and tested the level of TBSV repRNA accumulation in a high-throughput assay [50] . In this assay, we used yeast strains, in which the two wt eEF1A genes (TEF1/TEF2) were deleted from the chromosome, while the wt or mutated eEF1A was expressed from plasmids. Importantly, a given eEF1A mutant is the only source of eEF1A in the yeast cells used. Using the high-throughput assay, we identified one yeast strain (N21) expressing an eEF1A mutant that supported reduced TBSV repRNA replication, while the other three strains with eEF1A mutants (named C42, C53 and C62) showed increased level of repRNA accumulation ( Fig. 1A and S1B-G). Interestingly, the eEF1A mutants supporting increased steady-state level of repRNA accumulation did not increase the relative level of p33 and p92 pol replication proteins (Fig. 1A , bottom panel; S1D-E). Thus, these eEF1A mutants likely affect TBSV replication directly. Accordingly, affinity purification of the solubilized tombusvirus replicase complex from yeast cells, followed by in vitro replicase activity assay revealed that the replicase from C42, C53 and C62 mutant eEF1A-expressing yeast strains had ,2-fold increased activities when compared with wt eEF1A-expressing yeast strain (Fig. 1B , lanes 1-6 versus 7-8). The amounts of replication protein p33 and the co-purified eEF1A were comparable in the purified replicase samples (Fig. 1B, bottom panel) , indicating that the differences in replicase activities in the mutants are likely due to enhanced replicase functions, and not due to altered proteins levels in the replicase complexes. Testing the ability of C42, C53 and C62 mutant eEF1As to bind to the viral RNA or to the p33 and p92 pol replication proteins in vitro ( Fig. S2B -C) did not reveal significant differences between the mutants and the WT. This further supports that these eEF1A mutations likely increase the function of the viral replicase without altering the protein and RNA components in the replicase.