To test if the increased TBSV repRNA replication in vitro was due to enhanced (+) or (2)-strand synthesis, we analyzed the replication products under non-denaturing versus denaturing conditions (Fig. 2B) . These experiments showed that the amount of dsRNA [representing the 32 P-labeled (2)RNA product hybridized with the (+)RNA] increased ,3-fold in case of C42, C53 and C62 mutants (lanes 3-8, Fig. 2B ) in comparison with the wt (lanes 9-10). The dsRNA nature of these products was confirmed by the ssRNA-specific S1 nuclease digestion assay (Fig. 2C ). On the other hand, the ratio of dsRNA and ssRNA did not change in the various CFEs containing the eEF1A mutants or the wt (Fig. 2B) . These results are consistent with the model that the replicase complex carrying the eEF1A mutants increased mostly the level of (2)RNA production, which then led to proportionately higher level of (+)RNA progeny.