To further test the function of eEF1A in TBSV replication, we used chemical inhibitors of eEF1A, including Didemnin B (DB) and Gamendazole (GM). DB inhibits the activity of GTP-bound eEF1A during translation by binding to a pocket in eEF1A involved in the interaction with the aminoacylated tRNA and the nucleotide exchange factor eEF1Balpha [59, 60] . GM has been shown to inhibit the actin bundling function, while it does not inhibit protein translation or GTP binding functions of eEF1A [61] . We found that both DB and GM efficiently inhibited TBSV repRNA replication in the in vitro assay with CFE, which contains the endogenous eEF1A (Fig. 4A ). Time-course experiments revealed that the inhibition by DB was the most effective when the inhibitor was added at the beginning or during the first 10-15 min of the assay (Fig. 4B , lanes 2-5), while GM inhibited the cell-free replication of TBSV repRNA when added not only at the beginning, but up to 40 min after the start of the assay (lanes [12] [13] [14] [15] [16] [17] . It is known that the recruitment of the viral RNA and replication proteins as well as the assembly of the viral replicase complex take place during the first 40-60 min in the cell-free assay [31] . Since DB could inhibit translation, we also tested the effect of another translation inhibitor, namely cycloheximide, which did not affect TBSV repRNA replication in our assay (Fig. S3) . These data suggest that the inhibition by DB and GM is unlikely through decreased translation in the replication assay. Therefore, the above data are consistent with the model that DB and GM interfere with the assembly of the viral replicase complex in the CFE. Also, GM seems to be a more potent inhibitor of TBSV replication than DB.