The use of eEF1A inhibitors in the in vitro replicase assembly assay Purified Didemnin B (NSC 325319) was kindly provided by the Natural Products Branch, NCI (Bethesda, MD, USA), while Gamendazole was a generous gift from Dr. Tash (University of Kansas Medical Center). Both chemicals were dissolved in DMSO (the final concentration was 20 mM). The concentrations of chemical and time point of the addition of the chemicals to the in vitro reaction are indicated in the text. The cell-free TBSV replicase assay and the in vitro TBSV replicase assembly assay were performed according to [31] . Briefly, the purified recombinant TBSV p33, p92 pol and (+) repRNA were added to the cell-free reaction in the presence of 1.0 mM ATP and GTP in step 1. After incubation at 25uC for 1 h, the in vitro reactions were centrifuged 21,0006 g at 4uC for 10 min. The supernatant containing extra p33, p92 pol and repRNA, which were not bound to the membranes in the cell-free extract, was discarded, while the membrane pellet was re-suspended in a standard in vitro replicase assay buffer containing [ 32 P]-UTP and ATP, CTP, and GTP, and incubated at 25uC for 3 h [31] .