Since the assembly of the tombusvirus replicase also depends on events prior to the replicase assembly step, such as template RNA binding by the viral replication proteins/host proteins (such as eEF1A), and template recruitment to intracellular membranes [21, 52] , we also tested the effect of DB and GM on these processes as well based on purified recombinant eEF1A. We found that GM strongly interfered with the binding of eEF1A to the viral RNA in an EMSA assay (Fig. 4D, lanes 3-6 versus 2) , whereas DB did not affect the binding under the assay conditions (lanes 9-12). Since DB binds only weakly to eEF1A in solution, but it binds much more effectively to eEF1A in the presence of GTP and the ribosome [62] , we also performed in vitro co-purification experiments. First, 35 S-labeled eEF1A was produced in an in vitro translation system (containing ribosome and GTP) and, second, biotin-labeled viral (+)repRNA was added. After short incubation in the absence or presence of various amount of DB, we performed affinity-purification of the viral RNA. Phosphoimaging revealed that eEF1A was co-purified with the viral RNA and the amount of protein co-purified with the viral (+)repRNA was inhibited by increasing amount of DB in the assay (Fig. 4E , compare lane 1 with [2] [3] [4] [5] . This demonstrated that DB inhibits the binding of eEF1A to the viral repRNA. Moreover, both DB and GM interfered with the recruitment of the viral template RNA to the membrane of the CFE containing endogenous eEF1A (Fig. 4F,  lanes 5-8 versus 3-4) . On the other hand, DB and GM do not seem to affect the interaction between eEF1A and p33 or p92 replication proteins in vitro (Fig. S4B) . Altogether, these data suggest that inhibition of eEF1A function by DB and GM could block several steps during the assembly of the tombusvirus replicase complex, including template binding by eEF1A and viral RNA recruitment into replication.