To further test if DB and GM can interfere with the assembly of the tombusvirus replicase complex, we performed a two-step in vitro assembly/replication assay, also based on CFE containing endogenous eEF1A [31] . In this assay, first, we only provide ATP and GTP in addition to the replication proteins, the (+)repRNA and CFE, which can support the assembly of the replicase, but cannot perform RNA synthesis due to the lack of CTP and UTP [31] . After 1 hr incubation, once the replicase assembly had taken place, we collected the membrane fraction of the CFE by centrifugation and removed the supernatant containing the unbound p33, p92, repRNA as well as the cytosolic fraction of the CFE. Then we added all four rNTPs (including 32 P-labeled UTP) to the membrane fraction of the CFE to allow for RNA synthesis by the pre-assembled replicase complex (second step, Fig. 4C ) [31] . Interestingly, adding either DB or GM during the first step resulted in robust inhibition of TBSV repRNA synthesis during the second step of the assay (Fig. 4C, lanes 2-3 versus 1) , whereas providing the same amount of DB and GM at the beginning of the second step did not result in inhibition of repRNA replication (lanes 4-6) . These data support a model that DB and GM could inhibit the assembly of the tombusvirus replicase complex, but not the RNA synthesis by the already assembled replicase. Similarly, DB and GM failed to inhibit TBSV RNA synthesis in an in vitro assay with a highly purified RdRp from yeast (Fig. S4A) .