Similar to other positive-strand RNA viruses, tombusviruses are replicated by the membrane-bound viral replicase complex (VRC). The VRC consists of the p92 virus-coded RNA-dependent RNA polymerase (RdRp), the viral p33 RNA chaperone, and several co-opted host proteins. In order to become a functional RdRp after its translation, the p92 replication protein should be incorporated into the VRC, followed by its activation. We have previously shown in a cell-free yeast extract-based assay that the activation of the Tomato bushy stunt virus (TBSV) RdRp requires a soluble host factor(s). In this article, we identify the cellular heat shock protein 70 (Hsp70) as the co-opted host factor required for the activation of an N-terminally truncated recombinant TBSV RdRp. In addition, small-molecule-based blocking of Hsp70 function inhibits RNA synthesis by the tombusvirus RdRp in vitro. Furthermore, we show that neutral phospholipids, namely, phosphatidylethanolamine (PE) and phosphatidylcholine (PC), enhance RdRp activation in vitro. 1-7). Interestingly, the viral RdRp is assumed to be inactive in the cytosol to prevent the formation of a viral double-stranded RNA (dsRNA) intermediate that could trigger efficient gene silencing and RNAinduced innate immunity (8-10). Therefore, the VRC assembly, including the activation of the viral RdRp, could be a critical regulatory step during the infection cycle.The tombusvirus VRCs are housed in membranous spherule structures, which are membrane invaginations into peroxisomes or mitochondria in the cases of Tomato bushy stunt virus (TBSV) and Carnation Italian ringspot virus (CIRV), respectively (11-15). The tombusvirus VRCs contain the virus-coded p92 RdRp and p33 RNA chaperone and the dsRNA intermediate, as well as co-opted cellular proteins, such as the heat shock protein 70 (Hsp70) molecular chaperone (coded for by yeast [Saccharomyces cerevisiae] SSA1/2), glyceraldehyde-3-phosphate dehydrogenase (GAPDH [coded for by TDH2/3 in yeast]), Cdc34p E2 ubiquitinconjugating enzyme, eukaryotic translation elongation factor 1A (eEF1A), eEF1B␥, the Vps4p ESCRT (endosomal sorting complex required for transport) protein, and DDX3-like Ded1p and eIF4AIII-like RH2 DEAD box helicases (16-30). Hsp70, eEF1A, Cdc34p, and ESCRT proteins are involved in the assembly of the tombusviral VRC, while other subverted RNA-binding proteins (eEF1A, eEF1B␥, GAPDH, Ded1p, and RH2) facilitate viral RNA synthesis (1,3,18,31). Tombusvirus replication also depends on lipids, such as sterols and phospholipids, and oxysterol-binding