Abstract: Cytosolic chaperones are a diverse group of ubiquitous proteins that play central roles in multiple processes within the cell, including protein translation, folding, intracellular trafficking, and quality control. These cellular proteins have also been implicated in the replication of numerous viruses, although the full extent of their involvement in viral replication is unknown. We have previously shown that the heat shock protein (hsp) 40 chaperone encoded by the yeast YDJ1 gene facilitates RNA replication of Flock House virus (FHV), a well-studied and versatile model positive-sense RNA virus. To further explore the roles of chaperones in FHV replication, we examined a panel of 30 yeast strains with single deletions of cytosolic proteins that have known or hypothesized chaperone activity. We found that the majority of cytosolic chaperone deletions had no impact on FHV RNA accumulation, with the notable exception of J-domain-containing hsp40 chaperones, where deletion of APJ1 reduced FHV RNA accumulation by 60% while deletions of ZUO1, JJJ1, or JJJ2 markedly increased FHV RNA accumulation by 4- to 40-fold. Further studies using cross complementation and double-deletion strains revealed that the contrasting effects of J-domain proteins were reproduced by altering expression of the major cytosolic hsp70s encoded by the SSA and SSB families, and were mediated in part by divergent effects on FHV RNA polymerase synthesis. These results identify hsp70 chaperones as critical regulators of FHV RNA replication, and indicate that cellular chaperones can have both positive and negative regulatory effects on virus replication.