Efficient replication of hepatitis C virus (HCV) in cell lines requires the acquisition of host-specific adaptive mutations; however, the molecular basis for this adaptation has remained unsolved. Harak et al. report that adaptive mutations in the genes encoding viral NS5A and NS5B regulate the activity of the host phosphatidylinositol 4-kinase-α (PI4Kα; a kinase that alters the lipid composition of cellular membranes by converting phosphatidylinositol to phosphatidylinositol 4-phosphate (PI4P)). In the host, HCV NS5A and NS5B proteins interact with PI4Kα to generate PI4P-rich microenvironments that are permissive for viral replication; however, increased PI4Kα levels that exist in hepatoma cell lines restrict HCV replication. The authors found that loss-of-function adaptive mutations in NS5A and NS5B prevented overactivation of PI4Kα in cell lines, which promoted the replication of wild-type isolates in cell cultures.