Hepatitis C virus (HCV) RNA replication depends on viral protein association with intracellular membranes, but the influence of membrane composition on viral replication is unclear. We report that HCV RNA replication and assembly of the viral replication complex require geranylgeranylation of one or more host proteins. In cultured hepatoma cells, HCV RNA replication was disrupted by treatment with lovastatin, an inhibitor of 3-hydroxy-3-methyglutaryl CoA reductase, or with an inhibitor of protein geranylgeranyl transferase I, each of which induced the dissolution of the HCV replication complex. Viral replication was not affected by treatment of cells with an inhibitor of farnesyl transferase. When added to lovastatin-treated cells, geranylgeraniol, but not farnesol, restored replication complex assembly and viral replication. Inasmuch as the HCV genome does not encode a canonical geranylgeranylated protein, the data suggest the involvement of a geranylgeranylated host protein in HCV replication. Inhibition of its geranylgeranylation affords a therapeutic strategy for treatment of HCV infection.A pproximately 170 million people worldwide are persistently infected with hepatitis C virus (HCV), and these individuals account for a majority of all cases of chronic liver disease (1). The public health impact of HCV is compounded by the overall low response rate to current IFN-based therapies for treating HCV infection, underscoring the need for new therapeutic strategies to combat the HCV pandemic. HCV is a singlestranded positive sense RNA virus and member of the Flaviviridae (2). The 9.6-kb HCV genome encodes a single polyprotein that is posttranslationally processed into at least 10 individual structural and nonstructural (NS) viral proteins, the latter of which are sufficient to support HCV RNA replication (3). Current studies support a model in which HCV infection results in assembly of the viral RNA and NS proteins into a replication complex that associates with the host cell endoplasmic reticulum (ER). Viral-directed processes convert the ER into a membranous web conducive to virus replication (4-6). The cellular cofactors and membrane constituents that contribute to assembly and maintenance of the HCV replication complex are not known.Cell membrane composition is subject to modification through the mevalonate pathway, which produces cholesterol and nonsterol isoprenoid products (7). Two of the mevalonate-derived isoprenoids, farnesyl (15 carbons) and geranylgeranyl (20 carbons), are attached to membrane proteins via formation of a cysteine thioether (7,8). This process, called protein prenylation, targets certain proteins to cell membranes where they regulate many cellular functions, ranging from vesicle budding and fusion to growth. Therapeutic control of the mevalonate pathway has proven effective for the clinical treatment of hypercholesterolemia and is achieved in part through the use of statin compounds (7, 9). Statins block mevalonate production by inhibiting 3-hydroxy-3-methylglutaryl CoA reductase (HMG...