Hepatitis C virus (HCV) is a leading cause of chronic hepatitis, cirrhosis, and hepatocellular carcinoma. The absence of culture systems permissive for HCV replication has presented a major bottleneck to antiviral development. We sought to recapitulate the early steps in the life cycle of HCV by means of DNA-based expression of viral genomic sequences. Here we report expression of replicating HCV RNA by using a, to our knowledge, novel binary expression system in which cells were transfected with a T7 polymerase-driven full-length HCV cDNA plasmid containing a cis-acting hepatitis ⌬ ribozyme to control 3 cleavage, and infected with vaccinia-T7 polymerase. HCV genomic and replicative strand synthesis, in addition to protein synthesis, was detectable and depended on full-length HCV sequences. Moreover, the system was capable of generating HCV RNA quasispecies, consistent with the action of the low-fidelity HCV NS5B RNA polymerase. IFN-␣, but not ribavirin, directly inhibited the viral replicative cycle in these cells, identifying the virus itself and not solely the immune system as a direct target of IFN action. The availability of a cell-based test for viral replication will facilitate screening of inhibitory compounds, analysis of IFN-resistance mechanisms, and analysis of virus-host cell interactions.cell-based assay ͉ amantadine ͉ ribavirin I nfection with hepatitis C virus (HCV) is a leading cause of chronic liver disease throughout the world (1). Chronic infection nearly always follows acute exposure to HCV, and chronically infected persons develop cirrhosis and hepatocellular carcinoma at dramatically elevated rates (2). Considering both the failure of humoral immunity to prevent reinfection and the virus' propensity for sequence diversity (3, 4), prospects for the development of an HCV vaccine seem remote. Unfortunately, available antiviral therapies, including IFN-␣ and ribavirin (RBV), have limited effectiveness (5).The lack of tissue-culture systems permissive for HCV replication has limited the development of new treatments. Although self-replicating HCV RNA replicons may permit investigation of RNA replication inhibitors (6), this system is limited to expression of the nonstructural region of the genome and requires mutant viral sequences to replicate (7). The successful introduction of RNA transcripts from an infectious cDNA clone into chimpanzees has made possible the adaptation of clones to animal studies (8). However, the repetitive use of large animals to explore HCV biology is impractical.Although IFN directly inhibits the replication of many viruses and alters immune function (9), current evidence that it directly inhibits HCV replication is inferential. Sequence changes and protein interaction assays implicate several HCV proteins as particular targets of IFN (10, 11). Given the need to screen for antiviral compounds, we developed a system capable of recapitulating the early steps of the HCV life cycle by adapting the infectious cDNA clone with elements that permit HCV RNA replication in vivo....