Investigation of the hepatitis C virus (HCV) life cycle and the evaluation of novel antiviral strategies are limited by the lack of an efficient cell culture system. Therefore, continuous human cell lines inducibly expressing the entire HCV open reading frame were generated with use of a tetracyclineregulated gene expression system. HCV transgenes were found to be chromosomally integrated in a head-to-tail configuration. Northern blot analyses revealed a tightly regulated unspliced transcript of approximately 9 kilobases (kb). HCV structural and nonstructural proteins were faithfully processed, indicating that the cellular and viral proteolytic machineries and posttranslational modification pathways are fully functional in these cell lines. Steady state expression levels could be regulated over a broad range by the concentration of tetracycline present in the culture medium. Kinetic analyses revealed a half-life of less than 1 hour for the HCV RNA whereas a half-life of approximately 9.5, 12, 11, and 10 hours was found for core, NS3, NS4A, and NS5A proteins, respectively. Viral proteins were found to colocalize in the cytoplasm in a pattern characteristic of the endoplasmic reticulum. High-level expression of HCV proteins in the fully induced state was toxic to the cells. These cell lines provide a unique in vitro system to analyze structural and functional properties of HCV proteins, their interactions with cellular proteins and pathways, and the requirements for HCV morphogenesis. In addition, they should prove useful for the evaluation of novel antiviral strategies against hepatitis C in a well-defined and reproducible cellular context. (HEPATOLOGY 1998;28:192-201.) Hepatitis C virus (HCV) is the most common etiologic agent of posttransfusion and sporadic non-A, non-B hepatitis. 1,2 The majority of HCV infected individuals develop chronic infection which may progress to liver cirrhosis and eventually hepatocellular carcinoma. 3 HCV contains a singlestranded, positive-sense RNA genome of approximately 9,600 nucleotides in length. As in flavi-and pestiviruses, the viral genome comprises a 5Ј noncoding region, a long open reading frame (ORF) encoding a polyprotein precursor of 3,010 to 3,033 amino acids, and a 3Ј noncoding region. 4 The polyprotein precursor is co-and posttranslationally processed by cellular and viral proteases to yield the mature structural and nonstructural proteins (Fig. 1). [4][5][6] The structural proteins comprise the core protein and two envelope proteins, E1 and E2. These are followed by two viral proteases contained within NS2 and the aminoterminal one-third of NS3; a RNA helicase located within the carboxyterminal domain of NS3; NS4A, a cofactor for the NS3 protease; NS4B and NS5A, two proteins of as yet unknown function; and finally, a RNA-dependent RNA polymerase contained within NS5B. The structural proteins are believed to be processed by the signal peptidase of the endoplasmic reticulum (ER). Cleavage at the NS2/3 site is mediated by a viral protease composed of the region of ...