Hepatitis C represents a rapidly evolving worldwide virus plague. Hepatitis C virus (HCV), a small positive-sense RNA virus of the Flaviviridae family, is the major cause of hepatitis in the United States and often leads to chronic hepatitis, cirrhosis and hepatocellular carcinoma (HCC). Progress in our understanding of the virus and the development of effective treatment modalities have been hampered by lack of a reliable in vitro system for propagating HCV and of a convenient animal. Recently, we developed two long-term cell culture systems of HCV by transfection with infectious HCV RNA transcripts producing high levels of HCV. The viral protease NS3 cleaves the non-structural proteins of HCV and appears to be essential for HCV replication and packaging. Therefore, we generated several adenovirus constructs with the single.chain antibody (sFv) gene derived from human monoclonal antibody to the NS3' protease of HCV. We demonstrated that the anti-NS3/sFv are stably expressed in mammalian cells after adenovirus-mediated gene transfer and that the appropriate antibodies are produced and bind to NS3. It is our hypothesis that the transfected cell lines support HCV replication and virion formation in vitro comparable to in vivo infection and that intracellular sFv to the NS3 protease interferes with HCV propagation and/or packaging in vitro and in vivo. Accordingly, the specific aims of this proposal are: 1) to compare the production of genomic and replicative HCV RNA sequences and proteins in the transfected cell lines with that in infected human liver and HCC by quantitative assays; 2) to define the functional role of the conserved terminal 5' and 3' UTR elements in HCV replication and packaging; and 3) to determine whether sFv to NS3 protease will interfere with the NS3 functions, HCV replication, packaging and/or infectivity. The recent generation of HCV transgenic mice in our laboratory will allow in vivo testing of the anti-NS3/sFv. The attainment of these aims will provide insight into the mechanism of HCV replication and virion formation and will lead to the development of new treatment strategies against this significant human pathogen.