Hepatitis C virus infection causes long-term inflammation of the liver which frequently leads to chronic liver disease, liver fibrosis and hepatocellular carcinoma. Treatment for liver cancer and liver cirrhosis is transplantation. Therefore, development of novel therapies to eradicate hepatitis C can save some of these end stage liver diseases and hepatocellular carcinomas. Concepts of designing innovative anti-viral therapies, which will stop virus replication and basic studies on several aspects of virus replication, have been hampered due to the lack of a reliable cell culture model to replicate HCV. Recently, we reported replication of HCV in a human hepatocellular carcinoma cell line and in a lymphoblastoid cell line after transfecting them with full-length HCV RNA. We demonstrated that these two transfected cell lines produced infectious HCV particles for over 2 months. In addition, we showed that we could transmit HCV to a chimpanzee after intravenous inoculation of HCV derived from transfected HepG2 cells. Furthermore, avoid potential problems of low levels of HCV in the RNA transfection models, we developed an inducible model to study HCV replication and expression by biochemical means without the use of RT-PCR. On the basis of these preliminary results, we propose to extend our studies and establish infectious cell culture for HCV genotype la using a bonafide chimpanzee infectious clone prepared by Jens Bukh at the National Institute of Health. It is our hypothesis that efficient transfection of full-length HCV RNA from a chimpanzee infectious HCV clone to HepG2 cells will result in replication of HCV in cell culture. Establishment of such cell culture systems for la and lb strains of HCV should facilitate studies to test therapeutic potential of interferons, ribozymes and protease inhibitors. The non-structural protein NS3 possess multiple enzyme activities (protease, helicase and NTPase) which appear to be important for HCV replication. We generated several adenoviral constructs containing single-chain antibody fragments derived from human which target core, E2, NS3 and NS4 proteins. Furthermore, we postulate that intracellular expression of single-chain antibodies targeted to structural proteins should inhibit virus formation and those antibodies targeted to non-structural proteins, NS3 and NS4, may block protease, helicase and NTPase activities and thus interfere with the processing of HCV proteins and replication. To directly test our hypotheses, we have developed three Specific Aims: 1) To study replication, expression and morphogenesis of hepatitis C virus genotype la in human hepatocellular carcinoma cell line (HepG2) transfected with chimpanzee infectious clone and determine whether the level of replication can be comparable to that of infected human liver with or without hepatocellular carcinoma and utilize the HCV cell culture models to identify therapeutic potential of interferons, ribozymes and protease inhibitors that may block one or more steps of hepatitis C replication cycle. 2) To study the molecular biology of the protease, helicase and NTPase domains of the NS3 protein and its role in negative strand RNA synthesis using an inducible expression system that utilizes T7 RNA polymerase and a transcription plasmid. We propose to establish a more robust RNA replication and expression system as an alternative to low levels of HCV in the RNA transfection model. 3) To determine whether intracellular expression of single-chain antibodies (scFv) targeted to structural proteins, core and E2, interfere with virus morphogenesis and antibodies targeted to NS3 and NS4 proteins could inhibit protease, helicase and NTPase activities and replication of HCV. Accomplishment of these aims will promote development of novel therapies to treat chronic hepatitis C infection and may reduce the potential risk of developing liver cirrhosis and hepatocellular carcinoma.
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