Chronic hepatitis C virus (HCV) infection is associated with the development of cirrhosis, an elevated risk of hepatocellular carcinoma and is currently responsible for almost 30 percent of end-stage liver disease in need of transplantation in the United States. At present, no means of prevention of HCV infection exists and treatments are unsatisfactory. HCV is highly species-specific. Only humans and a few high primates are susceptible. The only animal model for HCV vaccine or antiviral research is the endangered chimpanzee. One of the major reasons that vaccine development and antiviral therapy are lacking for HCV stems from the fact that there are no satisfactory small animal or in vitro model systems for studying HCV replication. The applicant has recently reported a novel in vitro system for delivering a replication competent HBV to cells of hepatic origin by using an HBV recombinant baculovirus. In HBV baculovirus infected HepG2 cells, HBV transcripts, intracellular and secreted HBV antigens are produced and replication occurs as evidenced by the presence of high levels of intracellular replicative intermediates and protected HBV DNA in the medium. Covalently closed circular DNA is present indicating that, in this system, HBV core particles are capable of delivering newly synthesized HBV genomes back into the nucleus of infected cells. She has also demonstrated that the HBV recombinant baculovirus system can be used to monitor the effects of an antiviral on multiple aspects of the HBV life cycle include formation of newly synthesized CCC DNA as well as the effects on preexisting CCC DNA. Based on the success with generation and usage of the HBV recombinant baculovirus system, she will test in this proposal the following hypothesis: An HCV recombinant baculovirus can be generated which is replication competent in human hepatic cells in culture and can be used to study molecular aspects of HCV replication and effects of specific anitivirals on HCV replication.
The Specific Aims are: 1. To generate recombinant baculoviruses that contain all or part of the HCV cDNA under the control of mammalian promoters. 2. To test the ability of HCV-recombinant baculoviruses to express HCV gene products and replicate HCV in human cells of hepatic origin. 3. To use recombinant HCV baculovirus to evaluate the direct effect of interferon treatment on evolution of quasispecies during HCV replication in cells of hepatic origin.
