An estimated 170 million people worldwide are infected with the hepatitis C virus (HCV), a major cause of chronic hepatitis, liver cirrhosis and hepatocellular carcinoma. Recently an infectious HCV cell culture (HCVcc) system that recapitulates the complete virus life cycle has been described that allows virus morphogenesis to be studied for the first time. The HCV p7 protein is a small, integral membrane protein that oligomerizes in vitro to form amantadine sensitive ion channels. p7 is essential for infectivity of HCV in chimpanzees and contains genotype specific sequences that are important for this function, suggesting that p7 interacts with other virus proteins. The role of p7 during the virus life cycle, however, is unknown. In preliminary studies I have demonstrated that p7 is required for infectious HCVcc production and that it contains essential genotype specific sequences. In addition, I have genetically identified putative interactions between p7 and viral nonstructural proteins including NS2. In this application we will identify the stage of virus morphogenesis at which p7 acts as well as the importance of p7 processing intermediates for these functions. I will use mutagenesis to precisely define the essential genotype specific sequences in p7 and isolate second-site revertants to define the viral proteins that interact with p7. I will then examine these putative interactions using confocal fluorescence microscopy to first localize p7 in infected cells and then to determine whether p7 co-localizes with other viral proteins. The interactions of p7 with other viral proteins will be further examined by coimmunoprecipitation and FRET-based techniques. The experiments described in this application will provide much needed detail into the role(s) of p7 in infectious virus production and the participation of other viral proteins in this process. HCV is a major human pathogen for which there is no vaccine and only limited treatment options. The identification of novel targets is necessary for the development of more effective and satisfactory antiviral strategies. These studies of the HCV p7 protein and its essential role in the enigmatic process of infectious virus production may provide such targets.
