KSEF RDE: Development of Transgenic Hepatitis C virus Mouse Models

Hepatitis C virus (HCV) is a common cause of liver diseases such as chronic hepatitis, cirrhosis, and hepatocellular carcinoma (HCC). It affects approximately 4 million people in the U.S. and 170 million people worldwide. HCV infection is a major risk factor for the development of hepatocellular carcinoma (HCC). HCVassociated end-stage of liver diseases is the leading indication for liver transplantation. Advances on HCV research have been impeded by the lack of a robust cell culture system of HCV propagation and small animal models of HCV infection and replication. Recent breakthroughs have been the development of genomic and subgenomic HCV replicons and infectious HCV in cell culture, which allow genetic studies of the entire HCV life cycle. However, development of small animal models for the study of HCV replication, pathogenesis, and carcinogenesis has been proven difficult. Recently, we have demonstrated that a subgenomic HCV RNA of genotype 2a replicated efficiently in mouse embryonic fibroblasts (MEF) albeit very inefficiently in mouse hepatocytes. More importantly, we have demonstrated that the cDNA-derived HCV RNA genome by cellular Pol II polymerase transcription resulted in robust production of infectious virus. Recent work from Dr. Charlie Rice's group demonstrated that expression of human CD81 and occludin, both of which are HCV receptors/coreceptors, is essential and sufficient for infection of HCV pseudotyped particles (HCVpp) in mouse hepatocytes. These remarkable advances provide a firm foundation to develop transgenic mice for studying HCV infection, replication, pathogenesis, and carcinogenesis. The overall goal of this application is to develop HCV transgenic mouse models for the study of HCV infection, replication, pathogenesis, and carcinogenesis in vivo. Our specific aims are: 1) to identify cellular proteins important for efficient HCV RNA replication in mouse cells using multidisciplinary approaches, including genetic complementation, cross-linking, immunological, proteomics, and biochemical methodologies; 2) to develop transgenic mice that contain full-length cDNAs of luciferaseexpressing JFH1 HCV RNAs and transgenic mice expressing both human CD81 and occludin, both of which are essential and sufficient for infection of HCV pseudotyped particles (HCVpp) in mouse hepatocytes; and 3) to determine HCV infection and replication in transgenic mice using GFP- and luciferase-expressing infectious HCV in conjunction with administration of HCV NS3 protease- and NS58 RNA-dependent RNA polymerase (RdRp)- specific inhibitors and mouse interferon. Knowledge obtained and technologies developed in these studies will facilitate anti-HCV drug discovery and lead to identification of specific anti-HCV agents. In addition, advancement in knowledge and technologies from these studies will place us in a strong position to compete for additional federal and private-sector funding. Moreover, this application will likely result in intellectual property that will contribute to the economic development of the Commonwealth. KSEF