Hepatitis C Virus (HCV) is an enveloped, plus-stranded RNA virus with a high mutation rate that replicates in the cytoplasm of hepatocytes. HCV causes acute and chronic hepatitis and is the leading cause of hepatocellular carcinoma (HCC) in the United States. Most patients fail to clear the virus despite mounting polyclonal and multi-specific humoral and cellular immune responses against all of the HCV proteins. Despite its significance as a human pathogen, the mechanisms responsible for HCV persistence and immune evasion are not understood, and current therapies for the treatment of chronic HCV are lacking. There is neither a cure for chronic HCV infection nor is there a vaccine that protects against infection. Progress in these areas has been severely hindered by the absence of suitable research models that permit the study of HCV infection, replication, immunobiology, and pathogenesis. The long term goal of this work is to elucidate the molecular and cellular mechanisms leading to HCV clearance versus HCV persistence and pathogenesis in the host. The focus of this study is the development of a murine model of HCV replication. The strategy of directed evolution, taken from the disciplines of chemical and synthetic biology, will be used to develop HCV replicons that have been adapted in vivo to the murine liver. Past unsuccessful attempts to develop an HCV mouse model by generating transgenic mice bearing an HCV genomic cDNA and by transfecting mouse livers with in vitro-transcribed HCV (+)- strand RNA have utilized """"""""wild-type"""""""" HCV genomes isolated from natural infections or HCV variants adapted and isolated in cell culture through the use of a selectable marker. In no case has the HCV genome been """"""""optimized"""""""" to function in the murine liver. This study will use mutagenic PCR and DNA shuffling to form artificial quasispecies of HCV replicons in vitro. These pools of HCV variants will be delivered to the murine liver by hydrodynamic injection. The phenomenon of liver repopulation will then be used to amplify a sub-population of hepatocytes in which HCV is replicating. HCV species replicating in the liver will be isolated and characterized. The system developed will permit the study of HCV's intracellular interactions with its host in authentic hepatocytes within the context of the liver and will permit the dissection of the interactions between HCV and the host immune system. In addition, a mouse model of HCV replication will provide a valuable tool in evaluating the efficacy of potential antiviral therapeutics.
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