IN VITRO AND CELLULAR MODELS FOR HEPATITIS C VIRUS REPLICATION

丙型肝炎病毒（HCV）是慢性肝炎的主要原因， 肝衰竭和癌症的并发症。 研制有效的疫苗一直很困难。而且 由于缺乏模型， HCV系统。 研究HCV的模型系统的开发 分子疗法的复制和鉴定 代表着尚未解决的重大问题。HCV RNA依赖性RNA聚合酶 （RDRP）是HCV抗病毒药物的可能靶点。我们已经表示 重组HCV RDRP并将其作为活性酶分离。的 要检验的假设是体外系统模型可以 研究HCV复制的关键方面， 以HCV RNA依赖性RNA为靶点的分子疗法 聚合酶。 这项工作的总体目标是进一步净化和 描述HCV RNA依赖性RNA聚合酶（RDRP）的特征，并启动 研究以鉴定作为候选调节因子的RNA结合蛋白， HCV RDRP亚基。丙型肝炎病毒的进一步纯化、鉴定 RDRP和调节亚基的鉴定最终应该 允许更特异的基于分子的治疗慢性肝炎 C要发展。 具体目标： * 进一步纯化和鉴定HCV RDRP。 * 进行HCV RDRP的定点突变，以确定 特定氨基酸残基重要性和工程化活性酶 可以更快地净化。 * 为了识别细胞或病毒蛋白结合3'基因组RNA 结构基序并确定它们对HCV RDRP活性的影响（长- 术语）。

Hepatitis C virus (HCV) is a major cause of chronic hepatitis, with the complications of liver failure and cancer, throughout the world. Developing an effective vaccine has been difficult. Moreover, the identification of new antivirals has been limited by the lack of model HCV systems. Both the development of model systems to study HCV replication and identification of molecular based therapeutics represent major unsolved problems. The HCV RNA-dependent RNA polymerase (RDRP) is a likely target for HCV antivirals. We have expressed recombinant HCV RDRP and isolated it as an active enzyme. The hypothesis to be tested is that a model in vitro system can be developed to study key aspects of HCV replication and to identify molecular based therapeutics that target the HCV RNA-dependent RNA polymerase. The over-all objective of this work is to further purify and characterize the HCV RNA-dependent RNA polymerase (RDRP) and initiate studies to identify RNA binding proteins that are candidate regulatory subunits of HCV RDRP. The further purification, characterization of HCV RDRP and the identification of regulatory subunits should eventually permit more specific molecular based therapeutics for chronic hepatitis C to be developed. Specific Objectives: * To further purify and characterize the HCV RDRP. * To perform site directed mutagenesis of HCV RDRP to determine the importance of specific amino residues and to engineer an active enzyme that can be more rapidly purified. * To identify cellular or viral proteins bind the 3' genomic RNA structural motif and determine their effect on HCV RDRP activity (long- term).