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， 世界各地的肝功能衰竭和癌症并发症。 开发一种有效的疫苗一直很困难。此外， 由于缺乏模型，新的抗病毒药物的鉴定一直受到限制 丙型肝炎病毒系统。研究丙型肝炎病毒的模型系统的发展 分子治疗学的复制与鉴定 代表着尚未解决的重大问题。丙型肝炎病毒RNA依赖的RNA聚合酶 (RDRP)可能是丙型肝炎病毒抗病毒药物的靶点。我们已经表达了 重组丙型肝炎病毒RDRP，并将其分离纯化为活性酶。这个 需要检验的假设是体外模型系统可以 开发用于研究丙型肝炎病毒复制的关键方面并识别 靶向丙型肝炎病毒RNA依赖的RNA的分子疗法 聚合酶。 这项工作的总体目标是进一步净化和 丙型肝炎病毒RNA依赖的RNA聚合酶(RDRP)的特性和启动 识别候选调节RNA结合蛋白的研究 丙型肝炎病毒RDRP亚基。丙型肝炎病毒的进一步纯化和鉴定 RDRP和调节亚单位的确定最终应该 允许更多特定的基于分子的疗法治疗慢性肝炎 C有待开发。 具体目标： *进一步纯化和鉴定丙型肝炎病毒RDRP。 *对丙型肝炎病毒RDRP进行定点突变，以确定 特定氨基酸残基的重要性和设计有活性的酶 可以更快地提纯。 *识别与3‘基因组RNA结合的细胞或病毒蛋白 结构基序及其对丙型肝炎病毒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).