Hepatitis C virus genome structure: dynamic roles in replication and infectivity

项目摘要 丙型肝炎病毒（HCV）感染了美国的大量人口（约2%），在那里它仍然是一个流行病。 慢性肝病和癌症的主要原因。虽然有效的抗病毒药物最近已经成为 有效的，长期的对抗HCV相关疾病的成功需要清楚地了解病毒 复制机制。此外，HCV提供了一个重要的模型系统， 许多相关的正链RNA病毒的复制，包括登革热病毒和其他未满足的 威胁人类和动物健康。迄今为止，大多数关于HCV复制的工作都集中在活动上， 病毒的非结构（NS）蛋白或宿主因子在这一过程中的作用。很少有 HCV RNA基因组在复制中的作用或病毒与宿主之间的功能性相互作用 基因组和NS蛋白。这是一个关键的差距，因为HCV RNA和病毒之间的相互作用 蛋白质是字面上的“橡胶上路”，在病毒复制的关键事件发生。我们 将利用我们最近在NS蛋白质中定义分子网络的成功， 表征RNA和RNA-蛋白质复合物，我们将探索新的监测方法， 研究RNA在体内的相互作用，以达到以下三个目的：（一）确定RNA的功能性， HCV基因组内的RNA结构;（B）定义NS蛋白之间的分子相互作用 和RNA基因组，建立它们的相互作用位点和功能相关性， 病毒生命周期的各个阶段;（C）定义病毒的结构特征和功能属性。 复制前起始复合物（RPIC），启动病毒繁殖的整个过程。 这些目标将通过整合研究病毒的创新方法来实现 遗传学，RNA交联和测序，酶学和晶体学，并利用我们的 在病毒学和RNA生物化学方面的各自优势。通过关注分子间的相互作用 在基因组RNA和NS蛋白之间，这项工作在概念上是创新的。在开发工具的过程中， 需要推进这项研究，该项目也是技术创新，导致方法 以及将对RNA病毒研究界具有广泛用途的试剂。

Project Summary Hepatitis C virus (HCV) infects a large population within the United States (~2%), where it remains a major cause of chronic liver disease and cancer. While potent antiviral drugs have recently become available, long-term success in combating HCV-related disease requires a clear understanding of viral replication mechanisms. Furthermore, HCV provides an essential model system for understanding the replication of many related positive-strand RNA viruses, including dengue virus and other unmet threats to human and animal health. To date, most work on HCV replication has focused on activities of the viral nonstructural (NS) proteins or the role of host factors in this process. Very little has been done on role of the HCV RNA genome in replication or on the functional interactions between the viral genome and NS proteins. This is a critical gap, since the interaction between HCV RNA and viral proteins is literally where the “rubber hits the road”, and the key events in viral replication occur. We will leverage our recent success in defining molecular networks among the NS proteins, and in characterizing RNA and RNA-protein complexes, and we will exploit new approaches for monitoring RNA interactions in vivo in order to achieve the following three aims: (A) Determine the functional RNA structures within the HCV genome; (B) Define the molecular interactions between NS proteins and the RNA genome, establishing their interaction sites and functional relevance during sequential phases of the viral lifecycle; (C) Define the structural features and functional attributes of the Replication Pre-initiation Complexes (RPIC), which initiates the entire process of virus propagation. These objectives will be accomplished by integrating innovative approaches for studying viral genetics, RNA crosslinking and sequencing, enzymology and crystallography, and capitalizing on our respective strengths in virology and RNA biochemistry. By focusing on the molecular interplay between genomic RNA and NS proteins, the work is conceptually innovative. In developing the tools needed to advance this research, the project is also technologically innovative, resulting in methods and reagents that will be of widespread utility to the RNA virus research community.