Structure and function of the HCV replication complex

描述（由申请人提供）：丙型肝炎病毒影响约2%的美国人口，导致急性和慢性肝病，肝硬化，肝癌和肝功能衰竭。现有的治疗丙肝病毒的疗法耐受性差，而且仅对一小部分患者有效。因此，我们必须集中精力开发治疗丙型肝炎病毒感染的新疗法。这项工作的成功取决于对HCV基因表达和复制的分子机制的理解，因为参与这些过程的酶是药物设计的分子靶标。这些酶的研究，特别是在自然环境下的研究，由于缺乏强大的体外系统来研究完整复制复合体（RC）的组装和功能而受到阻碍。我们缺乏关于RC的结构信息，尽管对分离的复制酶（如非结构蛋白NS3和NS5B）进行了大量研究，但越来越多的证据表明，这些酶高度依赖于辅因子，并且在RC机器内操作时它们的功能不同。因此，为了开发与HCV复制酶相关的生物学和药理学检测方法，并获得有关HCV复制酶的新信息，我们建议分离HCV RC，并研究其作为完整复制全酶的结构和功能。将采用两种不同的、互补的方法来应对这一挑战：完整RC的体外翻译、加工和组装。2. 肝细胞系RC的分离纯化。丙型肝炎病毒治疗发展的第二个限制是没有关于丙型肝炎病毒研究中心结构的信息。我们不知道蛋白质是如何结合在一起的，RC是如何整合到膜上的，或者它是如何结合RNA基因组的。为了解决这些问题，我们项目的第二个目标是确定RC内分子间相互作用的位点，并建立RC复合体结构的三维模型。这项工作将为RC蛋白的协同活动提供新的见解，并将揭示蛋白质-蛋白质界面，可以作为开发小分子抑制剂的靶点。目前正在使用三种不同的方法来构建HCV RC的相互作用图：生物化学方法与高分辨率质谱相结合。2. 基因抑制分析鉴定偶联氨基酸。3. 从HCV序列比对中检测功能偶联残基的系统发育方法。这项工作的成功将取决于项目领导团队中代表的化学家、生物化学家和病毒遗传学家的协调工作。

DESCRIPTION (provided by applicant): Hepatitis C virus afflicts ~2% of the U.S. population, causing acute and chronic liver disease, cirrhosis, liver cancer, and liver failure. Available therapies for treating HCV are poorly tolerated and effective in only a fraction of patients. It is therefore vital that we focus on the development of new therapies to treat HCV infection. The success of this effort hinges on developing an understanding of the molecular mechanisms underlying HCV gene expression and replication, as the enzymes involved these processes are the molecular targets for drug design. The study of these enzymes, particularly in their natural context, has been hampered by the lack of robust in vitro systems to study the assembly and function of the intact replication complex (RC). We lack structural information on the RC and, despite numerous studies on the isolated replicative enzymes (such as the nonstructural proteins NS3 and NS5B) there is growing evidence that these enzymes are highly dependent on cofactors and that they function differently when operating within the RC machine. Therefore, to develop biologically and pharmacologically relevant assays and to learn new information about the HCV replicative enzymes, we propose to isolate the HCV RC and to study its structure and function as an intact replicative holoenzyme. This challenge will be confronted by using two different, complementary approaches: 1. In vitro translation, processing and assembly of the intact RC. 2. Isolation and purification of the RC from liver cell lines. A second limitation to the development of HCV therapeutics is that there is no information on the architecture of the HCV RC. We do not know how the proteins fit together, how the RC integrates into membranes or how it binds the RNA genome. To address these issues, a second goal of our project is to determine sites of intermolecular interaction within the RC and to build a three-dimensional model of the RC complex structure. This effort will provide new insights into the coordinated activities of the RC proteins and it will reveal protein-protein interfaces that can serve as targets for the development of small molecule inhibitors. Three different approaches are being used to build the interaction map for the HCV RC: 1. Biochemical methods combined with high-resolution mass spectrometry. 2. Genetic suppression analysis to identify coupled amino acids. 3. Phylogenetic methods to detect functionally coupled residues from HCV sequence alignments. The success of this effort will be contingent on the coordinated work of chemists, biochemists and viral geneticists that are represented in the project leadership team. PUBLIC HEALTH RELEVANCE: Hepatitis C virus (HCV) is a major cause of acute and chronic liver disease, cirrhosis, liver cancer, and liver failure. An HCV vaccine is not available, and current HCV therapies are inadequate. This proposal addresses the basic mechanisms of HCV genome replication, which are key targets for developing improved antiviral therapies.