HCV NS3: Biological, Biochemical and Structural Analysis

描述(申请人提供)：丙型肝炎病毒(丙型肝炎病毒)感染全球超过1.7亿人。它是美国肝脏疾病的主要原因，也是大多数肝脏移植的原因。目前对这种传染病的治疗是不够的，因此必须开发新的治疗方法。我们和其他人一样，已经获得了涉及病毒编码的所有已知非结构(NS)蛋白的多蛋白复合体的证据。NS3、NS4A、NS4B、NS5A和NS5B似乎在结构和功能上相互作用。我们将应用最先进的生化方法来研究NS3-4A的RNA解离、易位和RNA结合的机制，NS3-4A被认为是这种酶的生物相关形式。我们将在生物学上相关的丙型肝炎病毒复制子中检测NS3-4A与其他NS蛋白的潜在相互作用。我们发现NS3解旋酶极大地刺激了NS5B聚合酶的活性。这种功能相互作用的机制将被研究。我们最近已经解决了NS3解旋酶结构域的一种明显的二聚体形式的晶体结构，在这种结构中，酶的两个分子与同一条核酸链结合。我们将测试这种新结构揭示的相互作用的生物学和生化意义。我们的工作已经确定了复制丙型肝炎病毒基因组所需的NS3蛋白表面，但不是已知解旋酶结构域的一部分。这些表面可能参与高度特异的蛋白质-蛋白质相互作用，因此是破坏负责丙型肝炎病毒复制的多蛋白质复合体的目标。NS3与其他丙型肝炎病毒蛋白之间的相互作用位点将通过化学交联和质谱学来确定。我们发现NS5A与RNA和NS5B紧密结合。我们现在将研究NS5A、NS3和NS5B之间可能的功能相互作用，并试图解决NS5A的晶体结构。

DESCRIPTION (provided by applicant): Hepatitis C Virus (HCV) infects over 170 million persons worldwide. It is the leading cause of liver disease in the U.S. and is responsible for most liver transplants. Current treatments for this infectious disease are inadequate; therefore new therapies must be developed. We as well as others have obtained evidence for a multi-protein complex that involves all of the known nonstructural (NS) proteins encoded by the virus. NS3, NS4A, NS4B, NS5A, and NS5B appear to interact structurally and functionally. We will apply state-of-the art biochemical approaches for studying the mechanism of RNA unwinding, translocation, and RNA binding of NS3-4A, which is believed to be the biologically relevant form of this enzyme. We will examine potential NS3-4A interactions with other NS proteins in biologically relevant HCV replicons. We have found that the NS3 helicase stimulates the activity of the NS5B polymerase dramatically. The mechanism for this functional interaction will be investigated. We have recently solved the crystal structure of an apparent dimeric form of NS3 helicase domain in which two molecules of the enzyme are bound to the same strand of nucleic acid. We wilt test the biological and biochemical significance of the interactions revealed by this new structure. Our work has identified protein surfaces of NS3 that are required for replication of the HCV genome, but are not part of known helicase domains. These surfaces are likely to be involved in highly specific, protein-protein interactions and are therefore targets for disruption of multi-protein complexes that are responsible for HCV replication. Sites for interaction between NS3 and other HCV proteins will be determined by chemical crosslinking coupled with mass spectrometry. We have found that NS5A binds tightly to RNA and to NS5B. We will now examine possible functional interactions between NS5A, NS3, and NS5B and attempt to solve the crystal structure of NS5A.