HCV NS3: Biological, Biochemical and Structural Analysis

描述（由申请人提供）：丙型肝炎病毒（HCV）感染全球超过1.7亿人。它是美国肝脏疾病的主要原因，并负责大多数肝脏移植。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。我们和其他人已经获得了多蛋白复合物的证据，该复合物涉及病毒编码的所有已知非结构（NS）蛋白。NS 3、NS 4A、NS 4 B、NS 5A和NS 5 B似乎在结构和功能上相互作用。我们将应用最先进的生物化学方法来研究NS 3 -4A的RNA解旋、易位和RNA结合的机制，NS 3 -4A被认为是这种酶的生物学相关形式。我们将研究潜在的NS 3 -4A与其他NS蛋白在生物学相关的HCV复制子的相互作用。我们已经发现NS 3解旋酶显著地刺激NS 5 B聚合酶的活性。将研究这种功能相互作用的机制。我们最近已经解决了一个明显的二聚体形式的NS 3解旋酶结构域的晶体结构，其中两个分子的酶结合到同一链的核酸。我们将测试这种新结构所揭示的相互作用的生物学和生物化学意义。我们的工作已经确定了HCV基因组复制所需的NS 3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性的蛋白质-蛋白质相互作用，因此是破坏负责HCV复制的多蛋白质复合物的靶点。NS 3和其他HCV蛋白之间相互作用的位点将通过化学交联结合质谱法来确定。我们已经发现NS 5A与RNA和NS 5 B紧密结合。我们现在将研究NS 5A，NS 3和NS 5 B之间可能的功能相互作用，并试图解决NS 5A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.