HCV NS3 and NS5A: Biochemical Mechanisms and Biological Functions

HCV NS3 和 NS5A：生化机制和生物学功能

• 批准号: 7842164
• 负责人: Kevin Douglas Raney
• 金额: $ 38.9万
• 依托单位: UNIV OF ARKANSAS FOR MED SCIS
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-09-12 至 2010-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7842164
• 关键词: ATP Hydrolysis; Address; Amino Acid Motifs; Binding; Binding Sites; Biochemical; Biological; Biological Assay; Biological Process; Boxing; Chemicals; DNA; DNA biosynthesis; Data; Dimerization; Elements; Enzymatic Biochemistry; Equilibrium; Fluorescence; Genetic Recombination; Genome; Genomics; Goals; Hepatitis C virus; In Vitro; Investigation; Kinetics; Maps; Mass Spectrum Analysis; Metabolism; Methods; Modeling; Molecular; Molecular Motors; Motor; Mutate; Peptides; Phosphoproteins; Phosphorylation; Play; Process; Protein Binding; Protein Region; Proteins; Protocols documentation; RNA; RNA Binding; RNA Helicase; RNA Sequences; RNA Viruses; RNA replication; RNA-Binding Proteins; Reagent; Replicon; Resolution; Role; Site; Specificity; Structural Protein; Structure; System; Testing; Translations; Viral; Viral Proteins; Virus Replication; Work; abstracting; base; crosslink; dimer; helicase; in vivo; inorganic phosphate; member; monomer; prevent; protein protein interaction; viral RNA

HCV NS3 and NS5A: Biochemical Mechanisms and Biological Functions Abstract RNA metabolism requires the action of numerous ATP-dependent, molecular motor proteins that are believed to transport, remodel, and unwind secondary structures in RNA sequences. Many of these molecular motors are DEAD-box or closely related proteins. Positive strand RNA viruses such as the hepatitis C virus (HCV) require the activity of these proteins for RNA replication. Determination of the mechanism of these proteins is of fundamental importance to our understanding of viral replication as well as RNA metabolism in general. The long-term goal of this project is to determine the mechanism of RNA replication of HCV. The immediate focus of this proposal is on those proteins that bind and manipulate HCV RNA. Non-structural protein 3 (NS3) is an RNA motor protein (or helicase), that is necessary for HCV replication. Our data supports the hypothesis that NS3 exists in equilibrium between monomeric and oligomeric species and that its RNA unwinding activity increases with increasing oligomerization. We have devised new assays, reagents, and protocols that will allow us to test this hypothesis and to determine whether other HCV proteins such as NS5A can alleviate the need for oligomerization. We will examine helicase activity with high temporal and spatial resolution by using a new, chemical footprinting approach. We have studied the ATP hydrolysis cycle for NS3 and based on our preliminary data, we hypothesize that phosphate release limits the overall rate for RNA unwinding. We will examine protein motifs that are proposed to bind phosphate to test this hypothesis. Additionally, our work has identified NS5A as an RNA binding protein that also interacts directly with NS3. We will uncover the mechanism of NS5A RNA binding and its role in HCV replication through these studies. The interplay between the NS3 and NS5A will be examined in detail using new biochemical and biological approaches. In aim 1, we will Investigate the mechanism for unwinding and translocation of DNA and RNA by NS3 and relate the mechanism to ATP hydrolysis. In aim 2, we will determine the biochemical mechanism for RNA binding by NS5A and perform the first structure-function study of this protein. In aim 3, the molecular basis for NS3 interaction with NS5A will be examined by using a recently developed fluorescence assay along with our RNA unwinding, structural, and biological approaches. Specifc regions of protein-protien interactions will be identified by mass spectrometric methods.

HCV NS3和NS5A：生化机制和生物学功能 抽象的 RNA代谢需要大量依赖ATP的分子运动蛋白的作用 据信它可以在RNA序列中运输，重塑和放松二级结构。 这些分子电动机中的许多是死箱或密切相关的蛋白质。正链 RNA病毒（例如丙型肝炎病毒（HCV））需要这些蛋白质的活性RNA 复制。确定这些蛋白质的机制对于 我们对病毒复制以及RNA代谢的理解一般。长期目标 该项目的是确定HCV的RNA复制机制。直接的重点 该建议的是结合和操纵HCV RNA的蛋白质。非结构蛋白 3（ns3）是一种RNA运动蛋白（或解旋酶），是HCV复制所必需的。我们的数据 支持以下假设：NS3在单体和低聚之间的平衡中存在 物种和其RNA放松活性随着寡聚的增加而增加。我们有 设计了新的测定法，试剂和协议，使我们能够检验此假设和 确定其他HCV蛋白（例如NS5A）是否可以减轻对低聚的需求。 我们将通过使用新的，高时空和空间分辨率检查解旋酶活性 化学足迹方法。我们研究了NS3和基于的ATP水解周期 在我们的初步数据上，我们假设磷酸盐释放限制了RNA的整体速率 放松。我们将检查提议结合磷酸盐以对此结合的蛋白质基序 假设。此外，我们的工作已将NS5A确定为RNA结合蛋白 直接与NS3相互作用。我们将发现NS5A RNA结合的机制及其在 通过这些研究复制HCV。 NS3和NS5A之间的相互作用将是 使用新的生化和生物学方法详细检查。在AIM 1中，我们将 研究NS3对DNA和RNA放松和易位的机制，并相关 ATP水解的机制。在AIM 2中，我们将确定用于的生化机制 NS5A的RNA结合，并对该蛋白进行首次结构功能研究。在AIM 3中 NS3与NS5A相互作用的分子基础将通过使用最近开发的 荧光测定以及我们的RNA放松，结构和生物学方法。 蛋白质 - 培养基相互作用的指定区域将通过质谱法确定。