HCV NS3 and NS5A: Biochemical Mechanisms and Biological Functions

HCV NS 3和NS 5A的生化机制及生物学功能 摘要 RNA代谢需要许多ATP依赖的分子马达蛋白的作用 被认为在RNA序列中运输、重塑和展开二级结构。 许多这些分子马达是死亡盒或密切相关的蛋白质。正链 RNA病毒如丙型肝炎病毒（HCV）需要这些蛋白质的活性， 复制的确定这些蛋白质的作用机制对研究这些蛋白质的生物学特性具有重要意义。 我们对病毒复制和RNA代谢的理解。远景目标 本项目的主要目的是研究HCV RNA复制的机制。眼前的重点 这个建议的重点是那些结合和操纵HCV RNA的蛋白质。非结构蛋白 NS 3是HCV复制所必需的RNA马达蛋白（或解旋酶）。我们的数据 支持NS 3在单体和寡聚体之间平衡存在的假设 物种，其RNA解旋活性随着寡聚化的增加而增加。我们有 设计了新的检测方法、试剂和方案，使我们能够检验这一假设， 确定其他HCV蛋白如NS 5A是否可以减轻寡聚化的需要。 我们将使用一种新的， 化学足迹法我们研究了NS 3的ATP水解循环， 根据我们的初步数据，我们假设磷酸盐的释放限制了RNA的总速率， 放松我们将研究蛋白质基序，建议结合磷酸盐来测试这一点 假说.此外，我们的工作已经确定NS 5A作为RNA结合蛋白， 与NS 3直接交互。我们将揭示NS 5A RNA结合的机制及其在细胞凋亡中的作用。 HCV复制通过这些研究。NS 3和NS 5A之间的相互作用将是 使用新的生物化学和生物学方法进行详细研究。在目标1中， 探讨NS 3介导DNA和RNA解旋和转位的机制及其相关性 ATP水解的机制。在目标2中，我们将确定 NS 5A与RNA结合，并对该蛋白进行了首次结构-功能研究。在目标3中， NS 3与NS 5A相互作用的分子基础将通过使用最近开发的 荧光分析沿着我们的RNA解旋、结构和生物学方法。 蛋白质-蛋白质相互作用的特定区域将通过质谱方法鉴定。

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.