HCV NS3 and NS5A: Biochemical Mechanisms and Biological Functions

丙型肝炎病毒NS3和NS5A：生化机制和生物学功能 摘要 RNA新陈代谢需要许多依赖于ATP的分子马达蛋白的作用 它们被认为可以运输、重塑和解开RNA序列中的二级结构。 这些分子马达中的许多都是死盒或密切相关的蛋白质。正链 像丙型肝炎病毒(丙型肝炎病毒)这样的RNA病毒需要这些蛋白质的活性来获得RNA 复制。这些蛋白质的作用机制的确定对 我们对病毒复制以及RNA新陈代谢的总体理解。长期目标 本项目的目的是确定丙型肝炎病毒的RNA复制机制。眼下的焦点 这项提议的重点是那些结合和操纵丙型肝炎病毒RNA的蛋白质。非结构蛋白 3(NS3)是一种RNA马达蛋白(或解旋酶)，是丙型肝炎病毒复制所必需的。我们的数据 支持NS3在单体和低聚物之间平衡存在的假设 物种，其RNA解离活性随着齐聚程度的增加而增加。我们有 设计了新的分析、试剂和方案，使我们能够检验这一假说，并 确定NS5A等其他丙型肝炎病毒蛋白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NS3和NS3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的总体速率 放松。我们将研究被提议与磷酸盐结合的蛋白质基序来测试这一点 假设。此外，我们的工作已经确定NS5A是一种RNA结合蛋白，也可以 直接与NS3相互作用。我们将揭示NS5A RNA结合的机制及其在 通过这些研究复制丙型肝炎病毒。NS3和NS5A之间的相互作用将是 使用新的生化和生物学方法进行了详细的检查。在目标1中，我们将 探讨NS3解离和移位DNA和RNA的机制及相关 ATP水解酶的作用机理。在目标2中，我们将确定生物化学机制 与NS5A结合的RNA，并进行该蛋白质的第一次结构功能研究。在目标3中， NS3与NS5A相互作用的分子基础将通过使用最新开发的 荧光分析以及我们的RNA解离、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学方法识别。

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.