Mechanism of RNA Helicase Activity by DExH/D Proteins

说明(申请人提供)NS3解旋酶是丙型肝炎病毒(丙型肝炎病毒)复制机制的重要组成部分，丙型肝炎病毒是一种正链RNA病毒，对公众健康构成主要威胁。NS3是一种多功能分子马达，在DNA和RNA双链解离过程中能水解三磷酸核苷酸。它是解旋酶超家族2(SF2)的成员，属于DExH/D亚类，参与RNA代谢的方方面面，包括前mRNA剪接、RNA干扰、翻译、RNA降解以及多种形式的病毒复制。尽管DExH/D酶普遍存在，它们对高等生物体的生存至关重要，并且它们在许多人类病原体中扮演着重要的角色，但关于这一蛋白家族对RNA解离和核糖核蛋白重塑的分子机制的研究很少。人们对它们的核酸特异性、它们的转位方式、链置换的机制，或者ATP水解和解链过程中所消耗的功之间的耦合知之甚少。为了解决这些问题，我们建议使用NS3作为一个模型系统来探索DExH/D蛋白的行为。这是一个很好的原型，原因有很多：(A)NS3的结构特征已经确定，它是DExH/D成员的典型系统发育，(B)。它是一个大型核糖核蛋白机器的一部分，该机器包含调节其行为的辅因子(像大多数DExH/D蛋白质一样)，(C)。它具有强大的解卷反应，已进行了定性研究，(D)。有关NS3的信息可能对制定丙型肝炎病毒管理战略至关重要。我们提出了一个用于NS3解旋酶活性的生物物理表征的综合方案。我们将检测分离的NS3酶的行为及其与调节辅因子NS4A和NS5B的复合体的性质。还将探索NS3对DNA的解离，因为反应是机械上不同的，它可能为病毒的新陈代谢策略提供重要的见解。实验将通过应用监测组合底物文库解卷的新的预稳态动力学方法来进行。这一方法与耦合的ATPase分析、对化学修饰的双链底物的研究、时间分辨足迹和单分子方法将使我们能够表征NS3在各种大分子环境中的微观行为。这一结果将从根本上扩展我们对解旋酶机制的认识，并促进新的丙型肝炎病毒抑制剂的开发。

DESCRIPTION (provided by applicant) The NS3 helicase is an essential constituent of the replication machinery from Hepatitis C Virus (HCV), which is a positive-strand RNA virus that represents a major threat to public health. NS3 is a multifunctional molecular motor that hydrolyzes nucleotide triphosphates during the unwinding of both DNA and RNA duplexes. It is a member of helicase superfamily 2 (SF2), and it belongs to the DExH/D subgroup of enzymes that are involved in all aspects of RNA metabolism, including pre-mRNA splicing, RNA interference, translation, RNA degradation, and in many forms of viral replication. Despite the ubiquity of the DExH/D enzymes, their fundamental importance for viability of higher organisms, and their role in numerous human pathogens, there have been few studies on the molecular mechanisms for RNA unwinding and ribonucleoprotein remodeling by this family of proteins. Little is known about their nucleic acid specificity, their mode of translocation, the mechanism of strand displacement, or the coupling between ATP hydrolysis and work expended during unwinding. To address these issues, we propose to use NS3 as a model system for exploring the behavior of DExH/D proteins. It is an excellent prototype for numerous reasons: (a). NS3 has been structurally characterized and it is a phylogenetically typical of DExH/D member, (b). It is part of a large ribonucleoprotein machine that contains cofactors that modulate its behavior (like most DExH/D proteins), (c). It has a robust unwinding reaction that has been investigated qualitatively, (d). Information about NS3 is likely to be critical for the development HCV management strategies. We propose a comprehensive program for the biophysical characterization of NS3 helicase activity. We will examine behavior of the isolated NS3 enzyme and its properties in complex with modulatory cofactors NS4A and NS5B. DNA unwinding by NS3 will also be explored, as the reaction is mechanistically distinct and it may provide important insights into metabolic strategies of the virus. Experiments will be carried out by applying new pre-steady state kinetic methods that monitor the unwinding of combinatorial substrate libraries. This approach, together with coupled ATPase assays, studies on chemically modified duplex substrates, time-resolved footprinting and single molecule methods will allow us to characterize the microscopic behavior of NS3 in a variety of macromolecular contexts. The results will fundamentally extend our knowledge of helicase mechanism and facilitate the development of new HCV inhibitors.