Mechanism of RNA Helicase Activity by DExH/D Proteins

NS3解旋酶是丙型肝炎病毒（HCV）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，对公众健康构成重大威胁。NS3是一种多功能分子马达，在DNA和RNA双链解绕过程中水解核苷酸三磷酸。它是解旋酶超家族2 （SF2）的成员，属于DExH/D酶亚群，参与RNA代谢的各个方面，包括pre-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酶无处不在，它们对高等生物的生存能力至关重要，并且在许多人类病原体中发挥作用，但关于该蛋白家族RNA解绕和核糖核蛋白重塑的分子机制的研究很少。对于它们的核酸特异性、易位模式、链位移机制或ATP水解与解绕过程中所消耗的功之间的耦合，人们知之甚少。为了解决这些问题，我们建议使用NS3作为模型系统来探索DExH/D蛋白的行为。这是一个很好的原型，原因有很多：(a)。NS3已被结构表征，是DExH/D成员的系统发育典型，(b)。它是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助因子（如大多数DExH/D蛋白），(c)。它有一个强大的解绕反应，已定性研究，(d)。关于NS3的信息可能对HCV管理策略的制定至关重要。我们提出了一个全面的程序对NS3解旋酶活性的生物物理特性。我们将研究分离的NS3酶的行为及其与调节辅因子NS4A和NS5B复合物的性质。NS3的DNA解绕也将被探索，因为该反应在机制上是不同的，它可能为病毒的代谢策略提供重要的见解。实验将采用新的预稳态动力学方法来监测组合衬底库的展开。该方法与偶联atp酶测定、化学修饰双底物研究、时间分辨足迹和单分子方法一起，将使我们能够表征NS3在各种大分子环境下的微观行为。这些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HCV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.