MECHANISMS OF DENGUE VIRUS GENOME ENCAPSIDATION AND UNCOATING

 描述(申请人提供)：登革病毒是黄病毒家族的成员，是一种A类病原体，可导致人类最常见的节肢动物传播的病毒疾病。由于缺乏对病毒复制及其与宿主细胞相互作用的分子机制的深入了解，限制了抗病毒策略的发展。病毒基因组是 一种在病毒复制过程中扮演多种角色的RNA分子。它既是翻译的信使核糖核酸，又是核糖核酸扩增的模板，也是包埋的底物。在过去的十年里，关于DENV基因组复制的机制已经有了很大的了解。相比之下，在病毒颗粒形成过程中，基因组被衣壳蛋白招募，然后在感染期间从核衣壳释放到细胞质中，这是DENV和其他黄病毒正在研究的病毒生命周期的两个步骤。病毒衣壳是一种小的、高度碱性的蛋白质，它以低特异性结合核酸。此外，在病毒RNA中尚未发现包装信号。尽管如此，病毒基因组是包裹在颗粒内的唯一RNA。在这项提案中，我们将解决机制，并定义涉及登革热病毒基因组包裹和剥离的机制。为此，我们将结合我们在开发登革热病毒遗传工具方面的专业知识，将病毒基因组中重叠的功能与蛋白质组学方法和生化研究结合起来。在目标1中，我们将通过分析感染细胞中未被包裹的中间体和研究病毒成分在进入细胞过程中的命运来研究病毒基因组是如何释放到细胞质中的。此外，还将探索一种新的三维单粒子轨道跟踪方法来追踪感染过程中的衣壳蛋白。在目标2中，我们将使用基因工具分析衣壳蛋白对颗粒形成和感染性的要求。最近发现， 登革热病毒衣壳蛋白在感染细胞中遭受各种翻译后修饰。从感染细胞和病毒粒子中提纯的衣壳进行全面的质谱分析，结合重组病毒的设计，将用于确定衣壳结构特性的功能。在目标3中，我们将定义登革热病毒组装的蛋白质-蛋白质相互作用网络，并确定病毒蛋白NS3在这一过程中的功能。我们将在病毒感染的背景下使用最近开发的蛋白质组学平台来定义组装过程所需的宿主组件。剖析衣壳蛋白与宿主和病毒成分的多重功能和相互作用将有助于揭示登革热和其他黄病毒复制的基本方面。重要的是，拟议的研究将提供有关病毒过程的新信息，这些信息仍未被用于抗病毒干预。

 DESCRIPTION (provided by applicant): Dengue virus, a member of the Flaviviridae family, is a Category A pathogen that causes the most prevalent arthropod-borne viral illnesses in humans. The lack of in depth understanding of molecular mechanisms of viral replication and its interaction with the host cell, limits the development of antiviral strategies. The viral genome is an RNA molecule that plays multiple roles during viral replication. It serves as mRNA for translation, a template for RNA amplification, and substrate for encapsidation. A great deal has been learned in the last ten years about the mechanism of DENV genome replication. In contrast, the processes by which the genome is recruited by the capsid protein during viral particle formation and then released from the nucleocapsid into the cytoplasm during infection are two steps of the viral life cycle understudied for DENV and other flaviviruses. The viral capsid is a small highly basic protein that binds nucleic acids with low specificity. In addition, packaging signals have not been found in the viral RNA. Despite this, the viral genome is the only RNA encapsidated inside the particle. In this proposal, we will address mechanisms and define the machinery involved in dengue virus genome encapsidation and uncoating. To this end, we will combine our expertise in developing dengue virus genetic tools to dissociate overlapping functions in the viral genome together with proteomic approaches and biochemical studies. In Aim 1, we will investigate how the viral genome is freed into the cytoplasm by analyzing uncoating intermediates in infected cells and studying the fate of viral components during entry. In addition, a novel 3 dimensional single particle orbital tracking methods to trace the capsid protein during infection will be explored. In Aim 2, we will use genetic tools to dissec capsid protein requirements for particle formation and infectivity. It has been recently found that the dengue virus capsid protein suffers a variety of post-translational modification in infected cells. A comprehensive mass spectrometry analysis of capsid purified from infected cells and virions, together with the design of recombinant viruses will be used to define function of structural properties of capsid. In Aim 3, we will define the protein-protein interaction network fr dengue virus assembly and determine the function of the viral protein NS3 in this process. We will use a recently developed proteomic platform in the context of viral infections to define host components required for the assembly process. Dissecting the multiple functions and interactions of the capsid protein with host and viral components will shed light on fundamental aspects of dengue and other flavivirus replication. Importantly, the studies proposed will provide new information about viral processes still unexplored for antiviral intervention.