Dengue Virus Genome Encapsidation and Its Interplay with Host Lipid Droplets

描述（由申请人提供）：登革病毒是黄病毒科的一个成员，是A类病原体，可引起人类最常见的节肢动物传播的病毒性疾病。病毒基因组是在病毒复制期间起多种作用的RNA分子。它作为翻译的mRNA，RNA扩增的模板和底物。衣壳化过程是颗粒形态发生所必需的，并且涉及衣壳蛋白与病毒基因组的相互作用。黄病毒衣壳是结合任何核酸的高度碱性蛋白质，但只有病毒基因组被衣壳化。在感染过程中，病毒衣壳蛋白分布在被感染细胞的不同区室中。它与ER膜结合合成，然后积聚在核仁和脂滴表面。然而，仍然不清楚衣壳蛋白如何以及在何处募集病毒RNA以产生核衣壳颗粒。在这个计划中，我们将结合联合收割机分子生物学，生物化学和遗传学的方法与经典的病毒学研究，以确定登革病毒的机制及其与宿主细胞的相互作用。在目标1中，我们将使用新的遗传工具来剖析颗粒形成和感染性所需的衣壳蛋白。此外，我们将定义氨基酸和结构域的衣壳蛋白参与核酸结合和亚细胞定位。在目标2中，我们将使用细胞成像分析与受感染细胞的生化分离一起来确定核衣壳颗粒形成的位置以及ER、核仁和脂滴之间蛋白质的运输。病原体复制和脂滴代谢之间的联系是病毒和其他感染因子的一个新兴课题。我们最近报道登革病毒感染诱导脂滴的形成，这是病毒颗粒形成所必需的。在目标3中，我们建议通过感染和未感染细胞的蛋白质组学研究来研究病毒感染如何改变脂滴的蛋白质组成。这些研究将提供有关病毒复制的宿主因素和可能的干预新靶点的有价值的信息。在目标4中，我们将研究衣壳蛋白如何调节基因组结构，使用病毒粒子内的病毒RNA的高通量探测分析。剖析衣壳蛋白与宿主和病毒组分的多种功能和相互作用将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，这些信息尚未用于抗病毒干预。

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 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. The encapsidation process is essential for particle morphogenesis, and involves the interaction of the capsid protein with the viral genome. The flavivirus capsid is a highly basic protein that binds any nucleic acid, yet only the viral genome is encapsidated. During infection, the viral capsid protein distributes in different compartments of the infected cell. It is synthesized associated to the ER membrane and then accumulates in nucleolus and on the surface of lipid droplets. However, it is still unclear how and where the capsid protein recruits the viral RNA to produce the nucleocapsid particle. In this proposal, we will combine molecular biology, biochemistry, and genetic approaches with classical virology studies to define the mechanism of dengue virus encapsidation and its interplay with the host cell. In Aim 1, we will use novel genetic tools to dissect capsid protein requirements for particle formation and infectivity. In addition, we will define amino acids and domains of the capsid protein involved in nucleic acid binding and subcellular localization. In Aim 2, we will use cell imaging analysis together with biochemical fractionation of infected cells to define the location of nucleocapsid particle formation and the traffic of the protein between the ER, nucleolus, and lipid droplets. The link between pathogen replication and lipid droplet metabolism is an emerging topic for viruses and other infectious agents. We have recently reported that dengue virus infection induces the formation of lipid droplets, which is necessary for viral particle formation. In Aim 3, we propose to investigate how viral infection alters the protein composition of lipid droplets by proteomic studies of infected and uninfected cells. These studies will provide valuable information about host factors involved in viral replication and possible new targets for intervention. In Aim 4, we will investigate how the capsid protein modulates the genome structure using high throughput probing analysis of the viral RNA inside the virion. Dissecting the multiple functions and interactions of the capsid protein with host and viral components will shed light on a fundamental aspect of dengue and other flavivirus replication. Importantly, the studies proposed will provide new information about a viral process that has been unexplored for antiviral intervention.