Dengue Virus Genome Encapsidation and Its Interplay with Host Lipid Droplets

登革热病毒基因组衣壳化及其与宿主脂滴的相互作用

• 批准号: 8690755
• 负责人: Andrea Gamarnik
• 金额: $ 10.24万
• 依托单位: FUNDACION INSTITUTO LELOIR
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-07-15 至 2016-04-09
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8690755
• 关键词: Address; Amino Acids; Antiviral Agents; Arthropods; Binding; Biochemical; Biochemistry; Biological; Biology; Capsid; Capsid Proteins; Categories; Cell Extracts; Cell Nucleolus; Cells; Cellular biology; Chemicals; Code; Data; Dengue; Dengue Virus; Disease Outbreaks; Ensure; Epidemic; Family; Flaviviridae; Flavivirus; Fluorescent Probes; Fractionation; Genetic; Genetic Translation; Genome; Human; Image Analysis; Infection; Infectious Agent; Integration Host Factors; Intervention; Label; Life Cycle Stages; Light; Link; Lipids; Location; Medical; Membrane; Membrane Protein Traffic; Metabolism; Methods; Molecular Biology; Molecular Chaperones; Morphogenesis; Nuclear; Nucleic Acid Binding; Nucleic Acids; Nucleocapsid; Organelles; Pathogenesis; Peptides; Pharmaceutical Preparations; Play; Polymerase; Process; Property; Protein Binding; Proteins; Proteomics; Public Health; RNA; RNA amplification; RNA replication; Recruitment Activity; Reporting; Role; Signal Transduction; Site; Structure; Structure of thyroid parafollicular cell; Surface; System; Technology; Time; Translations; Vaccines; Viral; Viral Genome; Viral Physiology; Virion; Virus; Virus Diseases; Virus Replication; cellular imaging; citrate carrier; member; novel; particle; pathogen; protein transport; research study; tool; viral RNA; virology; virus genetics

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.

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。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。囊化过程是颗粒形态发生所必需的，涉及衣壳蛋白与病毒基因组的相互作用。黄病毒衣壳是一种高度碱性的蛋白质，可以与任何核酸结合，但只有病毒基因组被封装。在感染期间，病毒衣壳蛋白分布在感染细胞的不同隔间。它被合成并结合到内质网膜上，然后聚集在核仁和脂滴表面。然而，目前还不清楚衣壳蛋白如何以及在哪里招募病毒RNA来产生核衣壳颗粒。在这项建议中，我们将结合分子生物学、生物化学和遗传学方法与经典病毒学研究，以确定登革热病毒包膜的机制及其与宿主细胞的相互作用。在目标1中，我们将使用新的基因工具来剖析衣壳蛋白对颗粒形成和感染性的要求。此外，我们还将定义与核酸结合和亚细胞定位有关的衣壳蛋白的氨基酸和结构域。在目标2中，我们将使用细胞成像分析和感染细胞的生化分级来确定核衣壳颗粒形成的位置以及蛋白质在内质网、核仁和脂滴之间的运输。病原体复制和脂滴代谢之间的联系是病毒和其他感染性病原体面临的一个新课题。我们最近报道，登革热病毒感染诱导形成脂滴，这是病毒颗粒形成所必需的。在目标3中，我们建议通过对感染细胞和未感染细胞的蛋白质组学研究，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参与病毒复制的宿主因素和可能的新干预目标的有价值的信息。在目标4中，我们将通过对病毒粒子内的病毒RNA进行高通量探测分析来研究衣壳蛋白如何调节基因组结构。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一个基本方面。重要的是，拟议的研究将提供有关病毒过程的新信息，这一过程尚未被探索用于抗病毒干预。