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: 8145433
• 负责人: Andrea Gamarnik
• 金额: $ 10.26万
• 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 2015-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8145433
• 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. PUBLIC HEALTH RELEVANCE: Dengue outbreaks and epidemics are a tremendous public health problem around the world. In spite of the urgent medical need to control dengue infections, vaccines are still unavailable, and many aspects of dengue virus biology and pathogenesis remain elusive. In this proposal, we will investigate the mechanism of dengue virus encapsidation, one of the most obscure steps of the virus life cycle. We will combine genetic, biochemical, and cell biology approaches to dissect the functions of the viral capsid protein in infected cells and define the mechanism by which this protein recruits the viral genome during encapsidation.

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。 公共卫生相关性：登革热的爆发和流行是世界各地的一个巨大的公共卫生问题。尽管迫切需要控制登革热感染，但疫苗仍然不可用，并且登革热病毒生物学和发病机制的许多方面仍然难以捉摸。在本提案中，我们将研究登革热病毒壳体化的机制，这是病毒生命周期中最不起眼的步骤之一。我们将结合联合收割机遗传学、生物化学和细胞生物学的方法来剖析病毒衣壳蛋白在感染细胞中的功能，并确定这种蛋白在衣壳化过程中招募病毒基因组的机制。