Membrane Rearrangements in Flavivirus Infected Cells

黄病毒感染细胞的膜重排

• 批准号: 7876900
• 负责人: Richard J. Kuhn
• 金额: $ 19.06万
• 依托单位: PURDUE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-06-19 至 2012-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7876900
• 关键词: Antiviral Agents; Arboviruses; Architecture; Arthropods; Binding; Categories; Cells; Cellular Membrane; Cellular Structures; Cellular biology; Confocal Microscopy; Culicidae; DNA Sequence Rearrangement; Data; Data Analyses; Dengue Virus; Disease; Energy Transfer; Environment; Flavivirus; Flavivirus Infections; Fluorescence; Future; Gene Expression; Human; Imaging technology; Infection; Insecta; Intracellular Membranes; Knowledge; Life Cycle Stages; Lipid Bilayers; Lipids; Mass Spectrum Analysis; Measures; Mediating; Medical; Membrane; Methods; Modification; Molecular; Molecular Genetics; National Institute of Allergy and Infectious Disease; Pathway interactions; Plants; RNA Viruses; RNA replication; Reagent; Recruitment Activity; Regulation; Resolution; Role; Signal Pathway; Signal Transduction; Staging; Structure; System; Techniques; Therapeutic Intervention; Translating; Viral; Viral Genes; Viral Genome; Viral Proteins; Virion; Virus; Virus Assembly; Virus Diseases; West Nile virus; Yellow fever virus; base; combinatorial; design; fluorescence imaging; insight; interdisciplinary approach; lipid mediator; lipid metabolism; liquid chromatography mass spectrometry; member; membrane biogenesis; novel; novel therapeutics; particle; pathogen; public health relevance; research study; trafficking; transmission process; viral RNA

DESCRIPTION (provided by applicant): A hallmark of infection with positive strand RNA viruses is the impressive intracellular membrane rearrangements induced within host cells to facilitate replication of their viral genomes. This project will use a multidisciplinary approach to investigate the molecular mechanisms that drive intracellular membrane rearrangements during flavivirus infection. The focus of this project will be on dengue virus, an NIAID category A priority pathogen, because of its medical importance and the substantial data and reagents we and others have accumulated over the last several years. Dengue virus causes about 50-100 million infections per year making it the most important arbovirus worldwide. It belongs to the flavivirus genus, which comprises more than 70 members, many of which are important human pathogens. Infection of cells with dengue virus results in enhanced synthesis of new lipid-derived structures and extensive membrane rearrangements. It has been shown that these membranous structures are required for a productive dengue virus infection, however, the molecular requirements that underlie the formation of these structures remains largely unknown. This project will determine the membrane composition of these structures and the cellular signaling cascades and trafficking pathways recruited by dengue virus for their formation and function. A high-resolution mass spectrometry based approach has been developed to initially identify the lipid-mediators important for the formation of these intracellular membranous structures. Preliminary data from these analyses have indicated a clear difference between the overall lipid composition of dengue virus infected cells compared to uninfected cells. These observations will be extended to determine how these changes in lipid profiles translate to the observed ultrastructural modifications in dengue virus infected cells. High-resolution mass spectrometry analyses will be employed to identify the differentially expressed lipids. Specifically isolated membrane fractions will be analyzed from infected cells and used to determine the temporal changes in lipid profile and their accumulation in these structures during the course of infection. Using lifetime imaging technology and confocal microscopy, the temporal redistribution of key lipids into these membranous structures will be visualized and these will be correlated with the various stages of the viral life cycle including viral gene expression, viral RNA replication and virus assembly. Using molecular genetics we will define the viral gene products responsible for these rearrangements. Ultimately, the experiments proposed should provide a framework for understanding the virus-induced alterations in lipid metabolism and ultrastructure in dengue virus infected cells and provide the basis for future experiments detailing the lipid-mediated molecular interactions between virus and host. PUBLIC HEALTH RELEVANCE: The flaviviruses represent a large and significant set of viral pathogens that infect hundreds of millions of people each year. By discovering how dengue virus changes the lipid components of cells, it may be possible to better understand how the virus causes disease, and may give insight into how to design novel therapeutics that modify these changes to inhibit the virus.

描述（由申请人提供）：阳性链RNA病毒感染的一个标志是宿主细胞内诱导的令人印象深刻的细胞膜重排，以促进其病毒基因组的复制。本项目将采用多学科方法研究黄病毒感染过程中驱动细胞膜重排的分子机制。由于登革热病毒在医学上的重要性以及我们和其他人在过去几年中积累的大量数据和试剂，这个项目的重点将是登革热病毒，这是NIAID的A类优先病原体。登革热病毒每年造成约5000万至1亿例感染，使其成为世界上最重要的虫媒病毒。它属于黄病毒属，该属有70多个成员，其中许多是重要的人类病原体。感染登革病毒的细胞会导致新的脂质衍生结构的合成增强和广泛的膜重排。研究表明，这些膜状结构是产生性登革热病毒感染所必需的，然而，形成这些结构的分子要求在很大程度上仍然未知。该项目将确定这些结构的膜组成，以及登革热病毒招募的细胞信号级联和运输途径的形成和功能。一种基于高分辨率质谱的方法已经被开发出来，以初步确定对这些细胞内膜结构形成重要的脂质介质。这些分析的初步数据表明，登革热病毒感染细胞与未感染细胞的总体脂质组成存在明显差异。这些观察结果将被扩展，以确定这些脂质谱的变化如何转化为登革病毒感染细胞中观察到的超微结构修饰。高分辨率质谱分析将用于鉴定差异表达的脂质。将从感染细胞中分析特异性分离的膜组分，并用于确定感染过程中脂质谱的时间变化及其在这些结构中的积累。使用终身成像技术和共聚焦显微镜，关键脂质在这些膜结构中的时间重新分布将被可视化，这些将与病毒生命周期的各个阶段相关，包括病毒基因表达、病毒RNA复制和病毒组装。使用分子遗传学，我们将定义负责这些重排的病毒基因产物。最终，提出的实验应该为理解病毒诱导的登革热病毒感染细胞中脂质代谢和超微结构的改变提供一个框架，并为未来详细描述病毒与宿主之间脂质介导的分子相互作用的实验提供基础。公共卫生相关性：黄病毒是一大类重要的病毒病原体，每年感染数亿人。通过发现登革热病毒如何改变细胞的脂质成分，有可能更好地了解病毒如何引起疾病，并可能深入了解如何设计修改这些变化以抑制病毒的新疗法。

项目成果

Dengue virus infection perturbs lipid homeostasis in infected mosquito cells.

• DOI: 10.1371/journal.ppat.1002584
• 发表时间: 2012
• 期刊: PLoS pathogens
• 影响因子: 6.7
• 作者: Perera R;Riley C;Isaac G;Hopf-Jannasch AS;Moore RJ;Weitz KW;Pasa-Tolic L;Metz TO;Adamec J;Kuhn RJ
• 通讯作者: Kuhn RJ