Membrane Rearrangements in Flavivirus Infected Cells

描述(申请人提供)：感染正链RNA病毒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。该项目将使用多学科方法来研究在黄病毒感染过程中驱动细胞内膜重排的分子机制。该项目的重点将是登革热病毒，这是一种NIAID A类优先病原体，因为它在医学上具有重要意义，而且我们和其他人在过去几年中积累了大量数据和试剂。登革热病毒每年造成约5000-1亿人感染，使其成为全球最重要的虫媒病毒。它属于黄病毒属，由70多个成员组成，其中许多是重要的人类病原体。细胞感染登革病毒导致新的脂类衍生结构的合成增强和广泛的膜重排。已经证明，这些膜结构是产生登革热病毒感染所必需的，然而，这些结构形成的分子要求在很大程度上仍不清楚。该项目将确定这些结构的膜成分，以及登革热病毒招募的细胞信号级联和运输途径，以形成和发挥其功能。一种基于高分辨率质谱学的方法已经被开发出来，以初步识别对这些细胞内膜结构的形成至关重要的脂质介体。这些分析的初步数据表明，与未感染的细胞相比，感染登革热病毒的细胞的总体脂质成分存在明显差异。这些观察将扩大到确定这些脂类变化如何转化为在登革病毒感染细胞中观察到的超微结构改变。将使用高分辨率质谱分析来鉴定差异表达的脂类。具体地说，从感染细胞中分离出的膜部分将被分析，并用于确定在感染过程中脂类分布的时间变化及其在这些结构中的积累。利用寿命成像技术和共聚焦显微镜，关键脂类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不同阶段相关，包括病毒基因表达、病毒RNA复制和病毒组装。利用分子遗传学，我们将定义负责这些重排的病毒基因产物。最终，所提出的实验将为理解病毒诱导的登革病毒感染细胞脂代谢和超微结构的变化提供一个框架，并为未来详细描述病毒与宿主之间的脂质介导的分子相互作用的实验提供基础。与公共卫生相关：黄病毒代表了一大组重要的病毒病原体，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，可能会更好地理解病毒是如何致病的，并可能为设计新的疗法提供洞察，这些改变可以改变这些变化以抑制病毒。

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.