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Assembly of Influenza A viruses

甲型流感病毒的组装

基本信息

批准号：
7825455
负责人：
TORU TAKIMOTO
金额：
$ 22.98万
依托单位：
UNIVERSITY OF ROCHESTER
依托单位国家：
美国
项目类别：
财政年份：
2009
资助国家：
美国
起止时间：
2009-05-05 至 2011-04-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7825455
关键词：
Affect Amino Acids Antiviral Agents Binding Cell Nucleus Cell membrane Cells Code Complex Cytoplasm Data Development Epidemic Filament Fluorescence Genes Goals Grant Growth Human Imagery Immunofluorescence Immunologic In Vitro Infection Influenza Influenza A Virus, H5N1 Subtype Influenza A virus Label Lead Life Methods Molecular Morphology Mutate N-terminal Nucleocapsid Pathogenicity Phenotype Physiologic pulse Polymerase Process Production Proteins Reagent Recombinants Research Research Proposals Role Shapes Site Surface System Time Vertebral column Viral Viral Pathogenesis Viral Proteins Virion Virus Virus Assembly base design influenzavirus insight mutant pandemic disease pandemic influenza particle pathogen positional cloning prevent public health relevance recombinant virus research study respiratory trafficking transmission process

项目摘要

DESCRIPTION (provided by applicant): Influenza is a globally important respiratory pathogen that causes nearly annual epidemics and occasional pandemics. Recent spread of highly pathogenic avian influenza H5N1 viruses is a new threat that may lead to a deadly influenza pandemic. Influenza antiviral drugs are limited, although the need is urgent. Efficiency of progeny virus assembly and production is one of the critical factors of viral transmission and pathogenicity. However, the molecular mechanism of budding and virion formation is yet unclear. The goal of this research is to elucidate the mechanisms of virus assembly and progeny virion formation. Influenza viruses are assembled at the plasma membrane of infected cells by budding. Among the viral components, M1 proteins have been shown to be the key component in both bud formation, as well as the pinch-off that determines the size and shape of the virus particles. Our preliminary studies showed that the presence of specific NP prevented filament formation in infected cells, suggesting that an interaction between M1 and NP affects the budding and pinch- off process of influenza A viruses. In this research proposal, we hypothesize that appropriate M1-nucleocapsid (vRNP) interaction is required for efficient virus production and spherical virion formation. In Aim 1, we will identify M1-interacting domain(s) in NP and analyze the role of M1- NP interactions in viral assembly, production and morphology. Various recombinant influenza A viruses carrying chimeric or mutant NP genes will be generated by the reverse genetics system. In Aim 2, we will analyze the localization of vRNP in infected cells to determine if insufficient accumulation of vRNP at the plasma membrane due to the weak M1-vRNP interaction correlates with the morphological difference in progeny virions. We will utilize various approaches to chase vRNP distribution, including pulse-chase experiments and visualization of vRNPs in live cells using either FlAsH labeling or viral polymerase proteins fused to enhanced green fluorescence protein. Characterization of the role of M1-vRNP interaction in the virus assembly and budding processes is expected to unveil the mechanism of virion formation, which is a critical factor in virus transmission and pathogenicity. An understanding of the mechanism involved in efficient viral growth and spread should assist in the development of anti-viral strategies. PUBLIC HEALTH RELEVANCE: The efficiency of progeny virus assembly and production is one of the critical factors of transmission and pathogenicity of influenza A virus. However, the molecular mechanism of budding and virion formation is yet unclear. Studies proposed in this grant will unveil the process of virion formation and the role of M1-nucleocapsid interaction in virus assembly, thus yielding important implications for the design of antiviral reagents.

描述（由申请人提供）：流感是一种全球重要的呼吸道病原体，几乎每年都会引起流行病，偶尔也会引起大流行。最近高致病性禽流感H5N1病毒的传播是一种新的威胁，可能导致致命的流感大流行。抗流感病毒药物有限，尽管需求迫切。子代病毒的组装和生产效率是病毒传播和致病性的关键因素之一。然而，出芽和病毒体形成的分子机制尚不清楚。本研究的目标是阐明病毒组装和后代病毒粒子形成的机制。流感病毒通过出芽在受感染细胞的质膜上组装。在病毒组分中，M1蛋白已被证明是芽形成以及确定病毒颗粒大小和形状的夹断的关键组分。我们的初步研究表明，特异性NP的存在阻止了感染细胞中细丝的形成，表明M1和NP之间的相互作用影响了甲型流感病毒的出芽和夹断过程。在本研究提案中，我们假设有效的病毒生产和球形病毒粒子形成需要适当的M1-核衣壳（vRNP）相互作用。在目标1中，我们将确定NP中的M1相互作用结构域，并分析M1-NP相互作用在病毒组装、生产和形态学中的作用。将通过反向遗传学系统产生携带嵌合或突变NP基因的各种重组甲型流感病毒。在目的2中，我们将分析vRNP在感染细胞中的定位，以确定由于弱M1-vRNP相互作用导致的vRNP在质膜上的积累不足是否与子代病毒体的形态学差异相关。我们将利用各种方法来追逐vRNP分布，包括脉冲追逐实验和可视化vRNP在活细胞中使用FlAsH标记或病毒聚合酶蛋白融合增强的绿色荧光蛋白。M1-vRNP相互作用在病毒组装和出芽过程中的作用的表征有望揭示病毒体形成的机制，这是病毒传播和致病性的关键因素。了解病毒有效生长和传播的机制有助于抗病毒策略的制定。公共卫生相关性：子代病毒的组装和生产效率是影响甲型流感病毒传播和致病性的关键因素之一。然而，出芽和病毒体形成的分子机制尚不清楚。这项研究将揭示病毒粒子形成的过程以及M1-核衣壳相互作用在病毒组装中的作用，从而为抗病毒试剂的设计带来重要影响。