Assembly of Influenza A viruses

甲型流感病毒的组装

• 批准号: 7596835
• 负责人: TORU TAKIMOTO
• 金额: $ 19.25万
• 依托单位: UNIVERSITY OF ROCHESTER
• 依托单位国家: 美国
• 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/7596835
• 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

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。最近传播的高致病性禽流感H5N1病毒是一个新的威胁，可能导致致命的流感大流行。尽管需求迫切，但流感抗病毒药物有限。子代病毒组装和生产的效率是病毒传播和致病性的关键因素之一。然而，出芽和病毒粒子形成的分子机制尚不清楚。这项研究的目的是阐明病毒组装和子代病毒颗粒形成的机制。流感病毒通过出芽在受感染细胞的质膜上组装。在病毒成分中，M1 蛋白已被证明是芽形成以及决定病毒颗粒大小和形状的夹断的关键成分。我们的初步研究表明，特定 NP 的存在阻止了受感染细胞中细丝的形成，这表明 M1 和 NP 之间的相互作用影响甲型流感病毒的出芽和夹断过程。在本研究提案中，我们假设适当的 M1-核衣壳 (vRNP) 相互作用是高效病毒生产和球形病毒颗粒形成所必需的。在目标 1 中，我们将识别 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、生产和形态中的作用。反向遗传学系统将产生携带嵌合或突变NP基因的各种重组甲型流感病毒。在目标 2 中，我们将分析 vRNP 在受感染细胞中的定位，以确定由于 M1-vRNP 相互作用较弱而导致 vRNP 在质膜上积累不足是否与子代病毒粒子的形态差异相关。我们将利用各种方法来追踪 vRNP 分布，包括脉冲追踪实验以及使用 FlaAsH 标记或与增强型绿色荧光蛋白融合的病毒聚合酶蛋白对活细胞中 vRNP 进行可视化。表征 M1-vRNP 相互作用在病毒组装和出芽过程中的作用有望揭示病毒颗粒形成的机制，病毒颗粒形成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应有助于制定抗病毒策略。公共卫生相关性：子代病毒组装和生产的效率是甲型流感病毒传播和致病性的关键因素之一。然而，出芽和病毒颗粒形成的分子机制尚不清楚。该资助提出的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，从而对抗病毒试剂的设计产生重要影响。