Assembly of Influenza A viruses

描述(申请人提供)：流感是一种全球重要的呼吸道病原体，几乎每年都会导致流行病和偶尔的大流行。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。流感抗病毒药物有限，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的关键因素之一。然而，发芽和病毒粒子形成的分子机制尚不清楚。本研究的目的是阐明病毒组装和子代病毒粒子形成的机制。流感病毒通过萌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带的关键成分。我们的初步研究表明，特定NP的存在阻止了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了甲型流感病毒的萌发和夹持过程。在这项研究方案中，我们假设适当的M1-核衣壳(VRNP)相互作用是有效的病毒生产和球形病毒粒子形成所必需的。在目标1中，我们将确定NP中的M1相互作用结构域(S)，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在感染细胞中的定位，以确定由于M1-vRNP弱相互作用而导致的vRNP在质膜上积累不足是否与后代病毒粒子的形态差异有关。我们将利用各种方法来追踪vRNP的分布，包括脉冲追逐实验和使用闪光标记或融合到增强的绿色荧光蛋白的病毒聚合蛋白来显示活细胞中的vRNP。M1-vRNP相互作用在病毒组装和萌发过程中的作用的表征有望揭示病毒粒子的形成机制，而病毒粒子的形成是病毒传播和致病的关键因素。对病毒有效生长和传播所涉及的机制的了解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，发芽和病毒粒子形成的分子机制尚不清楚。这项拨款建议的研究将揭示病毒粒子的形成过程和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。

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.