Influenza Virus Assembly

描述（由申请人提供）：本研究的长期目标是在分子水平上了解流感病毒的组装。为此，我们建议阐明流感病毒RNA片段并入病毒体的机制，并评估选择性病毒RNA并入对产生新的流感病毒的贡献。在申请人的实验室中开发的基于质粒的反向遗传学系统允许完全从克隆的cDNA产生流感病毒。该技术用于鉴定介导vRNA病毒体掺入的片段特异性区域，为vRNA包装的选择性而非随机机制提供了第一个直接证据。目标1试图在这些区域内细化包装信号，而目标2确定这些信号之间的相互作用是否介导八个不同vRNA片段的集合。目的3试图阐明这些vRNA片段被整合到出芽病毒体中的机制。vRNA的病毒体掺入似乎涉及将病毒基因组片段隔离到出芽颗粒中的病毒结构蛋白。这种相互作用的候选介质（来自病毒体壳）是三种病毒跨膜蛋白HA、NA和离子通道蛋白（M2），它们可以通过其胞质尾执行这种功能;来自与每个vRNA片段相关的病毒核糖核蛋白复合物的候选介质包括三种聚合酶蛋白（PB 2、PBI、PA）。目的4将检验选择性vRNP掺入在新流感病毒株的重配和产生中起重要作用的假设。事实上，1957年和1968年的大流行性流感毒株以及1998年在北美猪群中出现的三重抵抗株的特征在于，除了HA和NA区段或HA区段之外，还将PB 1区段引入新宿主。因此，PB 1 vRNA和其他vRNA片段（例如，HA）可以控制它们优先并入病毒体。拟议的实验将为流感病毒的生命周期提供新的基本见解，特别是将多个vRNA片段有组织地招募到萌芽病毒体中的机制。如果选择性vRNA掺入显著有助于产生新的重组病毒，则将为开发抗病毒干预措施开辟新的途径，包括干扰有效vRNA病毒体掺入的化合物，或者由于掺入序列改变而不与田间菌株重新分类的活疫苗。

DESCRIPTION (provided by applicant): The long-term goal of this research is to understand the assembly of influenza viruses at the molecular level. To this end, we propose to elucidate mechanism(s) by which influenza virus RNA segments are incorporated into virions, and to assess the contribution of selective viral (v) RNA incorporation to the generation of new reassortant influenza viruses. A plasmid-based reverse genetics system, developed in the applicant's laboratory, allows influenza virus to be produced entirely from cloned cDNA. This technology was used to identify segment-specific regions that mediate vRNA virion incorporation, providing the first direct evidence for a selective rather than random mechanism of vRNA packaging. Aim 1 seeks to refine the packaging signals within these regions, while Aim 2 determines if interactions among these signals mediate the assembly of sets of eight different vRNA segments. Aim 3 attempts to elucidate the mechanism(s) by which these sets of vRNA segments are incorporated into budding virions. The virion incorporation of vRNAs appears to involve viral structural proteins that sequester the viral genome segments into budding particles. Candidate mediators of this interaction (from the virion shell) are the three viral transmembrane proteins HA, NA, and ion channel protein (M2), which may execute this function through their cytoplasmic tails; candidates, from the viral ribonucleoprotein complex associated with each vRNA segment include the three polymerase proteins (PB2, PBI, PA). Aim 4 will test the hypothesis that selective vRNP incorporation plays a significant role in the reassortment and generation of new influenza virus strains. Indeed, the 1957 and 1968 pandemic influenza strains, as well as a triple reassortant that appeared in the North American pig population in 1998, were characterized by the introduction of a PB1 segment, in addition to HA and NA segments or an HA segment, into a new host. Thus, interactions between the PB1 vRNA and other vRNA segments (e.g., HA) may govern their preferential incorporation into virions. The proposed experiments will provide new fundamental insights into the life cycle of influenza viruses, in particular the mechanisms for organized recruitment of multiple vRNA segments into budding virions. If selective vRNA incorporation contributes significantly to the generation of novel reassortant viruses, new avenues will be opened for the development of antiviral interventions, including compounds that interfere with efficient vRNA virion incorporation or perhaps live vaccines that do not reassort with field strains due to altered incorporation sequences.