Influenza Virus Assembly

描述（由申请人提供）：本研究的长期目标是了解流感病毒在分子水平上的组装。为此，我们建议阐明流感病毒RNA片段被整合到病毒粒子中的机制，并评估选择性病毒RNA整合对产生新的重组流感病毒的贡献。申请人实验室开发了一种基于质粒的反向遗传系统，可以完全从克隆的cDNA中产生流感病毒。该技术被用于鉴定介导vRNA病毒粒子整合的片段特异性区域，为vRNA包装的选择性而非随机机制提供了第一个直接证据。Aim 1旨在细化这些区域内的封装信号，而Aim 2则确定这些信号之间的相互作用是否介导了8个不同vRNA片段的集合。目的3试图阐明这些vRNA片段被纳入出芽病毒粒子的机制。vrna的病毒粒子结合似乎涉及病毒结构蛋白，该病毒结构蛋白将病毒基因组片段隔离到出芽颗粒中。这种相互作用的候选介质（来自病毒粒子外壳）是三种病毒跨膜蛋白HA、NA和离子通道蛋白（M2），它们可能通过细胞质尾部执行这种功能；与每个vRNA片段相关的病毒核糖核蛋白复合体的候选蛋白包括三种聚合酶蛋白（PB2， PBI， PA）。目的4将检验选择性vRNP结合在新流感病毒株的重组和产生中起重要作用的假设。事实上，1957年和1968年的流感大流行毒株，以及1998年在北美猪群中出现的三重重组毒株，其特点是除了HA和NA片段或HA片段外，还向新宿主引入了PB1片段。因此，PB1 vRNA与其他vRNA片段（如HA）之间的相互作用可能决定了它们优先并入病毒粒子。拟议的实验将为流感病毒的生命周期提供新的基本见解，特别是有组织地将多个vRNA片段招募到出芽病毒粒子的机制。如果选择性vRNA掺入对新型重组病毒的产生有重大贡献，将为开发抗病毒干预措施开辟新的途径，包括干扰vRNA病毒粒子有效掺入的化合物，或者可能由于掺入序列改变而不能与野毒株重组的活疫苗。

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.