Rotavirus Genome Replication and Virion Assembly

 描述(由申请人提供)：任何病毒的生命周期的中心是基因组复制和病毒粒子组装的过程。这两个事件必须在受感染的细胞内巧妙地协调，以最大限度地促进病毒繁殖，避免细胞固有防御。轮状病毒是一种无包膜、11个节段的双链RNA病毒，会导致幼儿严重腹泻。这些病毒也是研究病毒基因组复制和颗粒组装如何协调的有吸引力的实验模型，因为它们在生命周期中串联执行这些任务。具体地说，轮状病毒的组装被认为始于形成由病毒RNA聚合酶(VP1)和RNA封端酶(VP3)结合到正链RNA复制模板上的前核心复制中间产物(RIS)。在添加了外壳蛋白(VP2)、衣壳蛋白(VP6)和非结构蛋白(NSP2和NSP5)之后，11个前核心RI成为一个单一的、具有复制酶活性的核心RI，在这个核心RI中，正链RNA通过VP1介导的负链RNA合成转化为基因组片段。这种协同的复制酶组装机制要求VP1的酶活性受到其与核心RI蛋白相互作用的严格控制。然而，关于前核心RIS和核心RIS的结构组织的知识存在严重差距，因为这些复合体尚未被看到。此外，VP1及其辅助因子之间的多方面相互作用仍未明确。这项应用的总体目标是通过对RI及其蛋白质成分的详细结构、功能和遗传分析，揭示轮状病毒组装和基因组复制的早期阶段。在目标1中，将从轮状病毒感染的细胞中分离出天然的前核心RIS和核心RIS，并将使用免疫亲和捕获冷冻电子显微镜和单粒子图像重建来推断它们以前从未见过的大分子结构。在AIM 2中，将通过体外负链RNA合成、病毒样颗粒形成和免疫共沉淀试验来确定三个核心RI蛋白(VP1、VP2和NSP2)之间的相互作用界面。最后，在AIM 3中，将在轮状病毒感染细胞的背景下，使用反式互补和单基因反向遗传方法研究NSP2对核心RI形成和功能的影响，从而阐明这种非结构蛋白在复制酶组装过程中的作用。这一建议是创新的，因为它调查了轮状病毒基因组复制和病毒粒子组装的原始想法，目前关于这些想法的信息很少。这项工作意义重大，因为它将促进有针对性的下一代轮状病毒疫苗的开发，并可能增强我们将病毒载体设计为治疗人类疾病的治疗载体的能力。

 DESCRIPTION (provided by applicant): Central to the lifecycle of any virus are the processes of genome replication and virion particle assembly. These two events must be exquisitely coordinated within the infected cell to maximize viral multiplication and avert cell-intrinsic defenses. Rotaviruses are non-enveloped, eleven-segmented, double-stranded RNA viruses that cause severe diarrheal disease in young children. These viruses are also attractive experimental models to study how viral genome replication and particle assembly are coordinated, because they perform these tasks in tandem during their lifecycles. Specifically, rotavirus assembly is thought to begin with the formation of pre-core replication intermediates (RIs) comprised of the viral RNA polymerase (VP1) and RNA capping enzyme (VP3) bound to plus-strand RNA replication templates. Following the addition of the shell protein (VP2), the capsid protein (VP6), and nonstructural proteins (NSP2 and NSP5), eleven pre-core RIs become a single, replicase-competent core RI within which plus-strand RNAs are converted into genome segments by VP1-mediated minus-strand RNA synthesis. This concerted replicase-assembly mechanism requires that the enzymatic activity of VP1 be tightly controlled by its interactions with core RI proteins. Yet, critical gaps in knowledge exist about the structural organization of pre-core RIs and core RIs, as these complexes have not yet been seen. Moreover, the multifaceted interactions among VP1 and its co-factors remain poorly defined. The overall objective of this application is to shed new light on the early stages of rotavirus assembly and genome replication through the detailed structural, functional, and genetic analysis of RIs and their protein constituents. In AIM 1, native pre-core RIs and core RIs will be isolated from rotavirus-infected cells and their never-before-seen macromolecular architectures will be deduced using immunoaffinity-capture cryo-electron microscopy and single-particle image reconstruction. In AIM 2, interaction interfaces among three core RI proteins (VP1, VP2, and NSP2) will be defined using in vitro minus-strand RNA synthesis, virus-like particle formation, and co-immunoprecipitation assays. Finally, in AIM 3, the effects of interactions involving NSP2 on core RI formation and function will be studied in the context of rotavirus-infected cells using trans-complementation and single-gene reverse genetic approaches, thereby illuminating the role of this nonstructural protein during the replicase-assembly process. This proposal is innovative because it investigates original ideas about rotavirus genome replication and virion assembly for which little information currently exists. The work is significant because it will foster the development of targeted, next-generation rotavirus vaccines and may enhance our ability to engineer viral vectors as therapeutic delivery vehicles to treat human diseases.