Rotavirus Genome Replication and Virion Assembly

轮状病毒基因组复制和病毒粒子组装

• 批准号: 8862603
• 负责人: Sarah Marie McDonald Esstman
• 金额: $ 39.53万
• 依托单位: VIRGINIA POLYTECHNIC INST AND ST UNIV
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-01-15 至 2019-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8862603
• 关键词: Ablation; Antibodies; Antiviral Agents; Architecture; BCAR3 gene; Binding; Binding Sites; Biochemical; Biochemical Genetics; Biological Assay; Capsid Proteins; Cells; Child; Co-Immunoprecipitations; Complex; Computational algorithm; Core Assembly; Cryoelectron Microscopy; DNA-Directed RNA Polymerase; Data; Defect; Development; Diarrhea; Disease; Docking; Double Stranded RNA Virus; Double-Stranded RNA; Engineering; Enzymes; Event; Experimental Models; Fostering; Genes; Genetic; Genome; Human; Ice; Image; In Vitro; Infection; Intervention; Knowledge; Laboratories; Lesion; Life; Light; Maps; Measurable; Mediating; Methodology; Mono-S; Morphogenesis; Nonstructural Protein; Nucleic Acids; Plasmids; Polymerase; Population; Positioning Attribute; Process; Proteins; RNA Caps; RNA chemical synthesis; RNA replication; RNA-Protein Interaction; Relative (related person); Research; Resolution; Role; Rotavirus; Rotavirus Vaccines; Small Interfering RNA; Staging; Structure; System; Testing; Therapeutic; Vaccines; Viral; Viral Genome; Viral Proteins; Viral Vector; Virion; Virus; Virus-like particle; Work; cofactor; density; design; gain of function; genetic analysis; genetic approach; human disease; image reconstruction; improved; innovation; insight; mutant; next generation; novel vaccines; particle; premature; prevent; public health relevance; reconstruction; replicase; reverse genetics; viral RNA

 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.

 描述（由适用提供）：任何病毒生命周期的中心是基因组复制和病毒颗粒组装的过程。这两个事件必须在受感染的细胞中精确协调，以最大程度地提高病毒繁殖并避免细胞内部防御。轮状病毒是非发育的，十一分段的双链RNA病毒，可导致幼儿严重的腹泻病。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期间同时执行这些任务。具体而言，轮状病毒组装被认为是从核心复制中间体（RIS）的形成开始的，该病毒RNA聚合酶（VP1）和RNA限制酶（VP3）绑定到与链链RNA复制模板。在添加壳蛋白（VP2），衣壳蛋白（VP6）和非结构蛋白（NSP2和NSP5）之后，11个前核RI成为单一的，能力的核心RI，在该核心RI中，链链RNA在其中通过VP1介导的Mimused Mimus-Strand-Strand-Strandssttheiss Trandsttheiss rnas将基因组转化为基因组片段。这种一致的复制组装机制要求VP1的酶活性被其与核心RI蛋白的相互作用紧密控制。然而，关于核心前RI和核心RI的结构组织的关键差距存在，因为尚未看到这些复合物。此外，VP1及其副因素之间的多面相互作用的定义仍然很差。该应用的总体目的是通过详细的RIS及其蛋白质所构成的详细结构，功能和遗传分析来对轮状病毒组装和基因组复制的早期进行新的启示。在AIM 1中，将从轮状病毒感染的细胞中分离出天然核心RIS和核心RIS，并使用免疫亲和力的冷冻电子显微镜和单颗粒图像重建来推导其前所未有的大分子结构。在AIM 2中，将使用体外负链RNA合成，病毒样颗粒形成和共免疫沉淀分析来定义三种核心RI蛋白（VP1，VP2和NSP2）之间的相互作用接口。最后，在AIM 3中，将使用反式补充和单基因反向遗传方法研究涉及NSP2对核心RI形成和功能的相互作用对核心RI形成和功能的影响，从而阐明了复制酶 - 组装过程中这种非结构蛋白的作用。该提案具有创新性，因为它研究了有关轮状病毒基因组复制和病毒装置的原始想法，目前没有信息。这项工作很重要，因为它将促进靶向下一代轮状病毒疫苗的发展，并可能增强我们将病毒载体作为治疗性递送工具进行治疗的能力。