Defining the cis and trans acting factors required for assembly of infectious Blu

定义组装传染性 Blu 所需的顺式和反式作用因子

• 批准号: 9097511
• 负责人: POLLY ROY
• 金额: $ 27.24万
• 依托单位: LONDON SCH/HYGIENE & TROPICAL MEDICINE
• 依托单位国家: 美国
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-02-01 至 2018-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9097511
• 关键词: Address; Animals; Antiviral Agents; Arboviruses; Area; Bacteria; Biochemical; Bioinformatics; Biological Assay; Biological Models; Biology; Bluetongue; Bluetongue virus; Capsid; Cell Culture Techniques; Cell Line; Cells; Code; Competence; Complex; Computer Analysis; Core Assembly; Cryoelectron Microscopy; Culicoides (genus); Data; Development; Disease; Double Stranded RNA Virus; Double-Stranded RNA; Event; Family; Family Relationship; Family member; Funding; Genes; Genetic Transcription; Genome; Goals; Health; Human; Image; In Vitro; Infection; Insecta; Knowledge; Lead; Life Cycle Stages; Light; Methods; Modality; Molecular; Molecular Machines; Mutation; Nucleosome Core Particle; Orbivirus; Peptide Signal Sequences; Plants; Positioning Attribute; Precursor RNA; Process; Proteins; RNA; RNA Viruses; Recombinants; Reoviridae; Reovirus; Resolution; Role; Rotavirus; Signal Transduction; Staging; Structure; Surface; System; Technology; Tertiary Protein Structure; Trans-Activators; Transcript; United States National Institutes of Health; Vaccines; Viral; Viral Genome; Viral Proteins; Virion; Virus; Virus Diseases; Virus Replication; base; design; genomic RNA; image processing; inhibitor/antagonist; insight; man; member; mutant; new technology; novel; particle; pathogen; plant fungi; protein complex; research study; reverse genetics; tomography; tool; virus pathogenesis

DESCRIPTION (provided by applicant): The specific aims of this proposal are part of a long term goal to understand completely the infection course of a non-enveloped arthropod-borne virus, Bluetongue virus (BTV), a member of the Reoviridae family that include viruses infecting a wide range of hosts, including human, animals, plants, fungi, and bacteria. As such BTV shares a family relationship with several other scientifically and medically important viruses (e.g. Rotaviruses). BTV has been studied extensively as an agent of economically important disease and also, increasingly, as a model system for related viruses. Substantial progress has been achieved in the molecular, biochemical and structural fields; however, certain areas of BTV and related viruses' biology and structure remain unclear due to the unavailability of appropriate tools and assay systems. BTV and other members (rotavirus and reoviruses) are complex, multi-layered capsid viruses with 10-12 segmented double-stranded RNA genomes but the details of how virus genomes are packaged into their protective capsids or the mechanism by which a copy of each segment is incorporated for the virus to be viable remain elusive. In this proposal we aim to maintain and extend the leading position of BTV towards our long term goal of understanding, in totality, the infection course of the virus. In particular, this application wll utilize a groundbreaking technological advance (i.e., the first in vitro packaging system for a complex dsRNA virus) recently pioneered by us (through our current NIH funding), to understand the genome packaging and replication events of this complex capsid virus and by inference for all multi-segmented RNA viruses. In specific aim 1 we will determine the packaging order of ten ssRNAs and the signals involved in assembly of BTV particles. In specific aim 2 we will focus on identification of the protein and protein complexes essential for ssRNA packaging while in the specific aim 3, the replication competence of mutant constructs will be assessed in cell culture using our established reverse genetics system. Current data suggest that ssRNAs drive assembly of the BTV transcription complex (TC), leading to capsid (core) assembly and that packaged genome precursor RNA molecules are positioned at the 12 vertices of the assembling icosahedral capsid, precisely priming them for subsequent transcription. In specific aim 4 we will seek direct structural evidence of the TC organization at the vertices of the packaged particles, using both native and mutant particles to determine the consequences of mutation, using the state-of-the art single particle cryo-electron microscopy and cryo-tomography. Completion of these goals will pave the way for the rational design of inhibitors to block virus replication and provide lead compounds for related viruses of pathogenic significance to man and animals.

描述（由申请人提供）：该提案的具体目的是长期目标的一部分，即完全了解非发育的节肢动物传播病毒，Bluetongue病毒（BTV）的感染过程，Reoviridae家族的成员，包括人类，动物，植物，植物，Fungi和Fungi和Bacteria，包括宿主，包括广泛的宿主。因此，BTV与其他几种科学和医学上重要的病毒（例如轮状病毒）有家庭关系。 BTV已被广泛研究为经济上重要疾病的药物，并且越来越多地作为相关病毒的模型系统。在分子，生化和结构场中已经取得了实质性进步。但是，由于适当的工具和测定系统不可用，BTV及相关病毒的生物学和结构的某些领域尚不清楚。 BTV和其他成员（轮状病毒和旋孢子病毒）是复杂的多层衣壳病毒，具有10-12个分段的双链RNA基因组，但是将病毒基因组包装到其保护性胶囊中的细节或每个节段的副本以使病毒的副本包含在病毒中，以使其可行。在此提案中，我们旨在将BTV的领先地位维持和扩展到我们的长期理解，总体上是病毒的感染过程。特别是，该应用程序使用了开创性的技术进步（即，我们最近（通过我们当前的NIH资助）率先率先了解了这种复杂的Capsid病毒的基因组包装和复制事件，并通过针对所有多细分RNA的多段RNA病毒素来理解我们最近率先使用的复杂DSRNA病毒的第一个体外包装系统）。在特定目标1中，我们将确定十个SSRNA的包装顺序以及BTV颗粒组装所涉及的信号。在特定的目标2中，我们将重点介绍ssRNA包装必不可少的蛋白质和蛋白质复合物，而在特定目标3中，将使用我们既定的反向遗传学系统在细胞培养中评估突变构建体的复制能力。当前的数据表明，BTV转录复合物（TC）的SSRNA驱动组装，导致CAPSID（CORE）组装，并且包装的基因组前体RNA分子位于组装IcosaheDral Capsid的12个顶点，精确地启动它们以进行后续转录。在特定目标4中，我们将使用天然和突变粒子在包装颗粒的顶点上寻求TC组织的直接结构证据，并使用天然和突变粒子来确定突变的后果，并使用最先进的单个粒子冷冻电子微观显微镜和冷冻术。这些目标的完成将为抑制剂的合理设计铺平道路，以阻止病毒复制，并为对人和动物具有致病意义的相关病毒提供铅化合物。