ASSEMBLY OF INFECTIOUS BLUETONGUE VIRION FROM CDNA CLONE

来自 CDNA 克隆的传染性蓝舌病病毒粒子的组装

• 批准号: 6511549
• 负责人: POLLY ROY
• 金额: $ 25.11万
• 依托单位: UNIVERSITY OF ALABAMA AT BIRMINGHAM
• 依托单位国家: 美国
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-06-01 至 2005-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6511549
• 关键词: Baculoviridae; DNA directed RNA polymerase; Orbivirus; biotechnology; complementary DNA; genetic manipulation; genetic techniques; method development; molecular cloning; recombinant virus; tissue /cell culture; virus genetics

The generation of infectious virus particles depends on a number of specific events during the infectious processes in the host cells. Members of the Reoviridae family consist of 10-12 discrete segments of double-stranded (ds) RNA genome that are encapsidated by multi-layered protein components. The precise packaging of single copies of each RNA segment within each particle is a key step in the virus replication cycle and subsequent generation of infectious virus particles. Unlike other RNA viruses, very little advance has been made to date in understanding the replication processes of dsRNA viruses, despite extensive knowledge of virus structure and assembly. This is mainly due to the lack of a suitable system which allows genetic manipulation of these viruses. This proposal concerns establishment of such systems for a dsRNA virus, in particular, a model virus system, bluetongue virus (BTV), for which much is known, both at the structural and molecular genetic levels. Thus, the aims of the proposed research are to establish a suitable reverse genetics system. This will be achieved exploring systematically, four different approaches (including both established and novel systems) as it is not possible at this time to envisage which systems would be most efficient for BTV. Two of these will use established T7 RNA polymerase systems in mammalian cells and a third is a unique approach that uses insect cells expressing T7 RNA polymerase. The last system will exploit the native cellular RNA polymerase 1. The studies will take advantage of the extensive reagents and assay systems that are available in my laboratory, and exploit the versatile replication capabilities of BTV (virions for mammalian cultures and cores for insect cell cultures). Once established, the system will not only allow manipulation of BTV genomes and thus, to address important biological questions pertinent to BTV replication but will also give a way to establish similar systems for other members of the family including human rotavirus and reovirus.

感染性病毒颗粒的产生取决于宿主细胞感染过程中的许多特定事件。 呼肠孤病毒科的成员由 10-12 个离散的双链 (ds) RNA 基因组片段组成，这些片段被多层蛋白质成分包裹。 每个颗粒内每个 RNA 片段的单拷贝的精确包装是病毒复制周期和随后感染性病毒颗粒生成的关键步骤。 与其他 RNA 病毒不同，尽管人们对病毒结构和组装有了广泛的了解，但迄今为止在理解 dsRNA 病毒的复制过程方面进展甚微。 这主要是由于缺乏合适的系统来对这些病毒进行基因操作。 该提案涉及建立 dsRNA 病毒的此类系统，特别是模型病毒系统蓝舌病毒（BTV），该系统在结构和分子遗传水平上均已广为人知。因此，本研究的目的是建立合适的反向遗传学系统。 这将通过系统地探索四种不同的方法（包括已建立的系统和新颖的系统）来实现，因为目前不可能设想哪些系统对 BTV 最有效。其中两个将使用哺乳动物细胞中已建立的 T7 RNA 聚合酶系统，第三个是使用表达 T7 RNA 聚合酶的昆虫细胞的独特方法。 最后一个系统将利用天然细胞 RNA 聚合酶 1。这些研究将利用我实验室提供的广泛试剂和分析系统，并利用 BTV（用于哺乳动物培养的病毒颗粒和用于昆虫细胞培养的核心）的多功能复制功能。 一旦建立，该系统不仅可以操纵 BTV 基因组，从而解决与 BTV 复制相关的重要生物学问题，而且还将为该家族的其他成员（包括人类轮状病毒和呼肠孤病毒）建立类似的系统提供一种方法。