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: 6374603
• 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 2003-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6374603
• 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.

传染性病毒颗粒的产生取决于宿主细胞中传染性过程中的许多特定事件。 紫旋性科家族的成员由10-12个离散段的双链（DS）RNA基因组组成，这些基因组由多层蛋白质成分封装。 每个粒子中每个RNA片段的单个副本的精确包装是病毒复制周期的关键步骤，并随后产生传染性病毒颗粒。 与其他RNA病毒不同，尽管对病毒结构和组装有广泛的了解，但迄今为止，在理解DSRNA病毒的复制过程方面几乎没有进步。 这主要是由于缺乏合适的系统，该系统允许对这些病毒进行遗传操纵。 该建议涉及建立用于DSRNA病毒的系统，尤其是模型病毒系统，蓝肠病毒（BTV），在结构和分子遗传水平上，众所周知。因此，拟议的研究的目的是建立合适的反向遗传学系统。 这将是系统地探索的，这是四种不同的方法（包括已建立的系统和新型系统），因为目前不可能设想哪种系统对BTV最有效。其中两个将在哺乳动物细胞中使用已建立的T7 RNA聚合酶系统，第三种是使用表达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。研究将利用我的实验室中可用的广泛试剂和测定系统，并利用BTV的多功能复制能力（哺乳动物培养物的病毒元和核心细胞培养物的核心）。 建立后，该系统不仅将允许操纵BTV基因组，因此可以解决与BTV复制有关的重要生物学问题，而且还将为包括人的轮状病毒和依孢病毒在内的家庭其他成员建立类似的系统。