ASSEMBLY OF INFECTIOUS BLUETONGUE VIRION FROM CDNA CLONE

传染性病毒颗粒的产生取决于宿主细胞感染过程中的一些特定事件。呼肠孤病毒科的成员由10-12个离散的双链RNA基因组片段组成，这些片段被多层蛋白质成分封装。精确包装每个颗粒内每个RNA片段的单个拷贝是病毒复制周期和后续感染性病毒颗粒产生的关键步骤。与其他RNA病毒不同，尽管对病毒的结构和组装有广泛的了解，但迄今为止在了解dsRNA病毒的复制过程方面取得的进展很少。这主要是由于缺乏对这些病毒进行基因操作的合适系统。这一建议涉及为一种dsRNA病毒建立这样的系统，特别是在结构和分子遗传水平上都很了解的蓝舌病毒（BTV）模型病毒系统。因此，提出的研究目的是建立一个合适的反向遗传系统。这将通过系统地探索四种不同的方法（包括已建立的和新系统）来实现，因为目前还不可能设想哪种系统对BTV最有效。其中两种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。这些研究将利用我的实验室中可用的广泛试剂和分析系统，并利用BTV（哺乳动物培养的病毒粒子和昆虫细胞培养的核心）的多功能复制能力。一旦建立，该系统不仅将允许操纵BTV基因组，从而解决与BTV复制相关的重要生物学问题，而且还将为包括人类轮状病毒和呼肠孤病毒在内的其他家族成员建立类似系统提供一种方法。

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.