ASSEMBLY OF INFECTIOUS BLUETONGUE VIRION FROM CDNA CLONE

感染性病毒颗粒的产生取决于宿主细胞中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(DS)RNA基因组片段组成，这些片段由多层蛋白质组分包裹。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随后产生具有感染性的病毒颗粒的关键步骤。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。这主要是由于缺乏允许对这些病毒进行遗传操作的适当系统。这项建议涉及建立dsRNA病毒的这种系统，特别是蓝舌病毒(BTV)的模式病毒系统，该病毒在结构和分子遗传层面上都很为人所知。因此，本研究的目的是建立一个合适的反向遗传学体系。这将通过系统地探索四种不同的方法(包括现有的和新的系统)来实现，因为目前还不可能设想哪些系统对BTV来说是最有效的。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，第三个是一种独特的方法，使用表达T7 RNA聚合酶的昆虫细胞。最后一个系统将利用天然的细胞RNA聚合酶1。这些研究将利用我实验室现有的广泛试剂和检测系统，并利用BTV(用于哺乳动物培养的病毒粒子和用于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，而且还将为包括人类轮状病毒和呼肠孤病毒在内的家族其他成员建立类似的系统。

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.