ASSEMBLY OF INFECTIOUS BLUETONGUE VIRION FROM CDNA CLONE

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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.