The role of reovirus factories in viral RNA regulation.

描述(申请人提供)：非融合性哺乳动物正病毒是动物病毒(呼肠孤病毒科)家族的成员，在人类(轮状病毒)疾病和溶瘤病毒(呼肠孤病毒)中具有相当重要的作用。在呼肠孤病毒感染的早期，会形成小的、细胞质的、相致密的包涵体，随着感染的继续，包涵体的大小会增大。病毒蛋白以及部分和完全组装的病毒粒子和核心颗粒几乎完全定位于这些结构(称为病毒工厂)，这表明它们是病毒基因组复制和组装的场所。最近的研究表明，大多数参与病毒核心组装和基因组复制的病毒蛋白是通过与MNS蛋白的结合而被招募到工厂的。此外，在呼肠孤病毒感染期间，这些工厂被发现作为病毒(+)RNA转录的场所，这是一项由核心颗粒执行的任务。这项建议旨在研究呼肠孤病毒工厂在协调病毒RNA转录、翻译和复制中的作用。将对病毒工厂的起源进行调查，以确定病毒感染中病毒RNA转录的哪个点位于工厂的保护环境中，以及亲本病毒核心颗粒是否通过它们与细胞翻译蛋白的结合来指导工厂的发生。这些实验将通过标记亲本核心，然后通过免疫荧光、免疫电子显微镜和免疫沉淀研究它们在细胞内的定位来完成。利用RNAi技术，将确定在感染细胞中存在两个病毒RNA池的可能性，一个用于复制，另一个用于翻译。病毒RNA翻译的定位和与病毒工厂定位相关的细胞翻译因子将通过免疫荧光研究进行检查，以确定病毒工厂或工厂定位的蛋白质是否指导病毒RNA翻译。参与翻译的病毒蛋白将被确定。顺式作用的RNA序列在工厂内指导复制的作用将通过使用反向遗传学将突变引入病毒基因组来研究。这些实验旨在更好地了解病毒工厂在调节病毒RNA转录、翻译和复制中的作用，以便制定策略来阻断和利用呼肠孤病毒感染的这些方面。

DESCRIPTION (provided by applicant): The non-fusogenic mammalian orthoreoviruses are members of a family of animal viruses (Reoviridae) that are of considerable importance in human (rotavirus) disease and as oncolytic viruses (reoviruses). Early during reovirus infection small, cytoplasmic, phase-dense inclusions develop which grow in size as infection continues. Viral proteins, as well as partially and fully assembled virions and core particles localize almost exclusively to these structures (termed viral factories) suggesting they are the sites of viral genome replication and assembly. Recently, it has been shown that most viral proteins involved in virus core assembly and genome replication are recruited to the factories through an association with the MNS protein. Moreover, the factories have been found to function as sites for the transcription of viral (+)RNAs, a task that is carried out by core particles, during reovirus infection. This proposal is designed to investigate the role of the reovirus factory in coordinating viral RNA transcription, translation, and replication. The genesis of the viral factory will be investigated to determine at which point in viral infection transcription of viral RNA is located within the protective environment of the factory, and if parental viral core particles direct factory genesis through their association with cellular translation proteins. These experiments will be accomplished by labeling parental cores, then investigating their localization within cells by immunofluorescence, immunoelectron microscopy, and immunoprecipitation studies. Using RNAi technology, the possibility that there are two pools of viral RNA in infected cells, one for replication and one for translation, will be determined. The localization of viral RNA translation and cellular translation factors relative to the localization of viral factories will be examined by immunofluorescence studies to determine if the viral factory, or factory localized proteins direct viral RNA translation. Viral proteins involved in translation will be identified. The role of cis-acting RNA sequences in directing replication within the factory will be investigated by introducing mutations into the viral genome using reverse genetics. These experiments are designed to better understand the role of the viral factory in regulating viral RNA transcription, translation, and replication so that strategies can be developed to interdict and exploit these aspects of reovirus infection.