Research Training in Virus RNA Localization

说明(申请人提供)：呼肠孤病毒是呼肠孤病毒科节段dsRNA病毒家族的典型成员，包括重要的人类(轮状病毒)和兽医(蓝舌病)病原体。10个独特的正义病毒RNA被定位到病毒工厂并包装成子代核心颗粒的机制尚不清楚，将在本提案的实验中解决。呼肠孤病毒阳性RNA转录后所走的路径将通过原位杂交和感染细胞中溴代标记的病毒阳性RNA的免疫染色来检测。病毒RNA定位到病毒工厂的机制将通过将病毒或对照RNA显微注射到呼肠孤病毒感染的细胞或表达呼肠孤病毒RNA结合蛋白的转基因细胞中来检验。如果病毒RNA被专门定位到病毒工厂，那么涉及定位的反式和顺式作用因子就会被识别出来。还将研究RNA非特异性本地化到工厂的机制。了解RNA的定位过程可能会导致新的抗病毒策略的开发，并将有助于开发一个强大的dsRNA病毒的反向遗传学系统。

DESCRIPTION (provided by applicant): Reoviruses are prototypical members of the Reoviridae family of segmented dsRNA viruses that include important human (rotavirus) and veterinary (blue tongue disease) pathogens. The mechanisms by which 10 unique positive-sense viral RNAs are localized to viral factories for packaging into progeny core particles is poorly understood and will be addressed by the experiments in this proposal. The paths taken by reovirus positive-sense RNAs after they are transcribed will be examined by both in situ hybridization and immunostaining of bromouridine labeled viral positive-sense RNAs in infected cells. The mechanism for localization of viral RNAs to viral factories will be examined by microinjection studies of viral or control RNAs into reovirus infected cells, or transfected cells expressing reovirus RNA-binding proteins. If viral RNAs are specifically localized to viral factories, trans- and cis-acting factors involved in localization will be identified. Mechanisms for non-specific localization of RNA to factories will also be examined. Understanding the RNA localization process may lead to the development of new anti-viral strategies, and will aid in the development of a robust reverse genetics system for dsRNA viruses.