Mammalian Reovirus Post-transcriptional Gene Regulation

描述(由申请人提供)：该奖学金申请的主要目标是支持将重点放在剖析核糖核蛋白(RNP)复合体中的结构-功能关系的工作，该复合体介导具有双链A(DsRNA)基因组的病毒的转录后基因调控。哺乳动物呼肠孤病毒是一种已建立的dsRNA病毒复制模型系统，将用于这些研究。中心假说是，呼肠孤病毒RNA的调控机制涉及病毒RNA中特定的顺式作用元件与细胞和病毒RNA结合蛋白。提出了两个特定的目标，以获得对呼肠孤病毒RNA转录后控制的机械性洞察。在特定的目标1中，RNA编辑酶ADAR1在呼肠孤病毒感染周期中的作用将通过ADAR1缺陷的转化细胞、小鼠胚胎成纤维细胞和移植的大鼠神经元的原代培养来确定。将进行实验，以确定病毒RNA是否作为编辑底物，并评估呼肠孤病毒感染对编辑细胞RNA的影响。在特定目的2中，病毒RNA翻译调控元件将使用嵌合报告RNA来定义，该嵌合报告RNA包含荧光素酶开放阅读框架与病毒RNA 5‘和3’末端的保守序列融合在一起。报告RNA的活性将使用无细胞翻译系统、转化细胞和原代神经元培养来研究。含有翻译控制元件突变的病毒将被基于质粒的反向遗传学拯救，这些变化对病毒蛋白质合成和复制的影响将通过感染转化的细胞、小鼠胚胎成纤维细胞和移植的神经元来确定。与健康相关-本申请中提出的工作具有巨大的潜力，可以促进我们对促进宿主细胞损伤和疾病的病毒-细胞相互作用的理解。对病毒分子生物学进行研究的能力将有助于更好地了解病毒感染的发病机制和免疫反应。这项工作还将促进针对致病RNA病毒的病毒预防、诊断和治疗方式的发展。

DESCRIPTION (provided by applicant): The overarching goal of this fellowship application is to provide support for work which will focus on dissecting the structure-function relationships in ribonucleoprotein (RNP) complexes that mediate post-transcriptional gene regulation in viruses with double-strandedA (dsRNA) genomes. Mammalian reoviruses, an established model system of dsRNA virus replication, will be used for these studies. The central hypothesis is that the mechanism of reovirus RNA regulation involves specific cis-acting elements in viral RNA with cellular and viral RNA binding proteins. Two specific aims are proposed to obtain mechanistic insight into post-transcriptional control of reovirus RNA. In Specific Aim 1, the role of the RNA-editing enzyme, ADAR1, in the reovirus infectious cycle will be determined using ADAR1-deficient transformed cells, murine embryonic fibroblasts and primary cultures of explanted rat neurons. Experiments will be performed to determine whether viral RNAs serve as editing substrates and to assess the effect of reovirus infection on editing cellular RNAs. In Specific Aim 2, viral RNA translational regulatory elements will be defined using chimeric reporter RNAs containing the luciferase open reading frame fused to conserved sequences from the 5' and 3' terminal regions of viral RNAs. The activity of reporter RNAs will be studied using cell-free translation systems, transformed cells, and primary neuronal cultures. Viruses containing mutations in translational control elements will be rescued by plasmid-based reverse genetics, and effects of these changes on viral protein synthesis and replication will be determined by infecting transformed cells, murine embryonic fibroblasts, and explanted neurons. Health relevance- the work proposed in this application has great potential to advance our understanding of virus-cell interactions that promote cell injury and disease in the host. The ability to conduct research about the molecular biology of viruses will lead to a better understanding of pathogenesis and immune responses to viral infection. This work will also foster the development of viral preventative, diagnostic, and therapeutic modalities against pathogenic RNA viruses.