Mammalian Reovirus Post-transcriptional Gene Regulation

描述（由申请人提供）：本奖学金申请的总体目标是为研究工作提供支持，这些工作将集中在剖析具有双链da （dsRNA）基因组的病毒中介导转录后基因调控的核糖核蛋白（RNP）复合物的结构-功能关系。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。中心假设是呼肠孤病毒RNA调控的机制涉及病毒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的，以获得呼肠孤病毒RNA转录后控制的机制。在Specific Aim 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缺陷转化细胞、小鼠胚胎成纤维细胞和外植大鼠神经元的原代培养物来确定。实验将确定病毒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Specific Aim 2中，将使用嵌合报告RNA定义病毒RNA翻译调控元件，该报告RNA包含荧光素酶开放阅读框，融合了来自病毒RNA 5‘和3’末端区域的保守序列。报告rna的活性将通过无细胞翻译系统、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合成和复制的影响将通过感染转化细胞、小鼠胚胎成纤维细胞和外植神经元来确定。健康相关性-在本应用中提出的工作具有很大的潜力，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。开展病毒分子生物学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性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.