Mammalian Reovirus Post-transcriptional Gene Regulation

哺乳动物呼肠孤病毒转录后基因调控

• 批准号: 7754769
• 负责人: Charles Roy Martinez
• 金额: $ 3.48万
• 依托单位: VANDERBILT UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-09-01 至 2011-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7754769
• 关键词: ADAR1; Adenosine; Automobile Driving; Biological Models; Biology; Cells; Complex; Conserved Sequence; DNA; Development; Diagnosis; Diagnostic; Disease; Double-Stranded RNA; Ectopic Expression; Elements; Embryo; Engineering; Enzymes; Fellowship; Fibroblasts; Firefly Luciferases; Fostering; Functional RNA; Gene Expression; Gene Expression Regulation; Genome; Goals; Health; Imaging technology; Immune response; In Vitro; Indium; Infection; Inosine; L Cells; Lead; Life Cycle Stages; Luciferases; Mediating; Metabolic; Modality; Molecular Biology; Mus; Mutation; Nervous system structure; Neuraxis; Neurons; Nucleic Acid Regulatory Sequences; Open Reading Frames; Pathogenesis; Plasmids; Post-Transcriptional Regulation; Prevention; Process; Protein Biosynthesis; Protein Isoforms; Proteins; Proteomics; RNA; RNA Editing; RNA Interference; RNA Stability; RNA Viruses; RNA-Binding Proteins; Rattus; Regulation; Regulatory Element; Reoviridae Infections; Reovirus; Reporter; Research; Ribonucleoproteins; Rodent; Role; Structure-Activity Relationship; System; Therapeutic; Tissues; Transcript; Translational Regulation; Translations; Viral; Viral Genes; Viral Genome; Viral Proteins; Virus; Virus Diseases; Virus Replication; Virus-Cell Membrane Interaction; Work; base; cell growth regulation; cell injury; cell transformation; cell type; cellular imaging; cis acting element; dsRNA adenosine deaminase; gain of function; genetic regulatory protein; in vivo; injured; insight; intermolecular interaction; mutant; neurotropic; novel strategies; positional cloning; programs; research study; viral RNA; virus host interaction

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.

描述（由申请人提供）：该奖学金申请的首要目标是为以下工作提供支持：重点剖析核糖核蛋白（RNP）复合物中的结构-功能关系，该复合物介导具有双链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。核心假设是呼肠孤病毒RNA调节的机制涉及病毒RNA中具有细胞和病毒RNA结合蛋白的特异性顺式作用元件。提出了两个具体的目标，以获得呼肠孤病毒RNA的转录后控制机制的见解。在具体目标1中，RNA编辑酶ADAR 1在呼肠孤病毒感染周期中的作用将使用ADAR 1缺陷型转化细胞、鼠胚胎成纤维细胞和原代培养的大鼠神经元来确定。将进行实验以确定病毒RNA是否用作编辑底物，并评估呼肠孤病毒感染对编辑细胞RNA的影响。在特定目标2中，将使用嵌合报告RNA定义病毒RNA翻译调控元件，该嵌合报告RNA含有与病毒RNA 5'和3'末端区域的保守序列融合的荧光素酶开放阅读框。将使用无细胞翻译系统、转化细胞和原代神经元培养物研究报告RNA的活性。在翻译控制元件中含有突变的病毒将通过基于质粒的反向遗传学来拯救，并且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细胞、鼠胚胎成纤维细胞和神经元来确定。健康相关性-本申请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。进行病毒分子生物学研究的能力将导致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。