RNA processing in non-segmented minus-strand RNA viruses

非分段负链RNA病毒中的RNA加工

• 批准号: 6875438
• 负责人: Sean PJ Whelan
• 金额: $ 33.9万
• 依托单位: HARVARD MEDICAL SCHOOL
• 依托单位国家: 美国
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-03-01 至 2010-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6875438
• 关键词: DNA directed RNA polymerase; RNA virus; Vesiculovirus; binding sites; crosslink; enzyme activity; gene expression; guanosine monophosphate; messenger RNA; methylation; methyltransferase; polyadenylate; polymerase chain reaction; recombinant virus; site directed mutagenesis; virus genetics

DESCRIPTION (provided by applicant): The long-term objectives of my research are to understand the molecular mechanisms that regulate transcription in non-segmented negative-sense (NNS) RNA viruses. These viruses include several significant human, animal and plant pathogens such as the NIAID category A Ebola and Marburg viruses and the category C rabies and Nipah viruses. For many NNS RNA viruses there are no effective vaccines and antiviral drugs, and the development of such therapeutics demands an enhanced understanding of their biology. For decades, vesicular stomatitis virus (VSV) has been studied as a laboratory prototype of all the NNS RNA viruses. The advantages of VSV as a model system include its lack of serious pathogenicity for humans, ability to replicate in a wide range of cultured cells, well established in vitro systems to study RNA synthesis, and a robust reverse genetics system. For these reasons, studies on VSV have frequently provided novel insight into the biology of the less tractable NNS RNA viruses. This proposal aims to understand in mechanistic detail a key stage in viral gene expression, namely how viral mRNA's are processed. Current knowledge in this area indicates that the mechanism by which the viral mRNA's acquire their 5' cap structure is unique, suggesting that these reactions may represent an "Achilles Heel" to which novel broadly active antiviral drugs might be targeted. Using biochemical and genetic approaches we plan to map domains of the RNA polymerase essential for capping and methylation of the viral mRNAs, and determine the substrate requirements for these enzymatic activities. In specific aim 1, we will generate recombinant viruses to test the hypothesis that a transcript must be a minimal length to gain access to the capping machinery. In specific aim 2 we will test the hypothesis that the L protein subunit of the viral polymerase possess guanylyltransferase activity, and identify how the viral mRNA's are recognized for modification. In specific aim 3 we will test the hypothesis that the L protein subunit of polymerase contains two separate methyltransferase domains, and determine how these function to modify mRNA. In specific aim 4 we will test the hypothesis that the 5' mRNA processing events are essential for correct 3' end formation. These experiments should result in the functional assignment of guanylyltransferase and methyltransferase domains within an NNS RNA virus L protein, and thus identify novel targets for the development of antiviral drugs against emerging infectious and potential bio-weapons agents.

描述（由申请人提供）：我的长期研究目标是了解非分段负意义（NNS） RNA病毒转录调控的分子机制。这些病毒包括几种重要的人类、动物和植物病原体，如美国国家疾病预防控制研究所的A类埃博拉病毒和马尔堡病毒以及C类狂犬病和尼帕病毒。对于许多NNS RNA病毒，目前还没有有效的疫苗和抗病毒药物，开发这种治疗方法需要加强对其生物学的了解。几十年来，水疱性口炎病毒（VSV）作为所有NNS RNA病毒的实验室原型进行了研究。VSV作为模型系统的优势包括其对人类缺乏严重致病性，能够在广泛的培养细胞中复制，建立了良好的体外系统来研究RNA合成，以及强大的反向遗传系统。由于这些原因，对VSV的研究经常为不易处理的NNS RNA病毒的生物学提供新的见解。本研究旨在了解病毒基因表达的一个关键阶段，即病毒mRNA的加工过程。目前在这一领域的知识表明，病毒mRNA获得其5'帽结构的机制是独特的，这表明这些反应可能是新型广谱活性抗病毒药物的“阿喀琉斯之踵”。利用生化和遗传方法，我们计划绘制病毒mrna盖帽和甲基化所必需的RNA聚合酶结构域，并确定这些酶活性的底物需求。在具体目标1中，我们将生成重组病毒来验证转录物必须达到最小长度才能进入封盖机制的假设。在具体目标2中，我们将验证病毒聚合酶的L蛋白亚基具有guanylytransferase活性的假设，并确定病毒mRNA如何被识别以进行修饰。在具体目标3中，我们将测试聚合酶的L蛋白亚基包含两个独立的甲基转移酶结构域的假设，并确定这些结构域如何修饰mRNA。在具体目标4中，我们将验证5‘ mRNA加工事件对于正确的3’端形成至关重要的假设。这些实验将导致NNS RNA病毒L蛋白中guanylyltransferase和methyltransferase结构域的功能分配，从而为开发针对新出现的传染性和潜在生物武器制剂的抗病毒药物确定新的靶点。