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: 7024425
• 负责人: Sean PJ Whelan
• 金额: $ 33.1万
• 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/7024425
• 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）：我研究的长期目标是了解调节非分段负义（NNS）RNA 病毒转录的分子机制。这些病毒包括几种重要的人类、动物和植物病原体，例如 NIAID 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，没有有效的疫苗和抗病毒药物，此类疗法的开发需要加深对其生物学的了解。几十年来，水疱性口炎病毒（VSV）一直作为所有 NNS RNA 病毒的实验室原型进行研究。 VSV 作为模型系统的优点包括它对人类缺乏严重的致病性、在多种培养细胞中复制的能力、研究 RNA 合成的完善的体外系统以及强大的反向遗传学系统。由于这些原因，对 VSV 的研究经常为难以处理的 NNS RNA 病毒的生物学提供新的见解。该提案旨在详细了解病毒基因表达的关键阶段，即病毒 mRNA 的加工方式。目前该领域的知识表明，病毒 mRNA 获得 5' 帽结构的机制是独特的，这表明这些反应可能代表了新型广泛活性抗病毒药物可能针对的“致命弱点”。我们计划使用生化和遗传学方法来绘制对病毒 mRNA 加帽和甲基化至关重要的 RNA 聚合酶的结构域，并确定这些酶活性的底物要求。在具体目标 1 中，我们将生成重组病毒来测试转录本必须具有最小长度才能进入加帽机制的假设。在具体目标 2 中，我们将测试病毒聚合酶的 L 蛋白亚基具有鸟苷基转移酶活性的假设，并确定病毒 mRNA 如何被识别进行修饰。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，并确定它们如何发挥作用来修饰 mRNA。在具体目标 4 中，我们将测试以下假设：5' mRNA 加工事件对于正确的 3' 末端形成至关重要。这些实验应导致 NNS RNA 病毒 L 蛋白内鸟苷基转移酶和甲基转移酶结构域的功能分配，从而确定开发针对新出现的传染性和潜在生物武器的抗病毒药物的新靶点。