RNA processing in non-segmented minus-strand RNA viruses

描述（由申请人提供）：我研究的长期目标是了解非节段负义（NNS）RNA病毒中调节转录的分子机制。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类狂犬病病毒和尼帕病毒。对于许多NNS RNA病毒，没有有效的疫苗和抗病毒药物，并且这种治疗方法的开发需要对其生物学的进一步了解。几十年来，水泡性口炎病毒（VSV）一直被作为所有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其对人类缺乏严重的致病性，能够在广泛的培养细胞中复制，建立良好的体外系统来研究RNA合成，以及强大的反向遗传学系统。由于这些原因，对VSV的研究经常为较难处理的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如何加工。该领域的现有知识表明，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代表了新的广泛活性的抗病毒药物可能靶向的“阿喀琉斯之踵”。使用生物化学和遗传学的方法，我们计划映射的RNA聚合酶的结构域必不可少的病毒mRNA的加帽和甲基化，并确定这些酶活性的底物要求。在具体目标1中，我们将产生重组病毒以测试转录物必须具有最小长度以获得加帽机制的假设。在具体目标2中，我们将测试病毒聚合酶的L蛋白亚基具有鸟苷酰转移酶活性的假设，并鉴定病毒mRNA如何被识别用于修饰。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，并确定这些功能如何修饰mRNA。在具体目标4中，我们将检验5' mRNA加工事件对于正确的3'末端形成是必不可少的这一假设。这些实验应导致鸟苷酰转移酶和甲基转移酶结构域内的NNS RNA病毒L蛋白的功能分配，从而确定新的目标，为开发抗病毒药物对新兴的传染性和潜在的生物武器代理。

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.