RNA processing in non-segmented minus-strand RNA viruses

描述(申请人提供)：我研究的长期目标是了解调控非片段负义(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，如NIAID A类埃博拉和马尔堡病毒以及C类狂犬病和尼帕病毒。对于许多NNS RNA病毒来说，目前还没有有效的疫苗和抗病毒药物，这种疗法的发展需要对它们的生物学有更好的了解。几十年来，水疱性口炎病毒(VSV)一直被作为所有NNS RNA病毒的实验室原型进行研究。VSV作为模型系统的优势包括它对人类没有严重的致病性，能够在广泛的培养细胞中复制，建立了良好的体外研究RNA合成的系统，以及强大的反向遗传学系统。出于这些原因，对VSV的研究经常为不易驯服的NNS RNA病毒的生物学提供了新的见解。这项建议旨在从机制上详细了解病毒基因表达的一个关键阶段，即病毒mRNA是如何处理的。目前在这一领域的知识表明，病毒mRNA获得其5‘端帽子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抗病毒药物可能的靶向的“跟腱”。使用生化和遗传方法，我们计划绘制病毒mRNAs封端和甲基化所必需的RNA聚合酶的结构域，并确定这些酶活性的底物要求。在特定的目标1中，我们将产生重组病毒来测试这样一个假设，即转录本必须是最小长度才能进入封盖机制。在特定的目标2中，我们将检验病毒聚合酶的L蛋白亚基具有鸟苷酸转移酶活性的假设，并确定病毒mRNAs是如何被识别为修饰的。在特定的目标3中，我们将检验聚合酶的L蛋白亚基包含两个独立的甲基转移酶结构域的假设，并确定它们如何作用于修饰mR NA。在特定的目标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假设。这些实验将导致对NNSRNA病毒L蛋白中鸟苷酸转移酶和甲基转移酶结构域的功能分配，从而确定针对新出现的感染性和潜在生物武器制剂的抗病毒药物开发的新靶点。

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.