Novel antiviral targets in Ebola and Marburg virus polymerases

我们研究的长期目标是了解丝状病毒基因表达的机制。 我们目前对基因表达的许多了解都是从相关弹状病毒的研究结果推断出来的 和副粘病毒系统。在这些系统中的研究揭示了5‘的形成机制 MRNA帽结构和3‘PolyA尾巴是独特的，提示它们可能是有吸引力的靶点 抗病毒干预。在这项建议中，我们将建立在我们最近获得的一种新的体外试验的基础上 开发用来研究信使核糖核酸帽甲基化。我们将扩展这一系统，允许对每一步进行检查 重组埃博拉病毒和马尔堡病毒纯化的体外转录对丝状病毒mRNA合成的影响 重组成分。在目标1中，我们将重建mRNA帽加成和mRNA帽甲基化 纯化重组L蛋白和纯化的核糖核酸。我们将使用这个系统来定义L和 用于帽加成和mRNA帽甲基化的RNA。在目标2中，我们将重建从 纯化模板和重组聚合酶。我们将使用此系统通过以下方式确定机制 转录因子VP30的功能，决定聚合酶复合体如何组装，以及 确定c/S和Frans-action对信使核糖核酸合成的要求。在目标3中，我们将筛选小分子 文库以确定聚合酶的候选抑制物并确定它们的作用机制。这些 实验将导致对丝状病毒信使核糖核酸合成的新机制的理解，它们将揭示 病毒对信使核糖核酸帽加成和多聚A尾巴形成的要求。因此，这些研究将提供 关于抗病毒药物开发的新靶点以及确定候选药物的详细信息 丝状病毒聚合酶的小分子抑制剂。这项工作还将提供新的工具和试剂， 事实证明，这对我们了解丝状病毒聚合酶的结构和功能的长期目标是有用的。

The long-term objectives of our research are to understand the mechanisms of filovirus gene expression. Much of our current understanding of gene expression is extrapolated from findings in related Rhabdovirus and Paramyxovirus systems. Work in these systems has revealed that the mechanism of formation of the 5' mRNA cap structure and 3' poly A tail are unique and suggests that they may represent attractive targets for antiviral intervention. In this proposal, we will build upon a novel in vitro assay that we have recently developed to study mRNA cap methylation. We will extend this system to permit an examination of each step of mRNA synthesis in filoviruses by reconstituting Ebola and Marburg virus transcription in vitro from purified recombinant components. In aim 1, we will reconstitute mRNA cap addition and mRNA cap methylation from purified recombinant L protein and purified RNA. We will use this system to define the requirements in L and the RNA for cap addition and mRNA cap methylation. In aim 2, we will reconstitute mRNA synthesis from purified templates and recombinant polymerase. We will use this system to determine the mechanism by which the transcription factor VP30 functions, determine how the polymerase complex assembles, and define the c/s and frans-acting requirements for mRNA synthesis. In aim 3, we will screen small molecule libraries to identify candidate inhibitors of the polymerase and define their mechanism of action. These experiments will lead to a new mechanistic understanding of filovirus mRNA synthesis and they will reveal the viral requirements for mRNA cap addition and poly A tail formation. These studies will therefore provide detailed information regarding new targets for antiviral drug development as well as identifying candidate small molecule inhibitors of filovirus polymerases. This work will also provide new tools and reagents that will prove useful in our longer-term objective of understanding the structure and function of filovirus polymerases.