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理解大多是从相关弹状病毒的发现中推断出来的 和副粘病毒系统在这些系统中的工作已经揭示了5'的形成机制， mRNA帽结构和3' poly A尾是独特的，表明它们可能代表有吸引力的靶点， 抗病毒干预在这项提案中，我们将建立在一种新的体外试验，我们最近 用于研究mRNA帽甲基化。我们将扩大这一制度，允许对每一个步骤进行审查 通过在体外重建埃博拉和马尔堡病毒转录，从纯化的 重组成分。在目标1中，我们将从mRNA帽添加和mRNA帽甲基化中重建mRNA帽添加和mRNA帽甲基化。 纯化的重组L蛋白和纯化的RNA。我们将使用这个系统来定义L中的要求， 用于加帽和mRNA加帽甲基化的RNA。在目标2中，我们将重建mRNA合成， 纯化的模板和重组聚合酶。我们将使用这个系统来确定机制， 转录因子VP30的功能，决定聚合酶复合物如何组装， 定义mRNA合成的顺式和反式作用要求。在aim3中，我们将筛选小分子 文库以鉴定聚合酶的候选抑制剂并定义其作用机制。这些 实验将导致对丝状病毒mRNA合成的新机制的理解，它们将揭示 mRNA帽添加和聚腺苷酸尾形成的病毒要求。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， 证明有助于我们理解丝状病毒聚合酶的结构和功能的长期目标。

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.