Novel antiviral targets in Ebola and Marburg virus polymerases

埃博拉和马尔堡病毒聚合酶的新抗病毒靶点

• 批准号: 8233441
• 负责人: Sean PJ Whelan
• 金额: $ 32.46万
• 依托单位: HARVARD MEDICAL SCHOOL
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-03-01 至 2014-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8233441
• 关键词: Antiviral Agents; Biochemical; Biological Assay; Biology; Cells; Communicable Diseases; Complex; DNA-Directed RNA Polymerase; Development; Dissection; Ebola virus; Enzymes; Event; Filovirus; Frankfurt-Marburg Syndrome Virus; Future; Gene Expression; Genetic Transcription; In Vitro; Intervention; Lead; Messenger RNA; Methylation; Methyltransferase; Molecular; New England; Paramyxovirus; Poly(A) Tail; Polyadenylation; Polymerase; Process; Proteins; RNA; RNA Caps; RNA chemical synthesis; RNA-Directed RNA Polymerase; Reaction; Reagent; Recombinants; Research; Rhabdoviridae; Role; Signal Transduction; Structure; System; Testing; Therapeutic; Therapeutic Intervention; Vaccines; Viral; Viral Hemorrhagic Fevers; Viral Proteins; Virus; Work; base; biodefense; combat; design; drug development; in vitro Assay; in vivo; inhibitor/antagonist; mRNA capping; novel; reconstitution; research study; small molecule; small molecule libraries; tool; transcription factor; vaccine development

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.

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大部分理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。在这些系统中的工作揭示了 5' 的形成机制 mRNA 帽结构和 3' 聚 A 尾是独特的，表明它们可能代表有吸引力的目标 抗病毒干预。在本提案中，我们将基于我们最近开发的一种新型体外测定方法 开发用于研究 mRNA 帽甲基化。我们将扩展该系统以允许检查每个步骤 通过在体外从纯化的埃博拉病毒和马尔堡病毒转录中重建丝状病毒中的 mRNA 合成 重组成分。在目标 1 中，我们将重构 mRNA 帽添加和 mRNA 帽甲基化 纯化的重组L蛋白和纯化的RNA。我们将使用该系统来定义 L 和中的要求 用于帽添加和 mRNA 帽甲基化的 RNA。在目标 2 中，我们将重新构建 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 发挥作用，决定聚合酶复合物如何组装，以及 定义 mRNA 合成的顺式和反式作用要求。在目标3中，我们将筛选小分子 库来识别聚合酶的候选抑制剂并确定其作用机制。这些 实验将带来对丝状病毒 mRNA 合成的新机制的理解，并将揭示 病毒对 mRNA 帽添加和聚 A 尾形成的要求。因此，这些研究将提供 有关抗病毒药物开发新目标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，这对于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。