Structural basis of mRNA decapping in poxviruses

项目摘要/摘要 病毒已经发展出许多独特的策略来逃避宿主的免疫反应，以追求 共同的目标是：扩散。事实上，大多数病毒同时使用多种策略来实现这一目标。是这样的 痘病毒具有至少三种防止其宿主检测dsRNA的机制， 从而阻止宿主先天免疫感受器的激活。其中一种机制是使用病毒 编码脱壳酶D9和D10，通过去除保护性的5？帽清除积累的dsRNA 宿主和病毒的mRNAs，使它们被细胞内5？3？外切核糖核酸酶Xrn1降解。D9的事实是 和D10在病毒复制周期的不同阶段表达，早期研究表明它们识别 不同的情况表明，D9和D10具有不同的功能，可能针对不同的mRNAs 在感染期间。然而，尽管这些酶在宿主免疫逃避和 大量的文献描述了痘病毒的发病机制，但我们缺乏有效的分子理解 它们如何识别其被封顶的信使核糖核酸，并催化封盖的水解以逃避宿主的免疫反应。 这项研究计划寻求将生物化学、结构生物学和病毒学相结合，以建立 这些酶识别和降解其底物的分子基础，以及它们的底物如何 特异性在痘病毒的发病机制中起作用。以确定底物特异性是否在 底物结合或催化步骤、体外结合和活性分析将使用各种 与痘病毒感染期间存在的不同mRNAs相关的底物。这些分子决定因素 管理底物识别将使用高分辨率和低分辨率结构技术进行识别。这个 高分辨率和低分辨率技术的结合将建立对特定技术的更完整的理解 分子相互作用、构象变化和高阶组装对功能的贡献。 除了基于细胞的感染性分析外，还将使用体外结合和活性分析进行突变分析 用于将结构与表型联系起来，并验证结构的生化和生物学相关性 模特。最后，利用亲和纯化结合质谱法确定蛋白质-蛋白质相互作用伙伴。 光谱分析以确定底物是通过酶-底物结合本身还是蛋白质辅助因子来选择的 协助招募D9和D10来针对mRNAs。总之，这些研究将提供一种分子理解 底物识别和蛋白质与痘病毒解离酶的相互作用如何控制靶标 感染过程中信使核糖核酸的选择和帽子的切割。了解痘病毒解离酶的活性 分子水平是建立痘病毒感染期间mRNA稳定性综合模型的重要一步 可用于开发用于免疫治疗的痘病毒工具以及创造新的抗病毒药物 治疗作为对未来流行病和生物恐怖主义威胁的防御。

Project Summary / Abstract Viruses have developed many unique strategies to evade the host immune response in the pursuit of a common goal: to proliferate. In fact, most viruses use multiple tactics simultaneously to achieve this goal. Such is the case for poxviruses which have at least three mechanisms to prevent their hosts from detecting dsRNA, thereby preventing the activation of host innate immune sensors. One of these mechanisms is to use viral encoded decapping enzymes D9 and D10 to clear accumulating dsRNA by removing the protective 5¢ cap of host and viral mRNAs, committing them to degradation by cellular 5¢-3¢ exoribonuclease Xrn1. The fact that D9 and D10 are expressed at different stages of the viral replication cycle and early studies indicate they recognize capped mRNA differently suggests D9 and D10 have distinct functions, perhaps targeting different mRNAs during infection. However, despite the significant role these enzymes play in host immune evasion and the extensive body of literature describing poxvirus pathogenesis, we lack an effective molecular understanding of how they both recognize their capped mRNA and catalyze cap hydrolysis to evade the host immune response. This research plan seeks to combine biochemistry, structural biology and virology to establish the molecular basis with which these enzymes recognize and hydrolyze their substrates, and how their substrate specificity contributes to poxvirus pathogenesis. To determine if substrate specificity is conferred during substrate binding or the catalytic step, in vitro binding and activity assays will be performed using various substrates relevant to the different mRNAs present during poxvirus infection. The molecular determinants that govern substrate recognition will be identified using high- and low-resolution structural techniques. The combination of high- and low-resolution techniques will build a more complete understanding of the specific molecular interactions, conformational changes, and higher order assembly that contribute to function. Mutational analyses using in vitro binding and activity assays in addition to cell-based infectivity assays will be used to link structure to phenotype and to validate the biochemical and biological relevance of the structural model. Lastly, protein-protein interaction partners will be identified using affinity purification coupled with mass spectrometry to determine if substrates are selected by enzyme-substrate binding per se or if protein cofactors assist in recruiting D9 and D10 to target mRNAs. Together, these studies will provide a molecular understanding of how substrate recognition and protein-protein interactions with poxvirus decapping enzymes control target mRNA selection and cap cleavage during infection. Understanding poxvirus decapping enzyme activity at the molecular level is an important step toward a comprehensive model of mRNA stability during poxvirus infection that can be used in developing poxvirus tools for use in immunotherapy as well as to create novel antiviral therapeutics as a defense against future threats of epidemics and bioterrorism.