Structural basis of mRNA decapping in poxviruses

痘病毒 mRNA 脱帽的结构基础

基本信息

批准号：
9760335
负责人：
Jessica Peters
金额：
$ 6.12万
依托单位：
UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
依托单位国家：
美国
项目类别：
财政年份：
2019
资助国家：
美国
起止时间：
2019-06-01 至 2021-05-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9760335
关键词：
7-methylguanosine Active Sites Adopted Affinity Chromatography Antiviral Agents Binding Biochemical Biochemistry Biological Biological Assay Bioterrorism Cancerous Catalytic Domain Cells Coupled Data Development Double-Stranded RNA Enzymes Epidemic Fluorescence Polarization Future Gene Expression Genes Genetic Transcription Goals Hydrolysis Immune Immune Evasion Immune response Immunologics Immunology Immunotherapy In Vitro Infection Interferometry Knowledge Link Literature Mammals Mass Spectrum Analysis Measures Mediating Messenger RNA Modeling Molecular Molecular Biology Molecular Conformation Molecular Sieve Chromatography Mutation Analysis Oncolytic viruses Pathogenesis Pathogenicity Pharmacologic Substance Pharmacy (field)Phenotype Play Poxviridae Poxviridae Infections Proliferating Proteins Regulation Research Resolution Roentgen Rays Role Smallpox Structural Models Structure Substrate Interaction Substrate Specificity Techniques Therapeutic Thermodynamics Thin Layer Chromatography Time Treatment Efficacy Vaccinia virus Viral Viral Genes Viral Proteins Virulence Virus Virus Replication World Health Organization X-Ray Crystallography base cancer immunotherapy cofactor decapping enzyme enzyme activity enzyme substrate experimental study in vivo insight light scattering mRNA Decay mRNA Stability mRNA capping mRNA decapping novel nudix hydrolase poly A specific exoribonuclease prevent protein protein interaction recruit sensor stoichiometry structural biology tool virology

7-methylguanosine Active Sites Adopted Affinity Chromatography Antiviral Agents Binding Biochemical Biochemistry Biological Biological Assay Bioterrorism Cancerous Catalytic Domain Cells Coupled Data Development Double-Stranded RNA Enzymes Epidemic Fluorescence Polarization Future Gene Expression Genes Genetic Transcription Goals Hydrolysis Immune Immune Evasion Immune response Immunologics Immunology Immunotherapy In Vitro Infection Interferometry Knowledge Link Literature Mammals Mass Spectrum Analysis Measures Mediating Messenger RNA Modeling Molecular Molecular Biology Molecular Conformation Molecular Sieve Chromatography Mutation Analysis Oncolytic viruses Pathogenesis Pathogenicity Pharmacologic Substance Pharmacy (field)Phenotype Play Poxviridae Poxviridae Infections Proliferating Proteins Regulation Research Resolution Roentgen Rays Role Smallpox Structural Models Structure Substrate Interaction Substrate Specificity Techniques Therapeutic Thermodynamics Thin Layer Chromatography Time Treatment Efficacy Vaccinia virus Viral Viral Genes Viral Proteins Virulence Virus Virus Replication World Health Organization X-Ray Crystallography base cancer immunotherapy cofactor decapping enzyme enzyme activity enzyme substrate experimental study in vivo insight light scattering mRNA Decay mRNA Stability mRNA capping mRNA decapping novel nudix hydrolase poly A specific exoribonuclease prevent protein protein interaction recruit sensor stoichiometry structural biology tool virology

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项目摘要

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.

项目摘要 /摘要病毒已经制定了许多独特的策略来逃避宿主免疫反应，以追求共同目标：扩散。实际上，大多数病毒仅使用多种策略来实现这一目标。这样的痘病毒是至少具有三种机制来防止其宿主检测dsRNA的情况的情况。从而防止宿主先天免疫传感器的激活。这些机制之一是使用病毒编码的解次酶D9和D10通过去除受保护的5¢帽来清除累积的dsRNA 宿主和病毒mRNA，通过5¢-3¢exoriboneclelease xrn1承诺将其降解。 D9的事实和D10在病毒复制周期的不同阶段表达，早期研究表明他们认识到限制mRNA的限制表明D9和D10具有不同的功能，也许针对不同的mRNA 在感染期间。然而，尽管这些酶在宿主免疫进化中起着重要作用，描述痘病毒发病机理的广泛文献，我们缺乏对他们俩如何识别其限制的mRNA和催化帽水解以逃避宿主的免疫反应。该研究计划旨在结合生物化学，结构生物学和病毒学，以建立这些酶识别并水解其底物以及其底物的分子基础特异性有助于痘病毒发病机理。确定是否赋予底物特异性底物结合或催化步骤，体外结合和活性测定将使用各种与痘病毒感染过程中存在的不同mRNA相关的底物。分子决定素将使用高分辨率和低分辨率结构技术来确定底物识别。这高分辨率技术和低分辨率技术的结合将对特定有助于功能的分子相互作用，构象变化和高阶组装。除基于细胞的感染测定外，使用体外结合和活性测定的突变分析将是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型。最后，将使用亲和力纯化结合质量来鉴定蛋白质 - 蛋白质相互作用伴侣光谱法确定是否通过酶 - 底物结合选择底物本身或蛋白质辅因子是否选择底物协助招募D9和D10以靶向mRNA。这些研究将共同提供分子理解关于底物识别和蛋白质 - 蛋白质与痘病毒的相互作用如何控制酶控制目标感染过程中的mRNA选择和帽裂解。了解痘病毒在分子水平是朝着痘病毒感染过程中综合mRNA稳定模型的重要步骤可以用于开发用于免疫疗法的痘病毒工具以及创建新型防病毒软件治疗学是对情节和生物恐怖主义的未来威胁的辩护。