Enteroviral 2C protein as a therapeutic target

肠道病毒2C蛋白作为治疗靶点

• 批准号: 10609524
• 负责人: CRAIG E. CAMERON
• 金额: $ 74.79万
• 依托单位: UNIV OF NORTH CAROLINA CHAPEL HILL
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-04-13 至 2027-03-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10609524
• 关键词: ATP phosphohydrolase; Address; Biochemical; Biological; Biological Models; Biological Process; Cell model; Cells; Chemicals; Child; Chimeric Proteins; Code; Collaborations; Coxsackie Viruses; Cytoplasm; Data; Dependence; Development; Drug resistance; Electron Microscopy; Enterovirus; Enterovirus 68; Enterovirus 71; Enzymes; Evolution; Exhibits; Fluorescence Microscopy; Genetic Recombination; Genome; Guanidines; Hela Cells; Human; Human poliovirus; Hydantoins; Infant; Infection; Institution; Kinetics; Laboratories; Length; Lipids; Location; Mass Spectrum Analysis; Mediating; Membrane; Modeling; Molecular Target; Mutation; Nucleotides; Proteins; Public Health; Publishing; RNA; RNA-Directed RNA Polymerase; Reporting; Resistance; Risk; Role; Scanning; Serotyping; Site; Solubility; Specificity; Structure; Transmission Electron Microscopy; Tumor Cell Line; Vesicle; Virion; Virulent; Virus; Virus Diseases; biophysical properties; biophysical techniques; design; dimer; fitness; graduate student; helicase; inhibitor; lipidome; member; physical property; therapeutic target; viral RNA; viral fitness

Project Abstract Non-polio human enteroviruses (NPEVs) represent an existing and emerging threat to public health, especially to infants and children. With more than 100 NPEVs known and the high rate of mutation and recombination of these viruses, the risk for evolution of virulent strains is high. Because we cannot predict the serotype of any newly emerging strain, addressing this problem effectively will require pan-enterovirus solutions. All NPEVs encode an enzyme, which has been termed 2C. This enzyme is as well conserved as the enterovirus RNA- dependent RNA polymerase (RdRp). Indeed, inhibitors of 2C with activity against multiple enteroviruses have been known for decades, with even more reported over the past few years alone. A major obstacle to further development of these compounds is the absence of an established experimental framework to define mechanism of action and to guide design of pan-enterovirus activity. Two of the earliest inhibitors of 2C: guanidine hydrochloride (GuHCl); and 5-(3,4-dichlorophenyl)- methylhydantoin (hydantoin), revealed roles for 2C both in genome replication and virion assembly. Because 2C protein is a member of helicase superfamily 3, the prevailing view has been that 2C is a hexameric helicase that cooperates with RdRp by unwinding RNA structure during genome replication and by facilitating genome encapsidation during virion assembly. Unfortunately, only indirect evidence exists to support these views. Nearly five years ago, our laboratory initiated an effort to connect the biochemical and biophysical properties of 2C to its biological functions using poliovirus (PV) as our model. The advances made to date, all unpublished, have changed the way we think about structure-dynamics-function relationships of 2C, the subcellular locations in which 2C manifests its virion-assembly function, and the physical properties of the virus-induced membranes with which 2C must interact during infection. In this application, we propose to add Enterovirus A71 (EV-A71), Coxsackievirus B3 (CVB3), and Enterovirus D68 (EV-D68) to our studies of PV to establish unifying models for the biological functions of 2C that will provide a framework to establish 2C as a pan-enterovirus therapeutic target. We will do so by pursuing the following specific aims: Elucidate the quaternary structure and kinetic mechanism of 2C ATPase (Aim 1); Elucidate the impact of drug resistance on 2C ATPase activity and viral fitness (Aim 2), and Characterize sites contributing to genome encapsidation (Aim 3).

项目摘要 非脊髓灰质炎人类肠道病毒 (NPEV) 是对公共卫生的现有和新威胁，尤其是 给婴儿和儿童。已知 NPEV 超过 100 种，并且突变和重组率很高 这些病毒进化出强毒株的风险很高。因为我们无法预测任何血清型 新出现的菌株，有效解决这一问题将需要泛肠道病毒解决方案。所有 NPEV 编码一种酶，称为2C。这种酶与肠道病毒 RNA 一样保守。 依赖性 RNA 聚合酶 (RdRp)。事实上，具有抗多种肠道病毒活性的 2C 抑制剂已 几十年来人们都知道这一点，仅在过去几年就有更多的报道。进一步发展的主要障碍 这些化合物的开发缺乏确定的实验框架来定义机制 行动并指导泛肠道病毒活性的设计。 两种最早的 2C 抑制剂：盐酸胍 (GuHCl)；和5-(3,4-二氯苯基)- 甲基乙内酰脲（乙内酰脲）揭示了 2C 在基因组复制和病毒粒子组装中的作用。因为2C 蛋白质是解旋酶超家族 3 的成员，普遍的观点是 2C 是六聚体解旋酶 通过在基因组复制过程中解开 RNA 结构并促进基因组与 RdRp 合作 病毒粒子组装过程中的衣壳化。不幸的是，只有间接证据可以支持这些观点。 大约五年前，我们的实验室开始努力将生物化学和生物物理特性联系起来 使用脊髓灰质炎病毒（PV）作为我们的模型来研究 2C 的生物学功能。迄今为止取得的进展，全部未公开， 改变了我们思考 2C（亚细胞位置）的结构-动力学-功能关系的方式 其中2C体现了其病毒粒子组装功能，以及病毒诱导膜的物理特性 感染期间 2C 必须与之相互作用。 在此应用中，我们建议添加肠道病毒 A71 (EV-A71)、柯萨奇病毒 B3 (CVB3) 和肠道病毒 D68 (EV-D68) 参与我们的 PV 研究，为 2C 的生物功能建立统一模型，这将提供 建立2C作为泛肠道病毒治疗靶点的框架。我们将通过追求以下目标来做到这一点 具体目标：阐明2C ATPase的四级结构和动力学机制（目标1）；阐明 耐药性对 2C ATP 酶活性和病毒适应性的影响（目标 2），并表征有助于 基因组衣壳化（目标 3）。