Enteroviral 2C protein as a therapeutic target

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出现的威胁， 给婴儿和儿童。已知有100多种NPEV， 在这些病毒中，进化出强毒株的风险很高。因为我们无法预测任何 新出现的菌株，有效解决这一问题将需要泛肠道病毒的解决方案。所有NPEV 编码一种被称为2C的酶。这种酶和肠道病毒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， 几十年前就已经知道了，仅在过去几年就有更多的报道。进一步发展的主要障碍 这些化合物的发展是缺乏一个既定的实验框架来定义机制 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体组装中的作用。因为2C 蛋白质是解旋酶超家族3的成员，流行的观点是2C是六聚体解旋酶， 通过在基因组复制过程中解旋RNA结构和促进基因组复制， 病毒体组装过程中的解离。不幸的是，只有间接证据支持这些观点。 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， 以脊髓灰质炎病毒（PV）为模型，研究了2C的生物学功能。到目前为止所取得的进展，都没有公布， 改变了我们对2C结构-动力学-功能关系的看法，2C是亚细胞位置， 其中2C表现出其病毒体组装功能，以及病毒诱导膜的物理性质 在感染期间2C必须与其相互作用。 在本申请中，我们建议添加肠道病毒A71（EV-A71）、柯萨奇病毒B3（CVB 3）和肠道病毒 D 68（EV-D 68）的PV研究，以建立2C生物学功能的统一模型， 建立2C作为泛肠道病毒治疗靶点的框架。为此，我们将采取以下措施： 具体目的：阐明2C ATP酶的四级结构和动力学机制（目的1）;阐明2C ATP酶的四级结构和动力学机制（目的2）;阐明2C ATP酶的四级结构和动力学机制（目的3）。 耐药性对2C ATP酶活性和病毒适应性影响（目的2），并表征有助于 基因组测序（Aim 3）。

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).