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的威胁，特别是 对婴儿和儿童来说。已知的100多个NPEV和高突变率和重组率 这些病毒，进化成强毒株的风险很高。因为我们无法预测任何一种 新出现的毒株，有效地解决这一问题将需要泛肠道病毒解决方案。所有NPEV 编码一种被称为2C的酶。这种酶和肠道病毒RNA一样保守- 依赖RNA聚合酶(RdRp)。事实上，具有多种肠道病毒活性的2C抑制剂已经 几十年来一直为人所知，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确定机制的实验框架 并指导泛肠道病毒活性的设计。 2C最早的两种抑制剂：盐酸胍和5-(3，4-二氯苯基)- 甲基海因(海因)，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，流行的观点认为2C是一个六聚体解旋酶，它 通过在基因组复制过程中解开RNA结构并通过促进基因组与RdRp合作 病毒粒子组装过程中的包膜作用。不幸的是，只有间接证据支持这些观点。 近五年前，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，对其生物学功能进行了2摄氏度的研究。到目前为止所取得的进展，都是未发表的， 已经改变了我们对2C的结构-动力学-功能关系的思考方式，即亚细胞位置 其中2C显示其病毒粒子组装功能，以及病毒诱导的膜的物理性质 在感染过程中，2C必须与之相互作用。 在本申请中，我们建议添加肠道病毒A71(EV-A71)、柯萨奇病毒B3(CVB3)和肠道病毒 D68(EV-D68)为我们对光伏的研究建立统一的模型，为2C的生物学功能提供 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 具体目标：阐明2C-ATPase的四级结构和动力学机制(目标1)； 耐药性对2C-ATPase活性和病毒适合性的影响(目标2)，并表征有助于 基因组封装(目标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).