Understanding the fundamental enterovirus capsid assembly and maturation pathway.

项目摘要/摘要：肠道病毒(EnteroVirus，EV)是一类正向单链RNA病毒。最多的 研究得很好的是脊髓灰质炎病毒，但非脊髓灰质炎肠道病毒(NPEV)会导致严重的疾病，特别是在 年青的。这包括手足口病、松弛脊髓炎和脑炎。目前，据估计 在美国，NPEV造成了1000多万人感染和数万人住院 独自一人。此外，混合感染很常见，因此很可能会出现新的病毒，原因是 重组。因此，了解跨NPEV共享的关键事件是能够控制 目前的感染和对未来新出现的疾病的反应。这些关键事件之一就是多聚蛋白 正在处理。NPEV基因组编码一种多蛋白，该多蛋白被蛋白分解成成熟的病毒 子代病毒产生所需的蛋白质。多聚蛋白的结构区域被加工成 VP0、VP3和VP1。它们组装成一个原型，其中五个组装成五聚体。十二个五聚体 聚集在病毒基因组周围，产生一种被称为前病毒的结构。普罗瑞恩的集会 诱导一系列构象变化，导致VP0裂解成VP4和VP2。这种情况会发生 由于基因组被快速包裹，因此该病毒的存在时间很短，特征也很差。乳沟的分裂 VP0进入VP4和VP2以依赖于RNA的方式出现在组装的颗粒内，并且是先决条件 埃博拉病毒的传染性。理解前提条件并描述在此之前的关键构象变化 VP0裂解是本应用的重点。 我们认为，VP0成熟切割的机制在所有EVS中都是保守的，并且 了解这一过程将为未来疫苗和抗病毒疗法的发展提供信息。海流 VP0裂解和EV成熟的模型在很大程度上基于脊髓灰质炎病毒(PV)的数据，并提出了一种催化 RNA在VP0成熟中的作用。然而，我们假设基因组包装启动了构象 促进蛋白质类催化口袋形成的变化。我们建议识别变构 而稳定前病毒的催化变化将使我们能够捕捉到催化部位的分子细节 在原子分辨率下。使用一系列互补的技术和反复的方法，我们建议诱捕 组装中间体并表征介导VP0的关键残基和构象变化 在组装的病毒颗粒内发生裂解。我们最初将使用EVA71作为我们的NPEV和PAN-EV的原型 表型将通过核实ECHO病毒7、脊髓灰质炎病毒和EVD68的发现来确认。 我们的初步数据证明了产生突变稳定的前驱体的可行性。 适用于结构研究。更多的数据支持开发衣壳组装抑制剂的可操作性。 通过这些方法，我们将确定VP0裂解的保守机制，该机制将适用于 既治疗目前的NPEV感染，也治疗尚未出现的感染。

Project summary/Abstract: Enteroviruses (EVs) comprise a family of positive sense ssRNA viruses. The most well-studied is poliovirus, yet non-polio enteroviruses (NPEVs) cause serious disease, especially in the very young. This includes hand, foot and mouth disease, flaccid myelitis and encephalitis. Currently, it is estimated that NPEVs are responsible for over 10 million infections and tens of thousands of hospitalizations in the US alone. Furthermore, coinfections are common and therefore it is likely that new viruses will arise as a result of recombination. Understanding key events that are shared across NPEVs is therefore key to being able to control current infections and respond to emerging disease in the future. One of these key events is polyprotein processing. The NPEV genome encodes a polyprotein which is proteolytically cleaved into the mature viral proteins required for the generation of progeny virus. The structural region of the polyprotein is processed into VP0, VP3 and VP1. These assemble into a protomer, five of which assemble into a pentamer. Twelve pentamers coalesce around the viral genome, generating a structure termed the provirion. The assembly of the provirion induces a series of conformational changes which result in the cleavage of VP0 into VP4 and VP2. This occurs rapidly upon genome encapsidation, thus the provirion is short-lived and poorly characterized. The cleavage of VP0 into VP4 and VP2 occurs within the assembled particle in an RNA-dependent manner and is a prerequisite for EV infectivity. Understanding the provirion and characterising the key conformational changes which precede VP0 cleavage is the focus of this application. We propose that the mechanism of VP0 maturation cleavage is conserved across all EVs, and that understanding this process will inform the development of future vaccines and anti-viral therapies. The current model of VP0 cleavage and EV maturation is heavily based on data from poliovirus (PV), and suggest a catalytic role for RNA in VP0 maturation. However, we hypothesise that genome packaging initiates conformational changes which facilitate the formation of a proteinaceous catalytic pocket. We suggest that identifying allosteric and catalytic changes which stabilize provirions will allow us to capture the molecular details of the catalytic site at atomic resolution. Using a series of complementary techniques and a reiterative approach, we propose to trap assembly intermediates and characterize the critical residues and conformational changes that mediate VP0 cleavage within the assembled virus particle. We will initially use EVA71 as our prototype NPEV and pan-EV phenotypes will be confirmed by verifying findings in echovirus 7, poliovirus, and EVD68. Our preliminary data demonstrate the feasibility of generating mutationally-stabilized provirions with yields suitable for structural studies. Additional data support the tractability of developing inhibitors of capsid assembly. By these approaches we will define the conserved mechanism of VP0 cleavage which will be applicable to treating both current NPEV infections and those yet to arise.