Understanding the fundamental enterovirus capsid assembly and maturation pathway.

了解基本的肠道病毒衣壳组装和成熟途径。

• 批准号: 10450201
• 负责人: Nicola J Stonehouse
• 金额: $ 53.76万
• 依托单位: UNIVERSITY OF LEEDS
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-03-14 至 2027-02-28
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10450201
• 关键词: Antiviral Agents; Antiviral Therapy; Biochemical; Bioinformatics; Biological Assay; Capsid; Catalytic Domain; Chemical Structure; Chemicals; Classification; Clinical; Cryoelectron Microscopy; Data; Development; Disease; Echo Viruses; Encephalitis; Ensure; Enterovirus; Enterovirus Infections; Enzyme-Linked Immunosorbent Assay; Event; Family; Foundations; Future; Generations; Genetic Materials; Genetic Recombination; Genome; Hand, Foot and Mouth Disease; Hospitalization; Human poliovirus; In Vitro; Infection; Measures; Mediating; Modeling; Molecular; Molecular Conformation; Mutation; Myelitis; Outcome; Pathway interactions; Phenotype; Polyproteins; Process; Proteins; Protocols documentation; Protomer; Publishing; RNA; Resolution; Role; Sampling; Sedimentation process; Series; Structural Models; Structure; Study models; Techniques; Testing; Translating; Vaccines; Viral; Viral Genome; Viral Proteins; Virion; Virus; Virus Replication; Western Blotting; Work; antiviral drug development; base; co-infection; cost; design; inhibitor; mutant; novel; novel vaccines; particle; prototype; reconstruction; small molecule inhibitor; success; targeted treatment; therapeutic target; vaccine development; virus genetics

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.

项目摘要/摘要：肠道病毒（EV）由一个正义ssRNA病毒家族组成。最 研究最充分的是脊髓灰质炎病毒，但非脊髓灰质炎肠道病毒（NPEV）可引起严重疾病，特别是在非常 年轻这包括手足口病、弛缓性肌炎和脑炎。目前，据估计 在美国，NPEV造成了超过1000万例感染和数万例住院治疗， 一个人此外，合并感染很常见，因此可能会出现新的病毒， 重组因此，了解NPEV之间共享的关键事件是能够控制 目前的感染和应对未来出现的疾病。其中一个关键事件是多聚蛋白 处理. NPEV基因组编码一种多蛋白，该多蛋白被蛋白水解切割成成熟病毒 产生子代病毒所需的蛋白质。多蛋白的结构区域被加工成 VP 0、VP 3和VP 1。这些聚合物组成一个原聚体，其中五个聚合物组成一个五聚体。十二五聚体 在病毒基因组周围结合，产生一种称为前病毒体的结构。前病毒粒子的组装 诱导一系列构象变化，导致VP 0裂解为VP 4和VP 2。发生这种情况 因此，前病毒体是短寿命的，并且表征不佳。的裂解 VP 0到VP 4和VP 2的转化以RNA依赖性方式发生在组装的颗粒内，并且是一个先决条件 用于EV传染性。了解前病毒并描述之前的关键构象变化 VP 0切割是本申请的焦点。 我们认为VP 0成熟裂解的机制在所有EV中是保守的， 了解这一过程将为未来疫苗和抗病毒疗法的开发提供信息。当前 VP 0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，并表明 RNA在VP 0成熟中的作用。然而，我们假设，基因组包装启动构象 促进蛋白质催化口袋形成的变化。我们认为，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位点的分子细节 在原子分辨率。使用一系列的补充技术和一个迭代的方法，我们提出了陷阱 组装中间体，并表征介导VP 0的关键残基和构象变化 在组装的病毒颗粒内裂解。我们将首先使用EVA 71作为我们的原型NPEV和泛EV 将通过验证埃可病毒7型、脊髓灰质炎病毒和EVD 68中的发现来确认表型。 我们的初步数据证明了产生突变稳定的前病毒的可行性， 适合结构研究。其他数据支持开发衣壳组装抑制剂的易处理性。 通过这些方法，我们将确定VP 0切割的保守机制，这将适用于 治疗目前的NPEV感染和尚未出现的感染。