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：肠病毒（EVS）包括一个积极的ssRNA病毒家族。最多 研究充分是脊髓灰质炎病毒，但非polio肠病毒（NPEV）引起严重疾病，尤其是在 年轻的。这包括手，脚和口腔疾病，松弛的脊髓炎和脑炎。目前，估计 NPEV在美国负责超过1000万个感染和成千上万的住院 独自的。此外，共感染很常见，因此，由于 重组。因此，了解跨NPEV共享的关键事件是能够控制的关键 当前的感染并应对新兴疾病。这些关键事件之一是多蛋白 加工。 NPEV基因组编码蛋白水解裂解成成熟病毒的多蛋白 产生后代病毒所需的蛋白质。多蛋白的结构区域被加工到 VP0，VP3和VP1。这些组装成一个杂种，其中五个组装成五聚物。十二个五聚会 围绕病毒基因组结合，产生一种称为证明的结构。预言的组装 诱导一系列构象变化，从而导致VP0裂解为VP4和VP2。发生这种情况 基因组封装迅速，因此该预言是短暂的且表征不佳的。裂解 VP0中的VP4和VP2以RNA依赖性方式发生在组装粒子内，并且是先决条件 用于电动汽车感染性。理解证明并表征之前的关键构象变化 VP0裂解是此应用程序的重点。 我们建议VP0成熟的机理在所有电动汽车中都保存下来，并且 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。电流 VP0裂解和EV成熟的模型基于脊髓灰质炎病毒（PV）的数据，并提出了催化性的数据 RNA在VP0成熟中的作用。但是，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。我们建议确定变构 稳定证明的催化变化将使我们能够捕获催化位点的分子细节 在原子分辨率。使用一系列补充技术和一种重复性方法，我们建议陷阱 组装中间体并表征介导VP0的关键残基和构象变化 组装病毒颗粒内的切割。我们最初将使用EVA71作为我们的原型NPEV和PAN-EV 通过验证Echovirus 7，Poliovirus和EVD68中的发现，将确认表型。 我们的初步数据证明了以产量产生突变稳定的证明的可行性 适用于结构研究。附加数据支持开发帽子组装抑制剂的障碍。 通过这些方法，我们将定义VP0裂解的保守机制，该机制适用于 治疗当前的NPEV感染和尚未出现的感染。