Watching conformational rearrangements in picornavirus replication proteins

观察小核糖核酸病毒复制蛋白的构象重排

• 批准号: 10209169
• 负责人: David Douglas Boehr
• 金额: $ 38.02万
• 依托单位: PENNSYLVANIA STATE UNIVERSITY, THE
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-07-15 至 2025-07-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10209169
• 关键词: 1-Phosphatidylinositol 4-Kinase; 3-Dimensional; Active Sites; Affinity; Amino Acid Substitution; Antiviral Agents; Behavior; Binding; Binding Proteins; Biogenesis; Biological Assay; Biological Models; Biological Process; Biophysics; Capsid Proteins; Cells; Complement; Coronavirus; Crystallization; Development; Drug Targeting; Elements; Enterovirus; Enterovirus 68; Environment; Enzymes; Event; Family Picornaviridae; Funding; Genome; Grant; Heart; Human; Human poliovirus; Immune response; Lead; Life Cycle Stages; Lipid Binding; Lipids; Membrane; Membrane Proteins; Molecular Conformation; Motion; Mutagenesis; NMR Spectroscopy; Nucleotides; Organelles; Peptide Hydrolases; Peptides; Phosphatidylinositols; Play; Polymerase; Polyproteins; Process; Production; Protein Conformation; Proteins; Proteolysis; Proteolytic Processing; Proteome; RNA; RNA Binding; RNA Viruses; RNA replication; RNA-Directed RNA Polymerase; Replication-Associated Process; Rhinovirus; Ribonucleotides; Roentgen Rays; Role; Sampling; Specificity; Structure; Surface; System; Thermodynamics; Translation Process; Viral; Viral Proteins; Virus; Virus Diseases; Virus Inhibitors; Virus Replication; Work; attenuation; base; emerging pathogen; flexibility; inhibitor/antagonist; insight; nanosecond; novel; pathogenic virus; phosphodiester; protein function; vaccine development; viral RNA

Project Summary Some of the most important new and (re)emerging pathogens are positive-strand RNA viruses, including coronavirus and picornaviruses Enterovirus D68, Enterovirus A71 and even poliovirus. These viruses can directly use their RNA genome to guide the synthesis of a large polyprotein, which must then be proteolyzed into its component parts, including the capsid proteins and enzymes important for genome replication and encapsidation. Virus RNA genomes are rather small, and so these viruses have evolved strategies to essentially expand their functional proteomes. For example, the picornavirus 3C protein is a multi-functional protein that has protease activity, binds RNA control sequences important for coordinating replication and translation processes, and binds phosphoinositide lipids found in virus “replication organelles”, which act to protect the virus from host cell defenses. All of these activities are encoded within its small 20 kDa structure. Another strategy to expand functional protein content is for proteolytic precursors to have different functions than their fully processed counterparts. For example, 3C is also found as part of the 3CD protein, but the 3CD protein has different protease specificity, and different RNA and lipid binding affinities. The 3CD protein also has a 3D domain; the 3D protein is the RNA-dependent RNA polymerase but 3CD does not possess polymerase activity. By itself, 3CD also upregulates phosphoinositide lipid production and induces membrane proliferation, events important for replication organelle biogenesis. How the different and emergent functions of 3CD arise is poorly understood; X-ray crystal structures indicate that 3CD is merely a composite of the 3C and 3D proteins joined together by a small flexible linker. We propose that structural dynamics, that is, the ability to sample multiple structural conformations, is the missing ingredient in understanding virus protein function. We propose that 3C fluctuates among many conformations, providing 3C the ability to access and coordinate its many functions, and we propose that 3CD fluctuates into different conformations, providing it with alternative functions. These dynamic excursions can be further modified by interactions with RNA, lipids and protein binding partners to coordinate virus protein function. We will evaluate these protein structural dynamics through solution-state nuclear magnetic resonance spectroscopy, which provide atomic-level detail of protein motions from the picosecond to second timescales, and complement these studies with mutagenesis studies, functional assays and cell-based approaches to better understand the roles of protein structural dynamics in the virus life cycle. The completed work will provide new opportunities for rational anti-viral strategies, for example, by finding molecules that bind to alternative protein conformations and/or disrupt functionally-important motions, as already validated for 3D.

项目概要 一些最重要的新的和（重新）出现的病原体是正链 RNA 病毒， 包括冠状病毒和小核糖核酸病毒肠道病毒 D68、肠道病毒 A71 甚至脊髓灰质炎病毒。 这些病毒可以直接利用它们的RNA基因组来指导合成大的多蛋白，从而 然后必须被蛋白水解成其组成部分，包括衣壳蛋白和酶 对于基因组复制和衣壳化很重要。病毒RNA基因组相当小，因此 这些病毒已经进化出从本质上扩展其功能蛋白质组的策略。例如， 小核糖核酸病毒 3C 蛋白是一种多功能蛋白，具有蛋白酶活性，结合 RNA 控制 对于协调复制和翻译过程很重要的序列，并结合 病毒“复制细胞器”中发现的磷酸肌醇脂质，可保护病毒免受宿主侵害 细胞防御。所有这些活性都编码在其 20 kDa 的小结构中。另一种策略是 扩大功能蛋白含量是为了使蛋白水解前体具有与其自身不同的功能 完全加工的对应物。例如，3C 也被发现是 3CD 蛋白的一部分，但 3CD 蛋白质具有不同的蛋白酶特异性以及不同的RNA和脂质结合亲和力。 3CD 蛋白质也有3D结构域； 3D 蛋白是 RNA 依赖性 RNA 聚合酶，但 3CD 则不然 不具有聚合酶活性。 3CD 本身也上调磷酸肌醇脂质的产生 并诱导膜增殖，这是复制细胞器生物发生的重要事件。如何 对 3CD 出现的不同和新兴功能知之甚少； X射线晶体结构表明 3CD 只是 3C 和 3D 蛋白质通过一个小的柔性接头连接在一起的复合物。 我们认为结构动力学，即对多种结构构象进行采样的能力，是 了解病毒蛋白功能中缺失的成分。我们建议3C波动于 许多构象，为 3C 提供了访问和协调其许多功能的能力，我们 提出 3CD 波动成不同的构象，为其提供替代功能。这些 动态偏移可以通过与 RNA、脂质和蛋白质结合的相互作用来进一步修改 合作伙伴协调病毒蛋白功能。我们将评估这些蛋白质结构动力学 通过溶液态核磁共振波谱，提供原子级细节 从皮秒到秒时间尺度的蛋白质运动，并补充这些研究 诱变研究、功能测定和基于细胞的方法，以更好地了解 病毒生命周期中的蛋白质结构动力学。完成的工作将提供新的机遇 合理的抗病毒策略，例如，通过寻找与替代蛋白质结合的分子 构象和/或破坏功能上重要的运动，正如已针对 3D 进行验证的那样。