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.

项目摘要 一些最重要的新事物和新兴病原体是阳性RNA病毒， 包括冠状病毒和Picornaviruse肠病毒D68，肠病毒A71甚至脊髓灰质炎病毒。 这些病毒可以直接使用其RNA基因组来指导大型多蛋白的合成，该蛋白质的合成 然后必须将蛋白水解为其组件部分，包括衣壳蛋白和酶 对于基因组复制和封装很重要。病毒RNA基因组很小，因此 这些病毒已经发展了策略以从本质上扩展其功能蛋白质组。例如， Picornavirus 3C蛋白是一种具有蛋白酶活性的多功能蛋白，结合RNA控制 序列对于协调复制和翻译过程很重要，并结合 在病毒“复制细胞器”中发现的磷酸肌醇脂质，可保护病毒免受宿主的影响 细胞防御。所有这些活动都在其小20 kDa结构中编码。另一个策略 扩展功能蛋白含量是蛋白水解前体具有不同功能的功能 完全处理的对应物。例如，也发现3C是3CD蛋白的一部分，但3CD 蛋白质具有不同的蛋白质特异性，以及不同的RNA和脂质结合亲和力。 3CD 蛋白质也具有3D结构域； 3D蛋白是RNA依赖性的RNA聚合酶，但3CD DO 不具有聚合酶活性。本身，3CD还上调了磷酸肌醇脂质的产生 并诱导膜增殖，对复制细胞器生物发生重要的事件。如何 出现3CD的不同和紧急功能知之甚少。 X射线晶体结构表明 该3CD仅是3C和3D蛋白的复合材料，该蛋白会由一个小的柔性接头连接在一起。 我们建议结构动力学，即采样多种结构构象的能力，是 理解病毒蛋白功能的缺失成分。我们建议3C之间波动 许多构象，提供3C访问和协调其许多功能的能力，我们 建议3CD波动为不同的组成，从而提供了替代功能。这些 通过与RNA，脂质和蛋白质结合的相互作用，可以进一步修改动态游览 合作伙伴来协调病毒蛋白功能。我们将评估这些蛋白质结构动力学 通过溶液状态核磁共振光谱法提供了原子水平的细节 蛋白质运动从皮秒到第二时间尺度，并与这些研究补充 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会 例如，对于理性的抗病毒策略，通过寻找与替代蛋白结合的分子 构象和/或破坏功能至关重要的动作，如3D已验证。