PROTEOMICS ANALYSIS UNRAVELS THE FUNCTIONAL REPERTOIRE OF CORONAVIRUS NSP3

蛋白质组学分析揭示了冠状病毒 NSP3 的功能库

• 批准号: 8171341
• 负责人: PETER KUHN
• 金额: $ 0.08万
• 依托单位: UNIVERSITY OF WASHINGTON
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-09-01 至 2011-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8171341
• 关键词: Architecture; Bioinformatics; Cells; Computer Retrieval of Information on Scientific Projects Database; Coronaviridae; Coronavirus; Coronavirus Infections; Cysteine; Data; Enzymes; Escherichia coli; Funding; Grant; Growth; Infection; Institution; Mass Spectrum Analysis; Metal Ion Binding; Molecular Chaperones; Nonstructural Protein; Nucleic Acids; Paint; Papain; Peptide Hydrolases; Phosphotransferases; Process; Proteins; Proteomics; Publishing; RNA Recognition Motif; Recombinant Proteins; Research; Research Personnel; Resolution; Resources; Role; Severe Acute Respiratory Syndrome; Signal Transduction; Source; Staging; Surveys; Techniques; Tertiary Protein Structure; United States National Institutes of Health; Viral; Viral Matrix Proteins; Viral Proteins; Virion; Virus Assembly; base; novel; viral RNA

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Severe acute respiratory syndrome (SARS) coronavirus infection and growth are dependent on initiating signaling and enzyme actions upon viral entry into the host cell. Proteins packaged during virus assembly may subsequently form the first line of attack and host manipulation upon infection. A complete characterization of virion components is therefore important to understanding the dynamics of early stages of infection. Mass spectrometry and kinase profiling techniques identified nearly 200 incorporated host and viral proteins. We used published interaction data to identify hubs of connectivity with potential significance for virion formation. Surprisingly, the hub with the most potential connections was not the viral M protein but the nonstructural protein 3 (nsp3), which is one of the novel virion components identified by mass spectrometry. Based on new experimental data and a bioinformatics analysis across the Coronaviridae, we propose a higher-resolution functional domain architecture for nsp3 that determines the interaction capacity of this protein. Using recombinant protein domains expressed in Escherichia coli, we identified two additional RNA-binding domains of nsp3. One of these domains is located within the previously described SARS-unique domain, and there is a nucleic acid chaperone-like domain located immediately downstream of the papain-like proteinase domain. We also identified a novel cysteine-coordinated metal ion-binding domain. Analyses of interdomain interactions and provisional functional annotation of the remaining, so-far-uncharacterized domains are presented. Overall, the ensemble of data surveyed here paint a more complete picture of nsp3 as a conserved component of the viral protein processing machinery, which is intimately associated with viral RNA in its role as a virion component.

该子项目是利用该技术的众多研究子项目之一 资源由 NIH/NCRR 资助的中心拨款提供。子项目及 研究者 (PI) 可能已从 NIH 的另一个来源获得主要资金， 因此可以在其他 CRISP 条目中表示。列出的机构是 对于中心来说，它不一定是研究者的机构。 严重急性呼吸综合征（SARS）冠状病毒的感染和生长依赖于病毒进入宿主细胞后启动信号传导和酶的作用。在病毒组装过程中包装的蛋白质随后可能形成感染后的第一道攻击线和宿主操纵。因此，病毒体成分的完整表征对于了解感染早期阶段的动态非常重要。质谱分析和激酶分析技术鉴定出近 200 种整合的宿主和病毒蛋白。我们使用已发表的相互作用数据来识别对病毒粒子形成具有潜在意义的连接中心。令人惊讶的是，最具潜在连接的枢纽不是病毒 M 蛋白，而是非结构蛋白 3 (nsp3)，它是通过质谱鉴定的新型病毒体成分之一。基于新的实验数据和对冠状病毒科的生物信息学分析，我们提出了一种更高分辨率的 nsp3 功能域架构，决定了该蛋白质的相互作用能力。使用大肠杆菌中表达的重组蛋白结构域，我们鉴定了 nsp3 的两个额外的 RNA 结合结构域。这些结构域之一位于先前描述的 SARS 独特结构域内，并且有一个核酸伴侣样结构域位于木瓜蛋白酶样蛋白酶结构域的紧下游。我们还鉴定了一个新的半胱氨酸配位金属离子结合域。提出了对域间相互作用的分析和剩余的、迄今为止尚未表征的域的临时功能注释。总体而言，这里调查的数据整体描绘了 nsp3 作为病毒蛋白加工机制的保守组成部分的更完整的图景，它与病毒 RNA 作为病毒体组成部分的作用密切相关。