PROTEOMICS ANALYSIS UNRAVELS THE FUNCTIONAL REPERTOIRE OF CORONAVIRUS NSP3

这个子项目是许多研究子项目中的一个 由NIH/NCRR资助的中心赠款提供的资源。子项目和 研究者（PI）可能从另一个NIH来源获得主要资金， 因此可以在其他CRISP条目中表示。所列机构为 研究中心，而研究中心不一定是研究者所在的机构。 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和酶的作用。在病毒装配期间包装的蛋白质随后可在感染时形成攻击和宿主操纵的第一线。因此，病毒体组分的完整表征对于理解感染早期阶段的动力学是重要的。质谱和激酶分析技术鉴定了近200种掺入的宿主和病毒蛋白。我们使用已发表的相互作用数据来确定对病毒体形成具有潜在意义的连接中心。令人惊讶的是，具有最多潜在连接的枢纽不是病毒M蛋白，而是非结构蛋白3（nsp 3），这是质谱法鉴定的新型病毒体组分之一。基于新的实验数据和冠状病毒科的生物信息学分析，我们提出了一个更高分辨率的nsp 3功能域结构，决定了这种蛋白质的相互作用能力。利用大肠杆菌中表达的重组蛋白结构域，我们确定了两个额外的RNA结合结构域的nsp 3。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。我们还确定了一个新的半胱氨酸配位的金属离子结合域。域间的相互作用和临时功能注释的剩余，迄今未表征域的分析。总的来说，这里调查的数据集合描绘了一个更完整的图片nsp 3作为一个保守的组成部分的病毒蛋白质加工机制，这是密切相关的病毒RNA在其作为一个病毒粒子组件的作用。

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.