PROTEOMICS ANALYSIS UNRAVELS THE FUNCTIONAL REPERTOIRE OF CORONAVIRUS NSP3

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 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时的启动信号和酶作用。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，病毒粒子组成的完整特征对于了解感染早期的动态很重要。质谱学和激酶图谱技术鉴定了近200种结合的宿主和病毒蛋白。我们使用已发表的相互作用数据来确定对病毒粒子形成具有潜在意义的连接中心。令人惊讶的是，最有可能连接的集线器不是病毒M蛋白，而是非结构蛋白3(NSP3)，它是通过质谱学鉴定的新的病毒粒子成分之一。基于新的实验数据和整个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NSP3功能结构域结构，它决定了该蛋白的相互作用能力。利用在大肠杆菌中表达的重组蛋白结构域，我们鉴定了NSP3的另外两个RNA结合域。其中一个结构域位于先前描述的SARS特有结构域中，并且有一个核酸伴侣样结构域位于木瓜蛋白酶样酶结构域的下游。我们还鉴定了一个新的半胱氨酸配位的金属离子结合结构域。分析了域之间的相互作用和剩余的，到目前为止尚未表征的域的临时功能注释。总体而言，这里调查的数据集合描绘了一幅更完整的图景，将nsp3作为病毒蛋白质处理机制的保守组件，它作为病毒粒子组件与病毒RNA密切相关。

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.