X-ray Structural and Functional Studies of the Three Tandemly Linked Domains of Non-structural Protein 3 (nsp3) from Murine Hepatitis Virus Reveal Conserved Functions.
X-ray Structural and Functional Studies of the Three Tandemly Linked Domains of Non-structural Protein 3 (nsp3) from Murine Hepatitis Virus Reveal Conserved Functions.
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DOI：
10.1074/jbc.m115.662130
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发表时间：
2015-10-16
期刊：
影响因子：
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通讯作者：
Mesecar AD
中科院分区：
文献类型：
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作者：
Chen Y;Savinov SN;Mielech AM;Cao T;Baker SC;Mesecar AD
背景：冠状病毒nsp 3对病毒复制至关重要。结果：确定了MHV nsp 3的3个串联结构域的结构。结论：在MHV nsp 3中发现了一个新的结构域，MHV nsp 3的PLP 2结构域具有去泛素化和去ISGylating活性。意义：我们的研究为进一步研究冠状病毒nsp 3在病毒复制中的作用提供了平台。鼠肝炎病毒（MHV）长期以来一直是冠状病毒研究的模型系统。非结构蛋白3（nsp 3）是冠状病毒基因组中最大的非结构蛋白，它包含冠状病毒复制所需的多个功能结构域。尽管对MHV及其nsp 3结构域进行了大量的功能研究，但nsp 3中只有一个结构域，即小泛素样结构域1（Ubl 1）的结构已被确定。我们在这里报告的MHV nsp 3的三个串联连接的结构域的X射线结构，包括木瓜蛋白酶样蛋白酶2（PLP 2）催化结构域，泛素样结构域2（Ubl 2），和第三个域，我们称之为DPUP（域前Ubl 2和PLP 2）域。DPUP与严重急性呼吸综合征冠状病毒独特结构域C（SUD-C）具有密切的结构相似性，表明该结构域可能不是严重急性呼吸综合征冠状病毒所特有的。发现PLP 2催化结构域除了蛋白水解活性之外还具有去泛素化和去ISGylating异肽酶活性。生成了MHV PLP 2与泛素结合的计算衍生模型，并通过定点突变探测了泛素与PLP 2之间的潜在相互作用。这些研究大大扩展了我们对MHV nsp 3结构的了解，为进一步研究nsp 3结构域在MHV病毒复制中的作用提供了平台。
Background: Coronavirus nsp3 is essential for virus replication. Results: The structure of three tandemly linked domains of MHV nsp3 was determined. Conclusion: A new domain was discovered within MHV nsp3, and the PLP2 domain of MHV nsp3 possesses both deubiquitinating and deISGylating activities. Significance: Our studies provide a platform for further investigation of the role of coronaviral nsp3 in virus replication. Murine hepatitis virus (MHV) has long served as a model system for the study of coronaviruses. Non-structural protein 3 (nsp3) is the largest nsp in the coronavirus genome, and it contains multiple functional domains that are required for coronavirus replication. Despite the numerous functional studies on MHV and its nsp3 domain, the structure of only one domain in nsp3, the small ubiquitin-like domain 1 (Ubl1), has been determined. We report here the x-ray structure of three tandemly linked domains of MHV nsp3, including the papain-like protease 2 (PLP2) catalytic domain, the ubiquitin-like domain 2 (Ubl2), and a third domain that we call the DPUP (domain preceding Ubl2 and PLP2) domain. DPUP has close structural similarity to the severe acute respiratory syndrome coronavirus unique domain C (SUD-C), suggesting that this domain may not be unique to the severe acute respiratory syndrome coronavirus. The PLP2 catalytic domain was found to have both deubiquitinating and deISGylating isopeptidase activities in addition to proteolytic activity. A computationally derived model of MHV PLP2 bound to ubiquitin was generated, and the potential interactions between ubiquitin and PLP2 were probed by site-directed mutagenesis. These studies extend substantially our structural knowledge of MHV nsp3, providing a platform for further investigation of the role of nsp3 domains in MHV viral replication.