Daczkowski Courtney M, Goodwin Octavia Y, Dzimianski John V, Farhat Jonathan J, Pegan Scott D
Pharmaceutical and Biomedical Sciences Department, University of Georgia, Athens, Georgia, USA.
Pharmaceutical and Biomedical Sciences Department, University of Georgia, Athens, Georgia, USA
J Virol. 2017 Nov 14;91(23). doi: 10.1128/JVI.01067-17. Print 2017 Dec 1.
Middle East respiratory syndrome coronavirus (MERS-CoV) is an emerging human pathogen that is the causative agent for Middle East respiratory syndrome (MERS). With MERS outbreaks resulting in over 35% fatalities and now spread to 27 countries, MERS-CoV poses a significant ongoing threat to global human health. As part of its viral genome, MERS-CoV encodes a papain-like protease (PLpro) that has been observed to act as a deubiquitinase and deISGylase to antagonize type I interferon (IFN-I) immune pathways. This activity is in addition to its viral polypeptide cleavage function. Although the overall impact of MERS-CoV PLpro function is observed to be essential, difficulty has been encountered in delineating the importance of its separate functions, particularly its deISGylase activity. As a result, the interface of MERS-CoV and human interferon-stimulated gene product 15 (hISG15) was probed with isothermal calorimetry, which suggests that the C-terminal domain of hISG15 is principally responsible for interactions. Subsequently, the structure of MERS-CoV PLpro was solved to 2.4 Å in complex with the C-terminal domain of hISG15. Utilizing this structural information, mutants were generated that lacked appreciable deISGylase activity but retained wild-type deubiquitinase and peptide cleavage activities. Hence, this provides a new platform for understanding viral deISGylase activity within MERS-CoV and other CoVs. Coronaviruses, such as Middle East respiratory syndrome coronavirus (MERS-CoV), encode a papain-like protease (PLpro) that possesses the ability to antagonize interferon immune pathways through the removal of ubiquitin and interferon-stimulated gene product 15 (ISG15) from target proteins. The lack of CoV proteases with attenuated deISGylase activity has been a key obstacle in delineating the impact between deubiquitinase and deISGylase activities on viral host evasion and pathogenesis. Here, biophysical techniques revealed that MERS-CoV PLpro chiefly engages human ISG15 through its C-terminal domain. The first structure of MERS-CoV PLpro in complex with this domain exposed the interface between these two entities. Employing these structural insights, mutations were employed to selectively remove deISGylase activity with no appreciable impact on its other deubiquitinase and peptide cleavage biochemical properties. Excitingly, this study introduces a new tool to probe the pathogenesis of MERS-CoV and related viruses through the removal of viral deISGylase activity.
中东呼吸综合征冠状病毒（MERS-CoV）是一种新出现的人类病原体，是中东呼吸综合征（MERS）的致病因子。MERS疫情导致超过35%的死亡率，目前已传播到27个国家，MERS-CoV对全球人类健康构成了持续的重大威胁。作为其病毒基因组的一部分，MERS-CoV编码一种木瓜样蛋白酶（PLpro），该酶已被观察到作为一种去泛素酶和去ISG15酶来拮抗I型干扰素（IFN-I）免疫途径。这种活性是除其病毒多肽切割功能之外的。尽管观察到MERS-CoV PLpro功能的总体影响是必不可少的，但在确定其单独功能的重要性，特别是其去ISG15酶活性的重要性方面遇到了困难。因此，采用等温滴定量热法研究了MERS-CoV与人干扰素刺激基因产物15（hISG15）的相互作用界面，结果表明hISG15的C末端结构域主要负责相互作用。随后，解析了MERS-CoV PLpro与hISG15 C末端结构域复合物的结构，分辨率达到2.4 Å。利用这一结构信息，构建了缺乏明显去ISG15酶活性但保留野生型去泛素酶和肽切割活性的突变体。因此，这为理解MERS-CoV和其他冠状病毒内的病毒去ISG15酶活性提供了一个新平台。冠状病毒，如中东呼吸综合征冠状病毒（MERS-CoV），编码一种木瓜样蛋白酶（PLpro），该酶具有通过从靶蛋白上去除泛素和干扰素刺激基因产物15（ISG15）来拮抗干扰素免疫途径的能力。缺乏具有减弱的去ISG15酶活性的冠状病毒蛋白酶一直是确定去泛素酶和去ISG15酶活性对病毒宿主逃避和发病机制影响的关键障碍。在这里，生物物理技术表明，MERS-CoV PLpro主要通过其C末端结构域与人类ISG15结合。MERS-CoV PLpro与该结构域复合物的首个结构揭示了这两个实体之间的界面。利用这些结构见解，通过突变选择性地去除去ISG15酶活性，而对其其他去泛素酶和肽切割生化特性没有明显影响。令人兴奋的是，这项研究引入了一种新工具，通过去除病毒去ISG15酶活性来探究MERS-CoV和相关病毒的发病机制。
