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.

IMPORTANCE 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）的病原体。 MERS 疫情导致 35% 以上死亡，目前已蔓延至 27 个国家，对全球人类健康构成持续的重大威胁。作为病毒基因组的一部分，MERS-CoV 编码一种木瓜蛋白酶样蛋白酶 (PLpro)，据观察，该蛋白酶可充当去泛素酶和去ISG 酶，拮抗 I 型干扰素 (IFN-I) 免疫途径。该活性是其病毒多肽裂解功能的补充。尽管观察到 MERS-CoV PLpro 功能的总体影响至关重要，但在描述其单独功能的重要性时遇到了困难，特别是其去ISGylase 活性。结果，通过等温量热法探测了 MERS-CoV 和人干扰素刺激基因产物 15 (hISG15) 的界面，这表明 hISG15 的 C 末端结构域主要负责相互作用。随后，MERS-CoV PLpro 的结构被解析为与 hISG15 C 端结构域复合的 2.4 Å。利用该结构信息，产生了缺乏明显的去ISGylase活性但保留野生型去泛素酶和肽裂解活性的突变体。因此，这为了解 MERS-CoV 和其他 CoV 中的病毒 deISGylase 活性提供了一个新平台。

重要性冠状病毒，如中东呼吸综合征冠状病毒 (MERS-CoV)，编码一种木瓜蛋白酶样蛋白酶 (PLpro)，该蛋白酶具有通过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。目标蛋白质。缺乏 deISGylase 活性减弱的 CoV 蛋白酶一直是描述去泛素酶和 deISGylase 活性对病毒宿主逃避和发病机制影响的一个关键障碍。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 与该结构域复合的第一个结构暴露了这两个实体之间的界面。利用这些结构见解，利用突变选择性地去除去ISGylase活性，而对其其他去泛素酶和肽裂解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ISGylase活性来探究MERS-CoV和相关病毒的发病机制。