Coronavirus papain-like proteases (PLPs or PLpro), such as the one encoded in the genome of the infectious Middle East Respiratory Syndrome (MERS) virus, have multiple enzymatic activities that promote viral infection. PLpro acts as a protease and processes the large coronavirus polyprotein for virus replication. PLpro also functions as both a deubiquitinating (DUB) and deISGylating (deISG) enzyme and removes ubiquitin (Ub) and interferon-stimulated gene 15 (ISG15) from cellular proteins. Both DUB and deISG activities are implicated in suppressing innate immune responses; however, the precise role of each activity in this process is still unclear due in part to the difficulties in separating each activity. In this study, we determine the first structure of MERS PLpro in complex with the full-length human ISG15 to a resolution of 2.3 Å. This structure and available structures of MERS PLpro-Ub complexes were used as molecular guides to design PLpro mutants that lack either or both DUB/deISG activities. We tested 13 different PLpro mutants for protease, DUB, and deISG activitites using fluorescence-based assays. Results show that we can selectively modulate DUB activity at amino acid positions 1649 and 1653 while mutation of Val1691 or His1652 of PLpro to a positive charged residue completely impairs both DUB/deISG activities. These mutant enzymes will provide new functional tools for delineating the importance of DUB versus deISG activity in virus-infected cells and may serve as potential candidates for attenuating the MERS virus in vivo for modified vaccine design efforts.

中文翻译：

---

解偶联 MERS 病毒木瓜蛋白酶样蛋白酶的去糖化和去泛素化活性。


冠状病毒类木瓜蛋白酶（PLP 或 PLpro），例如传染性中东呼吸综合征 (MERS) 病毒基因组中编码的蛋白酶，具有多种促进病毒感染的酶活性。 PLpro 充当蛋白酶并处理大的冠状病毒多蛋白以进行病毒复制。 PLpro 还充当去泛素化 (DUB) 和去ISG 化 (deISG) 酶，并从细胞蛋白质中去除泛素 (Ub) 和干扰素刺激基因 15 (ISG15)。 DUB 和 deISG 活性均与抑制先天免疫反应有关；然而，每个活动在此过程中的确切作用仍不清楚，部分原因是很难区分每个活动。在这项研究中，我们确定了 MERS PLpro 与全长人类 ISG15 复合物的第一个结构，分辨率为 2.3 Å。 MERS PLpro-Ub 复合物的这种结构和可用结构被用作设计缺乏 DUB/deISG 活性之一或两者的 PLpro 突变体的分子指导。我们使用基于荧光的测定法测试了 13 种不同 PLpro 突变体的蛋白酶、DUB 和 deISG 活性。结果表明，我们可以选择性地调节氨基酸位置 1649 和 1653 处的 DUB 活性，而将 PLpro 的 Val1691 或 His1652 突变为带正电残基则完全损害 DUB/deISG 活性。这些突变酶将为描述病毒感染细胞中 DUB 与 deISG 活性的重要性提供新的功能工具，并可作为在体内减弱 MERS 病毒的潜在候选者，以改进疫苗设计工作。