The papain-like protease PLpro is an essential coronavirus enzyme that is required for processing viral polyproteins to generate a functional replicase complex and enable viral spread 1 , 2 . PLpro is also implicated in cleaving proteinaceous post-translational modifications on host proteins as an evasion mechanism against host antiviral immune responses 3 – 5 . Here we perform biochemical, structural and functional characterization of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) PLpro (SCoV2-PLpro) and outline differences with SARS-CoV PLpro (SCoV-PLpro) in regulation of host interferon and NF-κB pathways. SCoV2-PLpro and SCoV-PLpro share 83% sequence identity but exhibit different host substrate preferences; SCoV2-PLpro preferentially cleaves the ubiquitin-like interferon-stimulated gene 15 protein (ISG15), whereas SCoV-PLpro predominantly targets ubiquitin chains. The crystal structure of SCoV2-PLpro in complex with ISG15 reveals distinctive interactions with the amino-terminal ubiquitin-like domain of ISG15, highlighting the high affinity and specificity of these interactions. Furthermore, upon infection, SCoV2-PLpro contributes to the cleavage of ISG15 from interferon responsive factor 3 (IRF3) and attenuates type I interferon responses. Notably, inhibition of SCoV2-PLpro with GRL-0617 impairs the virus-induced cytopathogenic effect, maintains the antiviral interferon pathway and reduces viral replication in infected cells. These results highlight a potential dual therapeutic strategy in which targeting of SCoV2-PLpro can suppress SARS-CoV-2 infection and promote antiviral immunity. Biochemical, structural and functional studies on the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) papain-like protease PLpro reveal that it regulates host antiviral responses by preferentially cleaving the ubiquitin-like interferon-stimulated gene 15 protein (ISG15) and identify this protease as a potential therapeutic target for coronavirus disease 2019 (COVID-19).

中文翻译：

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类木瓜蛋白酶调节 SARS-CoV-2 病毒传播和先天免疫

木瓜蛋白酶样蛋白酶 PLpro 是一种必需的冠状病毒酶，它是加工病毒多蛋白以产生功能性复制酶复合物并使病毒能够传播 1 、 2 所必需的。PLpro 还涉及切割宿主蛋白质上的蛋白质翻译后修饰，作为针对宿主抗病毒免疫反应的逃避机制 3 – 5。在这里，我们对严重急性呼吸综合征冠状病毒 2 (SARS-CoV-2) PLpro (SCoV2-PLpro) 进行生化、结构和功能表征，并概述了与 SARS-CoV PLpro (SCoV-PLpro) 在调节宿主干扰素和 NF 方面的差异-κB 通路。SCoV2-PLpro 和 SCoV-PLpro 具有 83% 的序列同一性，但表现出不同的宿主底物偏好；SCoV2-PLpro 优先切割泛素样干扰素刺激的基因 15 蛋白 (ISG15)，而 SCoV-PLpro 主要针对泛素链。SCoV2-PLpro 与 ISG15 复合的晶体结构揭示了与 ISG15 氨基末端泛素样结构域的独特相互作用，突出了这些相互作用的高亲和力和特异性。此外，在感染后，SCoV2-PLpro 有助于将 ISG15 从干扰素反应因子 3 (IRF3) 上切割下来，并减弱 I 型干扰素反应。值得注意的是，用 GRL-0617 抑制 SCoV2-PLpro 会削弱病毒诱导的细胞致病作用，维持抗病毒干扰素途径并减少感染细胞中的病毒复制。这些结果突出了一种潜在的双重治疗策略，其中靶向 SCoV2-PLpro 可以抑制 SARS-CoV-2 感染并促进抗病毒免疫。生化、