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SARS冠状病毒基因组中非结构基因nsp3编码的木瓜样蛋白酶(PLpro)在病毒基因组复制及逃避宿主天然免疫中发挥重要作用,是研发抗病毒药物的重要靶标.SARS冠状病毒PLpro是一种病毒编码的去泛素化酶(DUB).为深入研究SARS冠状病毒PLpro对泛素样分子(ubiquitin-likeprotein,UBL)的DUB特性,本研究构建缺失PLpro N末端泛素样结构域(Ubl)和下游跨膜结构域(TM)的PLpro构建体(constructs),并构建3种缺失蛋白酶催化活性的突变体,检测PLpro对泛素样分子干扰素刺激基因15(ISG15)及SUMO-1的作用.实验结果表明,PLpro和PLpro-TM在细胞内具有很强的去ISG(DeISGylation)活性;缺失PLpro N末端泛素样结构域(Ubl)对PLpro的去ISG15活性没有影响;对PLpro蛋白酶活性位点C1651和H1812突变后,PLpro-TM的去ISG15活性消失,而对D1826位点突变后不影响此活性.PLpro不具有去SUMO(DeSUMOylation)活性,而PLpro-TM具有一定的去SUMO活性;PLpro催化活性相关的3个关键氨基酸残基Cys-His-Asp突变后对去SUMO活性有一定的影响.研究结果提示,SARS PLpro除了具有DUB的活性,还具有体内去ISG活性和去SUMO活性;PLpro蛋白酶活性与其去ISG活性之间有一定相关性;PLpro去SUMO-1活性具有TM依赖性.SARS冠状病毒PLpro对泛素样分子作用特性的研究为阐明病毒逃避宿主天然免疫机制和开发新型抗病毒药物提供重要的理论依据.
The papain-like protein (PLpro) encoded by the non-structural gene nsp3 in the SARS coronavirus genome plays an important role in viral genome replication and escape from host innate immunity and is an important target for developing antiviral drugs.SARS coronavirus PLpro is a virus-encoded To study the DUB characteristics of ubiquitin-likeprotein (UBL) in SARS coronavirus (PLpro), this study constructs a retroviral vector expressing Ubl with N-terminal ubiquitin-like domain (Ubl) PLpro constructs of the membrane domain (TM) were constructed and three mutants that lack the protease catalytic activity were constructed to test the effect of PLpro on the ubiquitin-like interferon-stimulating gene 15 (ISG15) and SUMO-1. The results showed that PLpro and PLpro-TM had a strong activity of de-ISG (DeISGylation) in cells. Lack of PLpro N-terminal ubiquitin-like domain (Ubl) had no effect on the activity of deproteinized ISG15 of PLpro. PLpro protease activity sites C1651 and After H1812 mutation, the disappearance of ISG15 activity of PLpro-TM disappeared, while it did not affect this activity after mutation of D1826 site.PLpro did not have the activity of de SUMO (DeSUMOylation), while PLpro-TM had some activity of de-SUMO; PLpro catalytic activity The mutation of Cys-His-Asp, a key amino acid residue of 3, had some effects on the activity of SUMO.The results suggest that SARS PLpro has the activities of in vitro GDNF removal and de-SUMO addition to DUB activity; PLpro protease activity And its activity to go to ISG have a certain correlation; PLpro to SUMO-1 activity has a TM-dependent.SARS coronavirus PLpro study on the role of ubiquitin-like molecules in order to elucidate the virus to evade host innate immune mechanisms and the development of new antiviral drugs Important theoretical basis.