SARS 冠状病毒非结构蛋白质NSP3突变体构建及其对类泛素分子DUB活性

SARS冠状病毒(SARS-CoV) 非结构蛋白NSP3编码的木瓜蛋白酶样蛋白酶(PLpro)对泛素样分子(Ubl) 具有去泛素化酶(DUB)活性，但目前有关NSP3 DUB活性研究的报道甚少. 本研究构建包含Nsp3基因 N末端不同结构域的突变体，并检测NSP3及其一系列突变体对类泛素分子ISG15和SUMO所修饰蛋白质分子的作用特性. 实验结果表明，NSP3及其突变体NSP3AD，NSP3AE，NSP3AF具有一定的去ISG15活性，而其突变体NSP3AC则没有去ISG15 (DeISGylation) 活性. 研究结果提示，SARS NSP3具有一定的体内去ISG15活性，并且这种活性主要依赖于Nsp3基因编码的PLpro. 但SARS NSP3及其突变体NSP3AC，NSP3AD，NSP3AE和NSP3AF并不具有去SUMO (DeSUMOylation) 活性. SARS冠状病毒NSP3对类泛素样分子作用特性的研究为后续NSP3的生物学特性及其对干扰素通路的调控研究奠定了基础.

Deubiquitinatinase Activities to Ubiquitin-like Proteins of Nonstructral Protein 3 from SARS Coronavirus

Our previous studies revealed that SARS papain-like protease (PLpro), one of the key domains contained in nonstructural protein 3 (NSP3), was a viral deubiquitinase (DUB) that played critical role in regulation of innate immunity. However, the characteristics and function of NSP3 acting as DUB remains unclear. To investigate the deubiquitinase (DUB) activity of SARS-CoV NSP3 to the ubiquitin-like proteins of ISG15 and SUMO, we cloned the NSP3 gene and constructed several NSP3 gene mutation constructs with deletions of different domains in the N-terminal of NSP3 gene, and then the DeISGylation and DeSUMOylating activities were assayed. SARS-CoV NSP3 has deISGylation activity and the activity depends on the catalytic sites of PLpro. Deletion of the PLpro or the domains downstream of PLpro abolished the deISGylation activity. NSP3 and its mutants did not have any effects on the cellular SUMO conjugated proteins. These results demonstrated that SARS-CoV NSP3 had DUB activity to ubiquitin-like protein of ISG15, and might provide the foundamental clues for SARS CoV countermeasure against host innate immunity.