Lindner Holger A, Fotouhi-Ardakani Nasser, Lytvyn Viktoria, Lachance Paule, Sulea Traian, Ménard Robert
Biotechnology Research Institute, National Research Council of Canada, 6100 Royalmount Avenue, Montreal, Quebec, Canada H4P 2R2.
J Virol. 2005 Dec;79(24):15199-208. doi: 10.1128/JVI.79.24.15199-15208.2005.
The severe acute respiratory syndrome coronavirus papain-like protease (SARS-CoV PLpro) is involved in the processing of the viral polyprotein and, thereby, contributes to the biogenesis of the virus replication complex. Structural bioinformatics has revealed a relationship for the SARS-CoV PLpro to herpesvirus-associated ubiquitin-specific protease (HAUSP), a ubiquitin-specific protease, indicating potential deubiquitinating activity in addition to its function in polyprotein processing (T. Sulea, H. A. Lindner, E. O. Purisima, and R. Menard, J. Virol. 79:4550-4551, 2005). In order to confirm this prediction, we overexpressed and purified SARS-CoV PLpro (amino acids [aa]1507 to 1858) from Escherichia coli. The purified enzyme hydrolyzed ubiquitin-7-amino-4-methylcoumarin (Ub-AMC), a general deubiquitinating enzyme substrate, with a catalytic efficiency of 13,100 M(-1)s(-1), 220-fold more efficiently than the small synthetic peptide substrate Z-LRGG-AMC, which incorporates the C-terminal four residues of ubiquitin. In addition, SARS-CoV PLpro was inhibited by the specific deubiquitinating enzyme inhibitor ubiquitin aldehyde, with an inhibition constant of 210 nM. The purified SARS-CoV PLpro disassembles branched polyubiquitin chains with lengths of two to seven (Ub2-7) or four (Ub4) units, which involves isopeptide bond cleavage. SARS-CoV PLpro processing activity was also detected against a protein fused to the C terminus of the ubiquitin-like modifier ISG15, both in vitro using the purified enzyme and in HeLa cells by coexpression with SARS-CoV PLpro (aa 1198 to 2009). These results clearly establish that SARS-CoV PLpro is a deubiquitinating enzyme, thereby confirming our earlier prediction. This unexpected activity for a coronavirus papain-like protease suggests a novel viral strategy to modulate the host cell ubiquitination machinery to its advantage.
严重急性呼吸综合征冠状病毒木瓜样蛋白酶（SARS-CoV PLpro）参与病毒多聚蛋白的加工，从而有助于病毒复制复合体的生物合成。结构生物信息学研究表明，SARS-CoV PLpro与泛素特异性蛋白酶——疱疹病毒相关泛素特异性蛋白酶（HAUSP）存在关联，这表明其除了在多聚蛋白加工中发挥作用外，还可能具有去泛素化活性（T. Sulea、H. A. Lindner、E. O. Purisima和R. Menard，《病毒学杂志》79:4550 - 4551，2005年）。为了证实这一预测，我们从大肠杆菌中过表达并纯化了SARS-CoV PLpro（氨基酸[aa]1507至1858）。纯化后的酶能够水解泛素-7-氨基-4-甲基香豆素（Ub-AMC），这是一种通用的去泛素化酶底物，其催化效率为13100 M⁻¹s⁻¹，比包含泛素C末端四个残基的小合成肽底物Z-LRGG-AMC高效220倍。此外，SARS-CoV PLpro被特异性去泛素化酶抑制剂泛素醛抑制，抑制常数为210 nM。纯化的SARS-CoV PLpro能够拆解长度为2至7个（Ub2 - 7）或4个（Ub4）单位的分支多聚泛素链，这涉及异肽键的切割。在体外使用纯化的酶以及在HeLa细胞中通过与SARS-CoV PLpro（aa 1198至2009）共表达，均检测到SARS-CoV PLpro对与泛素样修饰因子ISG15的C末端融合的蛋白质具有加工活性。这些结果清楚地表明SARS-CoV PLpro是一种去泛素化酶，从而证实了我们之前的预测。冠状病毒木瓜样蛋白酶的这种意外活性提示了一种新的病毒策略，即利用宿主细胞的泛素化机制为其自身谋利。
