The autocatalytic release of a putative RNA virus transcription factor from its polyprotein precursor involves two paralogous papain-like proteases that cleave the same peptide bond.
The autocatalytic release of a putative RNA virus transcription factor from its polyprotein precursor involves two paralogous papain-like proteases that cleave the same peptide bond.
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DOI：
10.1074/jbc.m104097200
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发表时间：
2001-08-31
期刊：
影响因子：
--
通讯作者：
Gorbalenya AE
中科院分区：
文献类型：
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作者：
Ziebuhr J;Thiel V;Gorbalenya AE
冠状病毒最大的复制蛋白在禽传染性支气管炎病毒（IBV）中被称为p195，在小鼠肝炎病毒中被称为p210 （p240）。在IBV中，一种含锌指的木瓜蛋白酶（PLpro）和在小鼠肝炎病毒中，两种相似的PLpro （PL1pro和PL2pro）可自催化地从前体pp1a和pp1ab中释放。含有plpro的蛋白最近被认为与控制冠状病毒亚基因组mRNA合成（转录）有关。通过比较序列分析，我们现在发现所有测序的冠状病毒各自的蛋白两侧都有两个保守的PL1pro切割位点，并且具有复杂的（多）结构域组织，PL1pro在IBV中失活。在此基础上，详细研究了人冠状病毒229ep195 /p210 N末端的加工过程。首先，在人冠状病毒229e感染的细胞中鉴定出一种87 kda蛋白（p87），该蛋白来源于p195/p210上游的pp1a/pp1ab区。其次，体外合成的代表pp1a不同部分的蛋白质在预测位点进行自催化处理。令人惊讶的是，PL1pro和PL2pro都在p87和p195/p210之间分裂。pl1pro介导的分裂缓慢且被PL2pro的非蛋白水解活性显著抑制。相比之下，PL2pro，其蛋白水解活性和特异性在本研究中被确定，在上游结构域存在的情况下有效地切割同一位点。第三，我们观察到重叠的底物特异性与PL1pro和PL2pro的平行进化之间存在相关性。总的来说，我们的研究结果表明，p195/p210的自动处理机制在冠状病毒中可能是保守的，在某种程度上是以前没有意识到的，其中PL2pro发挥了主要作用。冠状病毒的一个大子集可能使用两种蛋白酶来切割相同的位点，从而以一种独特的方式调节病毒基因组的表达。
The largest replicative protein of coronaviruses is known as p195 in the avian infectious bronchitis virus (IBV) and p210 (p240) in the mouse hepatitis virus. It is autocatalytically released from the precursors pp1a and pp1ab byone zinc finger-containing papain-like protease (PLpro) in IBV and by two paralogous PLpros, PL1pro and PL2pro, in mouse hepatitis virus. The PLpro-containing proteins have been recently implicated in the control of coronavirus subgenomic mRNA synthesis (transcription). By using comparative sequence analysis, we now show that the respective proteins of all sequenced coronaviruses are flanked by two conserved PLpro cleavage sites and share a complex (multi)domain organization with PL1pro being inactivated in IBV. Based upon these predictions, the processing of the human coronavirus 229E p195/p210 N terminus was studied in detail. First, an 87-kDa protein (p87), which is derived from a pp1a/pp1ab region immediately upstream of p195/p210, was identified in human coronavirus 229E-infected cells. Second, in vitro synthesized proteins representing different parts of pp1a were autocatalytically processed at the predicted site. Surprisingly, both PL1pro and PL2pro cleaved between p87 and p195/p210. The PL1pro-mediated cleavage was slow and significantly suppressed by a non-proteolytic activity of PL2pro. In contrast, PL2pro, whose proteolytic activity and specificity were established in this study, cleaved the same site efficiently in the presence of the upstream domains. Third, a correlation was observed between the overlapping substrate specificities and the parallel evolution of PL1pro and PL2pro. Collectively, our results imply that the p195/p210 autoprocessing mechanisms may be conserved among coronaviruses to an extent not appreciated previously, with PL2pro playing a major role. A large subset of coronaviruses may employ two proteases to cleave the same site(s) and thus regulate the expression of the viral genome in a unique way.