Human coronavirus 229E papain-like proteases have overlapping specificities but distinct functions in viral replication
Human coronavirus 229E papain-like proteases have overlapping specificities but distinct functions in viral replication
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DOI：
10.1128/jvi.02091-06
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发表时间：
2007-04-01
影响因子：
5.4
通讯作者：
Thiel, Volker
中科院分区：
文献类型：
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作者：
Ziebuhr, John;Schelle, Barbara;Thiel, Volker
冠状病毒异常大的RNA基因组的表达涉及多种调节机制，包括通过两种类型的半胱氨酸蛋白酶对大型复制酶多蛋白pp1a和pp1ab进行广泛的蛋白水解加工：胰凝乳蛋白酶样主要蛋白酶和木瓜蛋白酶样辅助蛋白酶(PL(pro)s)。在这里，我们通过两种旁系同源 PLpro 活性表征了人类冠状病毒 229E (HCoV-229E) 近氨基 pp1a/pp1ab 区域的蛋白水解加工。反向遗传学数据显示，替换 PL2(pro) 活性位点半胱氨酸是致命的。相比之下，PL1(pro) 活性被证明对于 HCoV-229E 病毒复制是可有可无的，尽管在细胞培养中数次传代后 PL1(pro) 活性位点替换恢复为野生型序列表明存在恢复 PL1(pro) 活性的选择压力。进一步的实验表明，PL1(pro)和PL2(pro)都能够切割nsp1-nsp2切割位点，而PL2(pro)切割该位点的效率较低。当nsp1-nsp2位点被短的自蛋白水解序列取代时，PL1(pro)阴性突变体基因型可以在细胞培养物中稳定维持，这表明观察到的PL1(pro)突变逆转的主要驱动力是有效nsp1-nsp2裂解的要求。数据表明，两种 HCoV-229E PLpro 平行同源物具有重叠的底物特异性，但在病毒复制中具有不同的功能。在两种蛋白酶活性严格控制的相互作用中，PL2(pro) 发挥着普遍且重要的蛋白水解作用，似乎在特定位点得到 PL1(pro) 旁系同源物的协助。讨论了主要 CoV 谱系中不同的氨基末端多蛋白加工途径的功能和进化意义。
Expression of the exceptionally large RNA genomes of CoVs involves multiple regulatory mechanisms, including extensive proteolytic processing of the large replicase polyproteins, pp1a and pp1ab, by two types of cysteine proteases: the chymotrypsin-like main protease and papain-like accessory proteases (PL(pro)s). Here, we characterized the proteolytic processing of the human coronavirus 229E (HCoV-229E) amino-proximal pp1a/pp1ab region by two paralogous PLpro activities. Reverse-genetics data revealed that replacement of the PL2(pro) active-site cysteine was lethal. By contrast, the PL1(pro) activity proved to be dispensable for HCoV-229E virus replication, although reversion of the PL1(pro) active-site substitution to the wild-type sequence after several passages in cell culture indicated that there was selection pressure to restore the PL1(pro) activity. Further experiments showed that both PL1(pro) and PL2(pro) were able to cleave the nsp1-nsp2 cleavage site, with PL2(pro) cleaving the site less efficiently. The PL1(pro)-negative mutant genotype could be stably maintained in cell culture when the nsp1-nsp2 site was replaced by a short autoproteolytic sequence, suggesting that the major driving force for the observed reversion of the PL1(pro) mutation was the requirement for efficient nsp1-nsp2 cleavage. The data suggest that the two HCoV-229E PLpro parallogs have overlapping substrate specificities but different functions in viral replication. Within the tightly controlled interplay of the two protease activities, PL2(pro) plays a universal and essential proteolytic role that appears to be assisted by the PL1(pro) paralog at specific sites. Functional and evolutionary implications of the differential amino-terminal polyprotein-processing pathways among the main CoV lineages are discussed.