Mouse hepatitis virus 3C-like protease cleaves a 22-kilodalton protein from the open reading frame 1a polyprotein in virus-infected cells and in vitro
Mouse hepatitis virus 3C-like protease cleaves a 22-kilodalton protein from the open reading frame 1a polyprotein in virus-infected cells and in vitro
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DOI：
10.1128/jvi.72.3.2265-2271.1998
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发表时间：
1998-03-01
影响因子：
5.4
通讯作者：
Denison, MR
中科院分区：
文献类型：
--
作者：
Lu, XT;Sims, AC;Denison, MR
小鼠肝炎病毒（MHV）的3C样蛋白酶（3CLpro）被预测切割803-kDa基因I多聚蛋白中的至少11个位点，导致蛋白酶、聚合酶和解旋酶蛋白的成熟。然而，这些切割位点中的大多数尚未被实验证实，并且这些蛋白尚未在体外或在病毒感染的细胞中被鉴定，我们使用特异性抗体来鉴定和表征MHV A59感染的DBT细胞中基因1表达的22-kDa蛋白（p1 a-22），翻译后立即开始从多聚蛋白加工p1 a-22，但有些加工持续了几个小时，从MIN感染细胞中纯化的p1 a-22的氨基末端测序显示，它在推定的3CLpro切割位点Gln_Ser（4014）处被切割（其中下划线表示切割位点），其位于3CLpro结构域和开放阅读框（ORF）1a的末端之间，基因1的该区域的亚克隆用于体外表达含有一个或多个3CLpro切割位点的多肽，重组3CLpro在体外切割这些底物证实了p1 a-22的氨基末端切割发生在Gln_Ser（4014）。我们证明了p1 a-22蛋白的羧基端切割发生在Gln_Asn（4208），这一序列没有被预测为MHV 3CLpro的切割位点。我们的结果证明了重组MW 3CLpro在鉴定和确认基因1多聚蛋白内的切割位点方面的有用性。基于我们的研究结果，我们预测，至少有7个成熟的蛋白质处理的ORF la多聚蛋白的3CLpro，并建议，额外的非典型切割位点可以使用3CLpro在基因1多聚蛋白的加工过程中。
The 3C-like proteinase (3CLpro) of mouse hepatitis virus (MHV) is predicted to cleave at least 11 sites in the 803-kDa gene I polyprotein, resulting in maturation of proteinase, polymerase, and helicase proteins, However, most of these cleavage sites have not been experimentally confirmed and the proteins have not been identified in vitro or in virus-infected cells, We used specific antibodies to identify and characterize a 22-kDa protein (p1a-22) expressed from gene 1 in MHV A59-infected DBT cells, Processing of p1a-22 from the polyprotein began immediately after translation, but some processing continued for several hours, Amino-terminal sequencing of p1a-22 purified from MIN-infected cells showed that it was cleaved at a putative 3CLpro cleavage site, Gln_Ser(4014) (where the underscore indicates the site of cleavage), that is located between the 3CLpro domain and the end of open reading frame (ORF) la, Subclones of this region of gene 1 were used to express polypeptides in vitro that contained one or more 3CLpro cleavage sites, and cleavage of these substrates by recombinant 3CLpro in vitro confirmed that amino-terminal cleavage of p1a-22 occurred at Gln_Ser(4014). We demonstrated that the carboxy-terminal cleavage of the p1a-22 protein occurred at Gln_Asn(4208), a sequence that had not been predicted as a site for cleavage by MHV 3CLpro, Our results demonstrate the usefulness of recombinant MW 3CLpro in identifying and confirming cleavage sites within the gene 1 polyprotein. Based on our results, we predict that at least seven mature proteins are processed from the ORF la polyprotein by 3CLpro and suggest that additional noncanonical cleavage sites may be used by 3CLpro during processing of the gene 1 polyprotein.