The 'cleavage' activities of foot-and-mouth disease virus 2A site-directed mutants and naturally occurring '2A-like' sequences
The 'cleavage' activities of foot-and-mouth disease virus 2A site-directed mutants and naturally occurring '2A-like' sequences
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DOI：
10.1099/0022-1317-82-5-1027
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发表时间：
2001-05-01
影响因子：
3.8
通讯作者：
Ryan, MD
中科院分区：
文献类型：
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作者：
Donnelly, MLL;Hughes, LE;Ryan, MD
口蹄疫病毒和心脏病毒2A多聚蛋白的2A/2B切割是由它们的2A蛋白在它们自己的C末端“去活”介导的。我们已经使用人工报告多聚蛋白系统分析了这种活性，所述人工报告多聚蛋白系统包括通过口蹄疫病毒（FMDV）2A连接到β-葡萄糖醛酸酶（GUS）的绿色荧光蛋白（GFP）-形成单个、长的、开放的阅读框。放射性标记分布的分析显示，高比例的体外翻译产物（类似于90%）是“切割”产物GUS和[GFP 2A]的形式。已经提出了替代模型来解释“切割”活性：宿主细胞蛋白酶的蛋白水解、自蛋白水解或翻译效应。为了研究这种切割事件的机制，使用编码定点突变体和天然存在的“2A样”序列的构建体来编程体外翻译系统并分析凝胶图谱。定点突变2A序列的分析表明，“切割”发生在所有候选亲核残基被取代的构建体中-天冬氨酸-12除外。然而，该残基在所有功能性“2A样”序列中不是保守的。在昆虫病毒多聚蛋白、C型轮状病毒的NS 34蛋白、锥虫属物种中的重复序列和真细菌α-葡糖苷酸酶序列（海栖热甲属aguA）中鉴定了“2A样”序列。除真细菌α-葡糖苷酸酶2A样序列外，所分析的所有2A样序列都有活性（不同程度）。因此，这种控制蛋白质生物发生的方法可能并不局限于小核糖核酸病毒科的成员。总之，这些数据提供了额外的证据，即FMDV 2A或“2A样”序列都不是自蛋白水解元件。
The 2A/2B cleavage of aphtho- and cardiovirus 2A polyproteins is mediated by their 2A proteins 'deaving' at their own C termini, We have analysed this activity using artificial reporter polyprotein systems comprising green fluorescent protein (GFP) linked via foot-and-mouth disease virus (FMDV) 2A to beta -glucuronidase (GUS) -forming a single, long, open reading frame. Analysis of the distribution of radiolabel showed a high proportion of the in vitro translation products (similar to 90%) were in the form of the 'cleavage' products GUS and [GFP2A], Alternative models have been proposed to account for the 'cleavage' activity: proteolysis by a host-cell proteinase, autoproteolysis or a translational effect. To investigate the mechanism of this cleavage event constructs encoding site-directed mutant and naturally occurring '2A-like' sequences were used to program in vitro translation systems and the gel profiles analysed. Analysis of site-directed mutant 2A sequences showed that 'cleavage' occurred in constructs in which all the candidate nucleophilic residues were substituted - with the exception of aspartate-12, This residue is not, however, conserved amongst all functional '2A-like' sequences. '2A-like' sequences were identified within insect virus polyproteins, the NS34 protein of type C rotaviruses, repeated sequences in Trypanosoma spp, and a eubacterial alpha -glucosiduronase sequence (Thermatoga maritima aguA), All of the 2A-like sequences analysed were active (to various extents), other than the eubacterial alpha -glucosiduronase 2A-like sequence. This method of control of protein biogenesis may well not, therefore, be confined to members of the Picornaviridae, Taken together, these data provide additional evidence that neither FMDV 2A nor '2A-like' sequences are autoproteolytic elements.