Donnelly Michelle L L, Hughes Lorraine E, Luke Garry, Mendoza Heidi, Ten Dam Edwin, Gani David, Ryan Martin D
Centre for Biomolecular Sciences, School of Biology, Biomolecular Sciences Building, University of St Andrews, North Haugh, St Andrews KY16 9ST, UK1.
The University of Birmingham, The School of Chemistry, Edgbaston, Birmingham B15 2TT, UK2.
J Gen Virol. 2001 May;82(Pt 5):1027-1041. doi: 10.1099/0022-1317-82-5-1027.
The 2A/2B cleavage of aphtho- and cardiovirus 2A polyproteins is mediated by their 2A proteins 'cleaving' 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 (approximately 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-glucosiduronasesequence(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.
口疮病毒和心病毒2A多聚蛋白的2A/2B切割是由其2A蛋白在自身C末端的“切割”介导的。我们使用人工报告多聚蛋白系统分析了这种活性，该系统包含通过口蹄疫病毒（FMDV）2A连接到β-葡萄糖醛酸酶（GUS）的绿色荧光蛋白（GFP）——形成一个单一的、长的开放阅读框。放射性标记分布分析表明，体外翻译产物中很大一部分（约90%）是“切割”产物GUS和[GFP2A]的形式。已经提出了替代模型来解释这种“切割”活性：由宿主细胞蛋白酶进行的蛋白水解、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序列分析表明，“切割”发生在所有候选亲核残基都被取代的构建体中——天冬氨酸-12除外。然而，该残基在所有功能性“2A样”序列中并不保守。在昆虫病毒多聚蛋白、C型轮状病毒的NS34蛋白、锥虫属中的重复序列以及一种真细菌α-葡萄糖醛酸酶序列（嗜热栖热菌aguA）中鉴定出了“2A样”序列。除了真细菌α-葡萄糖醛酸酶2A样序列外，分析的所有2A样序列都具有活性（程度不同）。因此，这种蛋白质生物合成的控制方法很可能并不局限于小核糖核酸病毒科的成员：综上所述，这些数据提供了额外的证据，表明FMDV 2A和“2A样”序列都不是自蛋白水解元件。
