Donnelly Michelle L L, Luke Garry, Mehrotra Amit, Li Xuejun, Hughes Lorraine E, 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):1013-1025. doi: 10.1099/0022-1317-82-5-1013.
The 2A region of the aphthovirus foot-and-mouth disease virus (FMDV) polyprotein is only 18 aa long. A 'primary' intramolecular polyprotein processing event mediated by 2A occurs at its own C terminus. FMDV 2A activity was studied in artificial polyproteins in which sequences encoding reporter proteins flanked the 2A sequence such that a single, long, open reading frame was created. The self-processing properties of these artificial polyproteins were investigated and the co-translational 'cleavage' products quantified. The processing products from our artificial polyprotein systems showed a molar excess of 'cleavage' product N-terminal of 2A over the product C-terminal of 2A. A series of experiments was performed to characterize our in vitro translation systems. These experiments eliminated the translational or transcriptional properties of the in vitro systems as an explanation for this imbalance. In addition, the processing products derived from a control construct encoding the P1P2 region of the human rhinovirus polyprotein, known to be proteolytically processed, were quantified and found to be equimolar. Translation of a construct encoding green fluorescent protein (GFP), FMDV 2A and beta-glucuronidase, also in a single open reading frame, in the presence of puromycin, showed this antibiotic to be preferentially incorporated into the [GFP2A] translation product. We conclude that the discrete translation products from our artificial polyproteins are not produced by proteolysis. We propose that the FMDV 2A sequence, rather than representing a proteolytic element, modifies the activity of the ribosome to promote hydrolysis of the peptidyl(2A)-tRNA(Gly) ester linkage, thereby releasing the polypeptide from the translational complex, in a manner that allows the synthesis of a discrete downstream translation product to proceed. This process produces a ribosomal 'skip' from one codon to the next without the formation of a peptide bond.
口蹄疫病毒（FMDV）多聚蛋白的2A区域仅18个氨基酸长。由2A介导的“主要”分子内多聚蛋白加工事件发生在其自身的C末端。在人工多聚蛋白中研究了FMDV 2A活性，其中编码报告蛋白的序列位于2A序列两侧，从而产生单个长开放阅读框。研究了这些人工多聚蛋白的自我加工特性，并对共翻译“切割”产物进行了定量。我们人工多聚蛋白系统的加工产物显示，2A N末端的“切割”产物摩尔数超过2A C末端的产物。进行了一系列实验来表征我们的体外翻译系统。这些实验排除了体外系统的翻译或转录特性作为这种不平衡的解释。此外，对编码人鼻病毒多聚蛋白P1P2区域的对照构建体的加工产物进行了定量，已知该区域可进行蛋白水解加工，结果发现其为等摩尔。在嘌呤霉素存在下，对同样在单个开放阅读框中编码绿色荧光蛋白（GFP）、FMDV 2A和β-葡萄糖醛酸酶的构建体进行翻译，结果表明这种抗生素优先掺入[GFP2A]翻译产物中。我们得出结论，我们人工多聚蛋白的离散翻译产物不是由蛋白水解产生的。我们提出，FMDV 2A序列并非代表蛋白水解元件，而是修饰核糖体的活性，以促进肽基（2A）-tRNA（Gly）酯键的水解，从而从翻译复合物中释放多肽，其方式是允许合成离散的下游翻译产物继续进行。这个过程导致核糖体从一个密码子“跳跃”到下一个密码子，而不形成肽键。
