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Abstract：
We engineered Saccharomyces cerevisiae to express structural proteins of foot-and-mouth disease virus (FMDV) and produce virus-like particles (VLPs). The gene, which encodes four structural capsid proteins (VP0 (VP4 and VP2), VP3, and VP1), followed by a translational \"ribosomal skipping\" sequence consisting of 2A and protease 3C, was codon-optimized and chemically synthesized. The cloned gene was used to transform S. cerevisiae 2805 strain. Western blot analysis revealed that the polyprotein consisting of VP0, VP3, and VP1 was processed into the discrete capsid proteins. Western blot analysis of 3C confirmed the presence of discrete 3C protein, suggesting that the 2A sequence functioned as a \"ribosomal skipping\" signal in the yeast for an internal re-initiation of 3C translation from a monocistronic transcript, thereby indicating polyprotein processing by the discrete 3C protease. Moreover, a band corresponding to only VP2, which was known to be non-enzymatically processed from VP0 to both VP4 and VP2 during viral assembly, further validated the assembly of processed capsid proteins into VLPs. Electron microscopy showed the presence of the characteristic icosahedral VLPs. Our results clearly demonstrate that S. cerevisiae processes the viral structural polyprotein using a viral 3C protease and the resulting viral capsid subunits are assembled into virion particles. KEY POINTS: • Ribosomal skipping by self-cleaving FMDV peptide in S. cerevisiae. • Proteolytic processing of a structural polyprotein from a monocistronic transcript. • Assembly of the processed viral capsid proteins into a virus-like particle.
摘要：
我们设计了酿酒酵母以表达口蹄疫病毒（FMDV）的结构蛋白并产生病毒样颗粒（VLP）。基因，编码四种结构衣壳蛋白（VP0（VP4和VP2），VP3和VP1),随后是由2A和蛋白酶3C组成的翻译“核糖体跳跃”序列，密码子优化和化学合成。克隆的基因用于转化酿酒酵母2805菌株。Western印迹分析显示,由VP0、VP3和VP1组成的多蛋白被加工成离散的衣壳蛋白。3C的蛋白质印迹分析证实了离散3C蛋白的存在，表明2A序列在酵母中充当“核糖体跳跃”信号，用于从单顺反子转录物内部重新开始3C翻译，从而表明通过离散3C蛋白酶的多蛋白加工。此外，一条仅对应于VP2的条带，已知在病毒组装过程中从VP0非酶处理到VP4和VP2，进一步验证了加工衣壳蛋白组装成VLP。电子显微镜显示存在特征性二十面体VLP。我们的结果清楚地表明,酿酒酵母使用病毒3C蛋白酶处理病毒结构多蛋白,并且所得的病毒衣壳亚基被组装成病毒体颗粒。关键点:•酿酒酵母中通过自切割FMDV肽的核糖体跳跃。•来自单顺反子转录物的结构多蛋白的蛋白水解加工。•将经加工的病毒衣壳蛋白组装成病毒样颗粒。
