Mechanism of virus attenuation by codon pair deoptimization

Codon pair deoptimization (CPD), also known as synthetic attenuation virus engineering (SAVE), is an attenuation strategy that is based on genetic recoding of virus genomes. The strategy has the potential to revolutionize the development of viral vaccines, because one can rapidly produce efficacious and non-reverting virus vaccines. Yet, the mechanism behind attenuation is unknown. The recoding rearranges the positions of synonymous codons in viral genomes to create suboptimal codon pairs. However, CPD also results in an unintentional increase of the frequency of CpG dinucleotides in recoded sequences, because suboptimal codon pairs often contain CpG dinucleotides at the codon pair boundary. We systematically dissected the contribution of underrepresented codon pairs from that of CpG dinucleotides by studying a series of recoded influenza A virus mutants in which the two features were independently varied. We show unequivocally that suboptimal codon pairs were responsible for attenuation of recoded influenza viruses in tissue culture and in vivo, while an increase of CpG dinucleotides had no effect. Next, we identified how suboptimal codon pairs induce attenuation. We show that suboptimal codon pairs reduce mRNA stability and reduce translation efficiency of recoded codon pair- deoptimized genes. This in turn decreases protein output and directly causes virus attenuation. Several recent studies showed that codon optimality is a major determinant of mRNA stability. Here we demonstrate that the identity of codon pairs is another critical determinant of mRNA stability, which profoundly affects protein output and, ultimately, fitness of recoded viruses. Our work explains why exchanging the positions of synonymous codons can have dramatic consequences on gene expression of recoded genes. Furthermore, our work demonstrates that codon pair (de)optimization can be employed to modulate mRNA stability and protein output of synthetic genes for a broad range of biotechnological and pharmaceutical applications.密码子对去优化（Codon pair deoptimization, CPD），又称合成减毒病毒工程（synthetic attenuation virus engineering, SAVE），是一种基于病毒基因组遗传重编码的减毒策略。该策略有望推动病毒疫苗研发的革命性变革，因为其可快速制备高效且无回复突变的病毒疫苗。然而，其背后的减毒机制尚未明确。该重编码手段通过重排病毒基因组中同义密码子的位置，生成次优密码子对。但CPD同时会导致重编码序列中CpG二核苷酸（CpG dinucleotides）的频率意外升高，因为次优密码子对往往在密码子对边界处包含CpG二核苷酸。本研究通过对一系列经重编码的甲型流感病毒突变株开展研究，独立调控这两个特征，从而系统解析了低代表性密码子对与CpG二核苷酸各自的贡献。研究结果明确证实，次优密码子对是导致重编码流感病毒在细胞培养体系及活体动物体内减毒的核心原因，而CpG二核苷酸频率升高并无此效应。随后，本研究进一步阐明了次优密码子对诱导病毒减毒的具体机制。研究表明，次优密码子对会降低密码子对去优化基因的mRNA稳定性，并减弱其翻译效率。这进而会减少蛋白质表达量，直接引发病毒减毒。多项近期研究已表明，密码子最优性是决定mRNA稳定性的关键因素之一。本研究证实，密码子对的组成特征是影响mRNA稳定性的另一关键因素，其可深刻调控蛋白质表达量，并最终影响重编码病毒的适合度。本研究解释了为何交换同义密码子的位置会对重编码基因的表达产生显著影响。此外，本研究证实，密码子对（去）优化手段可用于调控合成基因的mRNA稳定性与蛋白质表达量，适用于众多生物技术与制药应用场景。