| Summary: | Summary: Codon pair deoptimization is an efficient virus attenuation strategy, but the mechanism that leads to attenuation is unknown. The strategy involves synthetic recoding of viral genomes that alters the positions of synonymous codons, thereby increasing the number of suboptimal codon pairs and CpG dinucleotides in recoded genomes. Here we identify the molecular mechanism of codon pair deoptimization-based attenuation by studying recoded influenza A viruses. We show that suboptimal codon pairs cause attenuation, whereas the increase of CpG dinucleotides has no effect. Furthermore, we show that suboptimal codon pairs reduce both mRNA stability and translation efficiency of codon pair-deoptimized genes. Consequently, reduced protein production directly causes virus attenuation. Our study provides evidence that suboptimal codon pairs are major determinants of mRNA stability. Additionally, it demonstrates that codon pair bias can be used to increase mRNA stability and protein production of synthetic genes in many areas of biotechnology. : Codon pair deoptimization is a highly efficient virus attenuation strategy. It involves the recoding of viral genomes using underrepresented codon pairs. Groenke et al. show that suboptimal codon pairs are the primary cause of attenuation because they reduce mRNA stability, translation efficiency, and thus also protein production of recoded genes. Keywords: codon bias, codon pair bias, codon pair deoptimization, synthetic attenuated virus engineering, dinucleotide frequencies, recoding, influenza A virus, attenuation, mRNA stability
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