Effects of synonymous codons with optimization / deoptimization in nucleoprotein (NP) gene of influenza A virus on interaction between NP and tripartite motif protein 25 (TRIM25)

Nucleoprotein (NP), which performs multiple functions, participates in the life cycle of the influenza A virus (IAV). Although the synonymous codon usage of IAV NP exhibits a relatively variable pattern, it demonstrates a significant bias. Here, we first validated the directional interaction between IAV NP and tripartite motif protein 25 (TRIM25). Using the NP coding sequence of IAV PR8, we replaced all codon positions in the wild type NP coding sequence with either favorable or rare synonymous codons to construct two NP mutants: NPoptimal and NPrare. Compared to the mRNA stability and translation efficiency of the wild type NP, both NP mutants exhibit significant impairments, particularly NPoptimal. Furthermore, in comparison to the interaction between TRIM25 and wild type IAV NP, NPoptimal completely loses this biological function, whereas NPrare still binds to TRIM25, albeit with significant impairment. Moreover, the forward segment of IAV NP, identified as having a specific interaction with TRIM25, can be significantly impaired due to changes in synonymous codons. To some extent, these results address the question of why a strong usage bias of synonymous codons persists in the IAV NP gene, despite the variability in synonymous codon usage. Understanding the changes in synonymous codons that influence mRNA stability, translation speed, and folding-function provides new insights into the evolutionary mechanisms of IAV NP.