Phosphorylation of T425 and methylation of R426 synergistically regulate IBDV VP1 function and viral replication

Post-translational modifications (PTMs) are essential regulators of viral protein function and replication efficiency. Infectious bursal disease virus (IBDV) polymerase VP1 has been reported to undergo multiple PTMs; however, the interplay among different modifications remains poorly understood. In this study, we identified a novel phosphorylation site at threonine 425 (T425) adjacent to the previously characterized arginine 426 (R426) methylation site of VP1. Mass spectrometry and phospho-specific antibodies confirmed phosphorylation at T425 during IBDV replication. Functional assays demonstrated that phosphorylation at T425 enhances VP1 polymerase activity and promotes viral replication, whereas mutation of this site markedly impaired viral growth. Inhibition experiments further indicated that inhibitor IX significantly inhibit T425 phosphorylation. Importantly, we revealed a synergistic relationship between T425 phosphorylation and PRMT5-mediated R426 methylation, as loss of either modification attenuated the other. Recombinant IBDV harboring the T425A/R426A double mutation exhibited significantly reduced replication capacity compared to single mutants. Together, our findings uncover a previously unrecognized crosstalk between adjacent phosphorylation and methylation sites in VP1, providing new insights into the fine-tuned regulation of IBDV replication and offering potential targets for antiviral strategies and vaccine development.