Pioneering discovery: US3-phosphorylated sites on VP22 orchestrate duck plague virus release and pathogenicity.

Duck plague virus (DPV), a member of the Alphaherpesvirinae subfamily, causes severe infectious disease in waterfowl and Anseriformes birds. Although the VP22 protein (encoded by the UL49 gene) is known to play a pivotal role in secondary viral envelopment, its post-translational modifications and regulatory mechanisms during the viral life cycle remain poorly understood. In this study, we found that the conserved serine/threonine kinase US3 of DPV phosphorylates VP22. We identified eight serine residues (Ser37, Ser66, Ser68, Ser82, Ser114, Ser198, Ser226, and Ser227) as phosphorylation targets of the US3 kinase. This modification enhances the stability of the VP22 protein and positively regulates its expression. By constructing a phosphorylation-defective VP22 mutant virus, we found that impaired phosphorylation significantly inhibited viral particle release, reduced the accumulation of mRNAs for the majority of viral genes, and decreased viral replication efficiency in vitro. Animal challenge experiments further demonstrated that phosphorylation-defective VP22 attenuated viral replication in host tissues and markedly diminished viral virulence. Overall, our findings confirm that VP22 is a novel phosphorylation substrate of US3 in DPV and reveal its critical role in virion release and pathogenesis. These results provide new insights into herpesvirus pathogenic mechanisms and establish a theoretical basis for developing anti-herpesvirus therapeutics that target kinase-substrate interactions.