Identification of UL3.5 as a novel substrate of DPV US3 kinase whose phosphorylation at Ser64/Ser84 enhances protein stability and promotes viral release, and cell-to-cell spread.

Duck plague virus (DPV) is a major pathogen threatening waterfowl industry. As a conserved alphaherpesvirus kinase, DPV US3 participates in viral replication, while its functional substrates are largely uncharacterized. In this study, we verified that US3 directly binds and phosphorylates UL3.5 at Ser64 and Ser84. This post-translational modification inhibits protein degradation and prolongs the half-life of UL3.5. A phosphorylation-deficient recombinant virus was constructed, and phenotypic assays demonstrated that mutated virus had defective replication. Further tests indicated that UL3.5 phosphorylation mainly regulates viral genome replication, progeny release and cell-to-cell spread, rather than attachment and entry. Our findings expand the regulatory network of DPV US3 and uncover the biological function of UL3.5, laying a foundation for developing novel antiviral strategies against DPV.