Abstract

The tegument protein UL51 is conserved among herpesviruses, but the mechanisms underlying its role in viral pathogenesis remain unclear. In this study, using Anatid herpesvirus 1 (AnHV-1) as a model, we show that deletion of UL51 markedly attenuates viral replication and virulence in ducks. Immunoprecipitation coupled with LC–MS/MS identified interactions between pUL51 and multiple viral proteins, among which the association with pUL10 depends on pUL51 palmitoylation. This modification promotes pUL10 stability and its localization to the Golgi apparatus. Further analysis revealed that the palmitoylation of pUL51 is mediated by the host palmitoyltransferases DHHC9 and DHHC18 and is required to maintain pUL51 stability by preventing ubiquitin–proteasome-mediated degradation. Moreover, the deletion of pUL51 led to the accumulation of incompletely enveloped viral particles in the cytoplasm, and fewer viral particles were present within multivesicular bodies (MVBs), suggesting that pUL51 facilitates viral particle recruitment to MVBs for secondary envelopment. A palmitoylation-deficient (C9A) mutant of pUL51 also exhibited impaired viral replication, defective secondary envelopment, and attenuated virulence. These findings indicate that palmitoylation is a critical modification for pUL51 function, ensuring proper subcellular localization and promoting virion maturation, and highlight its potential as an antiviral target. The tegument protein UL51 is present in nearly all herpesviruses. Understanding its function and mechanism of action in viral replication and pathogenesis may provide a potential target for the development of future antiviral strategies. Using Anatid herpesvirus 1 (AnHV-1) as a model, we demonstrated that pUL51 is a key virulence factor. We show that pUL51 undergoes palmitoylation mediated by the host palmitoyltransferases DHHC9 and DHHC18, which enables its interaction with pUL10, promotes pUL10 stability, and facilitates its localization to the Golg