Marek's disease virus (MDV) is a potent oncogenic alphaherpesvirus that elicits a rapid onset of malignant T-cell lymphomas in chickens. Three MDV types, including GaHV-2 (MDV-1), GaHV-3 (MDV-2) and MeHV-1 (HVT), have been identified and all encode a US3 protein kinase. MDV-1 US3 is important for efficient virus growth in vitro. To study the role of US3 in MDV replication and pathogenicity, we generated MDV-1 US3-null virus and chimeric viruses by replacing MDV-1 US3 with MDV-2 or HVT US3. Using MD as a natural virus-host model, we showed that both MDV-2 and HVT US3 partially rescued the growth deficiency of MDV-1 US3-null virus. In addition, deletion of MDV-1 US3 attenuated the virus resulting in higher survival rate and lower MDV specific tumor incidence, which could be partially compensated by MDV-2 and HVT US3. We also identified chicken histone deacetylase 1 (chHDAC1) as a common US3 substrate for all three MDV types while only US3 of MDV-1 and MDV-2 phosphorylate chHDAC2. We further determined that US3 of MDV-1 and HVT phosphorylate chHDAC1 at serine 406 (S406), while MDV-2 US3 phosphorylates S406, S410, and S415. In addition, MDV-1 US3 phosphorylates chHDAC2 at S407, while MDV-2 US3 targets S407 and S411. Furthermore, biochemical studies show that MDV US3 mediated phosphorylation of chHDAC1 and 2 affect their stability, transcriptional regulation activity, and interaction network. Using a class I HDACs specific inhibitor, we showed that MDV US3 mediated phosphorylation of chHDAC1 and 2 is involved in regulation of virus replication. Overall, we identified novel substrates for MDV US3 and characterized the role of MDV US3 in MDV pathogenesis. Author summary: Marek's disease virus (MDV) is a highly contagious and oncogenic avian alphaherpesvirus that causes T-cell lymphomas in chickens. Alphaherpesviruses encoded US3 is a multifunctional protein kinase involved in viral replication, apoptosis resistance, and cell-to-cell spread. In this study, we evaluated the importance of MDV US3 in regulating MDV replication and pathogenesis in chickens. Our results provide first evidence that MDV US3 protein kinase is involved in the replication and pathogenicity of MDV in its natural host. We also identified chicken histone deacetylase 1 and 2 (chHDAC1 and 2) as novel substrates of US3 for MDV and characterized the potential impacts of MDV US3 induced phosphorylation in their protein stability, transcriptional regulation and protein interactions; to our knowledge, this is the first comparative study of the functions of US3 from all three MDV types. This is an important finding towards a better understanding of the functions of alphaherpesviruses encoded US3 protein kinase. [ABSTRACT FROM AUTHOR]