Marek's disease (MD) is a neoplastic disease of chickens caused by Marek's disease virus (MDV), a member of the Alphaherpesviridae subfamily. Three MDV serotypes, including MDV-1, MDV-2 and HVT, have been identified and all encode a US3 serine/threonine protein kinase. Functions of US3 have been extensively studied in other alphaherpesviruses; however, the functions and substrates of MDV US3 have not been studied in detail. MDV-1 also encodes a Meq oncoprotein that is critical for MDV-1 induced T cell transformation. In the first part, we explored the cellular signaling pathways regulated by MDV-1 US3 using luciferase reporter systems and identified CREB signaling pathway was highly activated by MDV-1 US3. Our results further demonstrated that MDV-1 US3 phosphorylates chicken CREB (chCREB) and increases the binding of phospho-CREB (pCREB) at the promoter of CREB responsive cellular and viral genes to activate their transcription. Additionally, we demonstrated that MDV- 1 US3 interacts with and phosphorylates Meq. In the second part, we identified more cellular substrates of MDV US3 and characterized the role of MDV US3 in MDV replication and pathogenesis. Our results showed that MDV US3 phosphorylates and interacts with chicken histone deacetylase 1 (chHDAC1) and chHDAC2, and we determined the effect of MDV US3 mediated phosphorylation in regulating protein stability, transcriptional regulation, and protein interactions of chHDAC1 and 2. In addition, we demonstrated that MDV-2 and HVT US3 could partially restore the growth deficiency of MDV-1 US3 null virus, and compensate the role of MDV-1 US3 in MDV pathogenesis. In the third part, we examined the interactions between Meq and chHDAC1 and 2. Our immunofluorescence assay and immunoprecipitation assay showed that Meq co-localizes and interacts with chHDAC1 and 2, and Meq is a component of chHDAC1 and 2 associated CoREST, NuRD and Sin3 repressor complexes. In addition, our results showed that Meq induces the degradation of chHDAC1, chHDAC2, and global ubiquitinated proteins via a proteasome dependent pathway. In conclusion, this study identified novel MDV US3 substrates, characterized the functions of MDV US3 in viral replication and pathogenesis, and investigated the interplay between MDV Meq with host repressors and proteasome degradation pathway.
