Role of Marek's Disease Virus (MDV)-Encoded US3 Serine/Threonine Protein Kinase in Regulating MDV Meq and Cellular CREB Phosphorylation

Abstract

Marek's disease (MD) is a neoplastic disease of chickens caused by Marek's disease virus (MDV), a member of the subfamily Alphaherpesvirinae Like other alphaherpesviruses, MDV encodes a serine/threonine protein kinase, U_S3. The functions of U_S3 have been extensively studied in other alphaherpesviruses; however, the biological functions of MDV U_S3 and its substrates have not been studied in detail. In this study, we investigated potential cellular pathways that are regulated by MDV U_S3 and identified chicken CREB (chCREB) as a substrate of MDV U_S3. We show that wild-type MDV U_S3, but not kinase-dead U_S3 (U_S3-K220A), increases CREB phosphorylation, leading to recruitment of phospho-CREB (pCREB) to the promoter of the CREB-responsive gene and activation of CREB target gene expression. Using U_S3 deletion and U_S3 kinase-dead recombinant MDV, we identified U_S3-responsive MDV genes during infection and found that the majority of U_S3-responsive genes were located in the MDV repeat regions. Chromatin immunoprecipitation sequencing (ChIP-seq) studies determined that some U_S3-regulated genes colocalized with Meq (an MDV-encoded oncoprotein) recruitment sites. Chromatin immunoprecipitation-PCR (ChIP-PCR) further confirmed Meq binding to the ICP4/LAT region, which is also regulated by U_S3. Furthermore, biochemical studies demonstrated that MDV U_S3 interacts with Meq in transfected cells and MDV-infected chicken embryonic fibroblasts in a phosphorylation-dependent manner. Finally, in vitro kinase studies revealed that Meq is a U_S3 substrate. MDV U_S3 thus acts as an upstream kinase of the CREB signaling pathway to regulate the transcription function of the CREB/Meq heterodimer, which targets cellular and viral gene expression.IMPORTANCE MDV is a potent oncogenic herpesvirus that induces T-cell lymphoma in infected chickens. Marek's disease continues to have a significant economic impact on the poultry industry worldwide. U_S3 encoded by alphaherpesviruses is a multifunctional kinase involved in the regulation of various cellular pathways. Using an MDV genome quantitative reverse transcriptase PCR (qRT-PCR) array and chromatin immunoprecipitation, we elucidated the role of MDV U_S3 in viral and cellular gene regulation. Our results provide insights into how viral kinase regulates host cell signaling pathways to activate both viral and host gene expression. This is an important step toward understanding host-pathogen interaction through activation of signaling cascades.

Keywords: CREB; Marek’s disease virus; Meq; US3 protein kinase; herpesviruses; transcription regulation.