The Epstein-Barr Virus Protein Kinase BGLF4 Integrates DNA Damage Response and Mitotic Phosphorylation Signaling to Promote Virus Replication

摘要

The Epstein-Barr virus (EBV) protein kinase BGLF4 plays a critical role in the viral life cycle by reprogramming the cellular micro-environment through phosphorylation of both viral and host proteins. In vitro studies have identified a variety of BGLF4 substrates. However, the specificity of BGLF4 site selection and the complete range of functional outcomes of BGLF4 activity remain unknown. To provide functional insight into the host proteins regulated by BGLF4, we utilized stable isotope labeling of amino acids in cell culture (SILAC)-based quantitative proteomics to compare site-specific phosphorylation and individual protein levels in BGLF4-induced and vector control Akata cells. More than 4,000 unique phosphorylation events were identified. BGLF4 up-regulated the phosphorylation of more than 1,700 unique sites on 823 phospho-proteins. In general, phosphorylation up-regulation occurred in the absence of changes in the protein abundance. Motif analysis of the 823 phospho-proteins indicated enrichment in cyclin dependent kinase 1 (CDK1)/BGLF4, Ataxia telangiectasia mutated (ATM) and Ataxia telangiectasia and Rad3-related (ATR) proteins and Aurora kinase substrates while functional analyses revealed enrichment in pathways related to the DNA damage response, mitosis and cell cycle. Particularly notable in these categories were proteins involved in DNA repair and in the spindle assembly checkpoint. Increased phosphorylation of nuclear pore proteins and phosphorylation-mediated down-regulation of phosphatase activity were also detected. Changes such as activation of ATM and inactivation of the protein phosphatase PP1 would be anticipated to impact both the DNA damage response and mitotic kinase signaling suggesting integration of these pathways is an novel mechanism by which BGLF4 promotes efficient virus replication. EBV protein kinase BGLF4 impacts multiple processes and pathways to manipulate the intracellular environment and foster viral replication.