title: Phosphoproteomic Profiling Reveals Epstein-Barr Virus Protein Kinase Integration of DNA Damage Response and Mitotic Signaling.
abstract: Epstein-Barr virus (EBV) is etiologically linked to infectious mononucleosis and several human cancers. EBV encodes a conserved protein kinase BGLF4 that plays a key role in the viral life cycle. To provide new insight into the host proteins regulated by BGLF4, we utilized stable isotope labeling by amino acids in cell culture (SILAC)-based quantitative proteomics to compare site-specific phosphorylation in BGLF4-expressing Akata B cells. Our analysis revealed BGLF4-mediated hyperphosphorylation of 3,046 unique sites corresponding to 1,328 proteins. Frequency analysis of these phosphosites revealed a proline-rich motif signature downstream of BGLF4, indicating a broader substrate recognition for BGLF4 than its cellular ortholog cyclin-dependent kinase 1 (CDK1). Further, motif analysis of the hyperphosphorylated sites revealed enrichment in ATM, ATR and Aurora kinase substrates while functional analyses revealed significant enrichment of pathways related to the DNA damage response (DDR), mitosis and cell cycle. Phosphorylation of proteins associated with the mitotic spindle assembly checkpoint (SAC) indicated checkpoint activation, an event that inactivates the anaphase promoting complex/cyclosome, APC/C. Furthermore, we demonstrated that BGLF4 binds to and directly phosphorylates the key cellular proteins PP1, MPS1 and CDC20 that lie upstream of SAC activation and APC/C inhibition. Consistent with APC/C inactivation, we found that BGLF4 stabilizes the expression of many known APC/C substrates. We also noted hyperphosphorylation of 22 proteins associated the nuclear pore complex, which may contribute to nuclear pore disassembly and SAC activation. A drug that inhibits mitotic checkpoint activation also suppressed the accumulation of extracellular EBV virus. Taken together, our data reveal that, in addition to the DDR, manipulation of mitotic kinase signaling and SAC activation are mechanisms associated with lytic EBV replication. All MS data have been deposited in the ProteomeXchange with identifier PXD002411 (http://proteomecentral.proteomexchange.org/dataset/PXD002411).
authors.affiliation: Department of Oncology, Johns Hopkins University School of Medicine, Baltimore, Maryland, United States of America.|Philips Institute for Oral Health Research, VCU School of Dentistry, Virginia Commonwealth University, Richmond, Virginia, United States of America.|Massey Cancer Center, Virginia Commonwealth University, Richmond, Virginia, United States of America.
authors.forename: Renfeng
authors.initials: R
authors.lastname: Li
authors.affiliation: Department of Oncology, Johns Hopkins University School of Medicine, Baltimore, Maryland, United States of America.
authors.forename: Gangling
authors.initials: G
authors.lastname: Liao
authors.affiliation: McKusick-Nathans Institute of Genetic Medicine, Johns Hopkins University School of Medicine, Baltimore, Maryland, United States of America.|Institute of Bioinformatics, International Technology Park, Bangalore, India.
authors.forename: Raja Sekhar
authors.initials: RS
authors.lastname: Nirujogi
authors.affiliation: Institute of Bioinformatics, International Technology Park, Bangalore, India.
authors.forename: Sneha M
authors.initials: SM
authors.lastname: Pinto
authors.affiliation: McKusick-Nathans Institute of Genetic Medicine, Johns Hopkins University School of Medicine, Baltimore, Maryland, United States of America.
authors.forename: Patrick G
authors.initials: PG
authors.lastname: Shaw
authors.affiliation: McKusick-Nathans Institute of Genetic Medicine, Johns Hopkins University School of Medicine, Baltimore, Maryland, United States of America.
authors.forename: Tai-Chung
authors.initials: TC
authors.lastname: Huang
authors.affiliation: Wilmer Institute, Johns Hopkins University School of Medicine, Baltimore, Maryland, United States of America.
authors.forename: Jun
authors.initials: J
authors.lastname: Wan
authors.affiliation: Massey Cancer Center, Virginia Commonwealth University, Richmond, Virginia, United States of America.
authors.forename: Jiang
authors.initials: J
authors.lastname: Qian
authors.affiliation: Institute of Bioinformatics, International Technology Park, Bangalore, India.
authors.forename: Harsha
authors.initials: H
authors.lastname: Gowda
authors.affiliation: McKusick-Nathans Institute of Genetic Medicine, Johns Hopkins University School of Medicine, Baltimore, Maryland, United States of America.
authors.forename: Xinyan
authors.initials: X
authors.lastname: Wu
authors.affiliation: Philips Institute for Oral Health Research, VCU School of Dentistry, Virginia Commonwealth University, Richmond, Virginia, United States of America.
authors.forename: Dong-Wen
authors.initials: DW
authors.lastname: Lv
authors.affiliation: Philips Institute for Oral Health Research, VCU School of Dentistry, Virginia Commonwealth University, Richmond, Virginia, United States of America.
authors.forename: Kun
authors.initials: K
authors.lastname: Zhang
authors.affiliation: Philips Institute for Oral Health Research, VCU School of Dentistry, Virginia Commonwealth University, Richmond, Virginia, United States of America.|McKusick-Nathans Institute of Genetic Medicine, Johns Hopkins University School of Medicine, Baltimore, Maryland, United States of America.
authors.forename: Srikanth S
authors.initials: SS
authors.lastname: Manda
authors.affiliation: Philips Institute for Oral Health Research, VCU School of Dentistry, Virginia Commonwealth University, Richmond, Virginia, United States of America.|McKusick-Nathans Institute of Genetic Medicine, Johns Hopkins University School of Medicine, Baltimore, Maryland, United States of America.|Diana Helis Henry Medical Research Foundation, New Orleans, Louisiana, United States of America.
authors.forename: Akhilesh
authors.initials: A
authors.lastname: Pandey
authors.affiliation: Department of Oncology, Johns Hopkins University School of Medicine, Baltimore, Maryland, United States of America.
authors.forename: S Diane
authors.initials: SD
authors.lastname: Hayward
mesh_terms: D000595:Amino Acid Sequence; D002460:Cell Line; D002853:Chromatography, Liquid; D004249:DNA Damage / Q000502:physiology*; D020031:Epstein-Barr Virus Infections / Q000378:metabolism*; D015967:Gene Expression Regulation, Viral; D004854:Herpesvirus 4, Human / Q000378:metabolism*; D006801:Humans; D015151:Immunoblotting; D008938:Mitosis / Q000502:physiology*; D008969:Molecular Sequence Data; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D040901:Proteomics / Q000379:methods; D015398:Signal Transduction / Q000502:physiology; D053719:Tandem Mass Spectrometry; D014764:Viral Proteins / Q000378:metabolism*; D014779:Virus Replication / Q000502:physiology*
chemical_list: D014764:Viral Proteins; C406063:BGLF4 protein, Epstein-Barr virus; D017346:Protein Serine-Threonine Kinases