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).
Highlights
Infection with Epstein-Barr virus (EBV), a ubiquitous herpesvirus, is associated with malignant disease, including Burkitt lymphoma, nasopharyngeal carcinoma, gastric carcinoma, and posttransplant lymphoproliferative disease [1,2]
Epstein-Barr virus (EBV) is a herpesvirus that is associated with B cell and epithelial human cancers
Previous work on BGLF4 has largely focused on its cyclin-dependent kinase 1 (CDK1)-like activity
Summary
Infection with Epstein-Barr virus (EBV), a ubiquitous herpesvirus, is associated with malignant disease, including Burkitt lymphoma, nasopharyngeal carcinoma, gastric carcinoma, and posttransplant lymphoproliferative disease [1,2]. BGLF4 phosphorylates EBV latency and lytic proteins to regulate their transactivation activity [13,14,15] and expression [16,17,18]. Protein SUMOylation is modified by BGLF4 in a kinase activity and SUMO-binding dependent manner [28,29] and BGLF4 phosphorylates IRF3 and UXT to suppress host immune responses and NF-κB signaling, respectively [30,31,32]. Taken together, these studies indicate that BGLF4 affects many aspects of the cellular environment. A global overview of BGLF4 regulated signaling events in cells is still lacking
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