EBNA3C-Mediated Regulation of Aurora Kinase B Contributes to Epstein-Barr Virus-Induced B-Cell Proliferation through Modulation of the Activities of the Retinoblastoma Protein and Apoptotic Caspases

Abstract

Epstein-Barr virus (EBV) is an oncogenic gammaherpesvirus that is implicated in several human malignancies, includingBurkitt's lymphoma (BL), posttransplant lymphoproliferative disease (PTLD), nasopharyngeal carcinoma (NPC), and AIDSassociated lymphomas. Epstein-Barr nuclear antigen 3C (EBNA3C), one of the essential EBV latent antigens, can induce mammalian cell cycle progression through its interaction with cell cycle regulators. Aurora kinase B (AK-B) is important for cell division, and deregulation of AK-B is associated with aneuploidy, incomplete mitotic exit, and cell death. Our present study shows that EBNA3C contributes to upregulation of AK-B transcript levels by enhancing the activity of its promoter. Further, EBNA3C also increased the stability of the AK-B protein, and the presence of EBNA3C leads to reduced ubiquitination of AK-B. Importantly, EBNA3C in association with wild-type AK-B but not with its kinase-dead mutant led to enhanced cell proliferation, and AK-B knockdown can induce nuclear blebbing and cell death. This phenomenon was rescued in the presence of EBNA3C. Knockdown of AK-B resulted in activation of caspase 3 and caspase 9, along with poly(ADP-ribose) polymerase 1 (PARP1) cleavage, which is known to be an important contributor to apoptotic signaling. Importantly, EBNA3C failed to stabilize the kinasedead mutant of AK-B compared to wild-type AK-B, which suggests a role for the kinase domain in AK-B stabilization and downstream phosphorylation of the cell cycle regulator retinoblastoma protein (Rb). This study demonstrates the functional relevance of AK-B kinase activity in EBNA3C-regulated B-cell proliferation and apoptosis.

The EBNA3 Family: Two Oncoproteins and a Tumour Suppressor that Are Central to the Biology of EBV in B Cells

“…However, during primary infection, EBNA3C might also have a direct effect on the ATM/CHK2 signalling pathway or some component(s) of the DDR complex at the site of DNA strand breaks (Nikitin et al 2010). Although EBNA3C can apparently associate with CHK2 and/or histone H2AX (Choudhuri et al 2007;Jha et al 2013), the possibility that these interactions are involved in the attenuation of the DDR early after infection requires further investigation. Only a systematic genetic analysis of EBNA3C and its actions early after infection will unravel the DDR and the p16 INK4a -mediated senescence response and establish what relative contributions each make to the inhibition of B cell transformation.…”

“…Several reports during the past decade indicate that EBNA3C can physically associate with a variety of other factors involved in the regulation of cell cycle progression and/or the G1/S checkpoint-most of these appear to be via amino acids located at the N-terminus of EBNA3C (aa100-200). The factors include the ubiquitin ligase SCF SKP2 , the tumour suppressor retinoblastoma (RB), the oncoprotein MYC, MDM2, p53, cyclin A, cyclin D1, E2F1, CHK2 and Aurora kinase B (Bajaj et al 2008;Choudhuri et al 2007;Jha et al 2013;Kashuba et al 2008;Knight et al , 2005aSaha et al 2009Saha et al , 2011Saha et al , 2012Yi et al 2009). It remains to be established, by reverse genetic studies of mutants that specifically and independently disrupt these binding sites, which of these interactions are functionally important in vitro during B cell transformation or in vivo during the establishment of persistence and/or in B cell lymphomagenesis.…”

Epstein-Barr Virus Essential Antigen EBNA3C Attenuates H2AX Expression

“…EBNA3C formed a stable complex with H2AX through interactions with its amino-terminal first 100 amino acid residues. Interestingly, we know that the N-terminal region of EBNA3C interacts with various cell cycle regulatory molecules, such as cyclin D1, cyclin A, p53, Mdm2, E2F1, IRF4, and Aurora kinase B (16,30,32,34,35).…”

“…All construct sequences were further confirmed by DNA sequencing at the core facility at the University of Pennsylvania. All transfection procedures were described previously (30).…”

“…EBNA3C is a strong transcription factor capable of regulating transcription of both viral and cellular genes (16,30,32,35). To determine if H2AX was directly modulated by EBNA3C at the transcript level, we evaluated H2AX transcripts in EBV-transformed and EBNA3C stable cell lines compared to EBV-negative B cells.…”