Epstein-Barr Virus Essential Antigen EBNA3C Attenuates H2AX Expression

Abstract

Epstein-Barr virus (EBV) latent antigen EBNA3C is implicated in B-cell immortalization and linked to several B-cell malignancies. Deregulation of H2AX can induce genomic instability with increased chromosomal aberrations, which ultimately leads to tumorigenesis. Here we demonstrated that EBNA3C can attenuate H2AX expression at the transcript and protein levels. A reduction of total H2AX levels was clearly observed upon infection of primary B cells with wild-type EBV but not with EBNA3C knockout recombinant EBV. H2AX also interacted with EBNA3C through its N-terminal domain (residues 1 to 100). Furthermore, H2AX mutated at Ser139 failed to interact with EBNA3C. Luciferase-based reporter assays also revealed that the binding domain of EBNA3C is sufficient for transcriptional inhibition of the H2AX promoter. EBNA3C also facilitated H2AX degradation through recruitment of components of the ubiquitin proteasome pathway. We further demonstrated that knockdown of H2AX in lymphoblastoid cell lines (LCLs) led to the upregulation of the Bub1 oncoprotein and downregulated expression of p53. Overall, our study provides additional insights into EBV-associated B-cell lymphomas, which are linked to the regulation of the DNA damage response system in infected cells. The importance of these insights are as follows: (i) EBNA3C downregulates H2AX expression at the protein and transcript levels in epithelial cells, B cells, and EBV-transformed LCLs, (ii) EBNA3C binds with wild-type H2AX but not with the Ser139 mutant of H2AX, (iii) the N terminus (residues 1 to 100) of EBNA3C is critical for binding to H2AX, (iv) localization of H2AX is predominantly nuclear in the presence of EBNA3C, and (v) H2AX knocked down in LCLs led to enhanced expression of Bub1 and downregulation of the tumor suppressor p53, which are both important for driving the oncogenic process.

Regulation of DNA Damage Signaling and Cell Death Responses by Epstein-Barr Virus Latent Membrane Protein 1 (LMP1) and LMP2A in Nasopharyngeal Carcinoma Cells

“…Therefore, many DNA viruses have evolved mechanisms to antagonize the DDR pathway in order to prevent activation of cell death pathways (53). Establishment of EBV latency in B cells and subsequent outgrowth of LCLs requires the attenuation of DDR signaling by EBNA3C (27,28). However, NPC tumors do not express EBNA3C or other C promoter (Cp) transcripts characteristic of type III latency in LCLs.…”

“…Phosphorylation of H2AX can be critical for the maintenance of latent gammaherpesvirus infections (25,26). In contrast, EBV-mediated B cell immortalization requires the attenuation of DDR by EBNA3C, which may be mediated by downregulation of H2AX expression (27,28).…”

Histone loaders CAF1 and HIRA restrict Epstein-Barr virus B-cell lytic reactivation

“…It also remains possible that they indirectly control EBV genome expression through effects host transcription factor expression, which then secondarily regulate the EBV genome. Further underscoring the intricate relationship between EBV and histone biology, EBNA3C downregulates the histone H2A variant H2AX shortly after primary B-cell infection at the mRNA and protein levels (74).…”

Epstein–Barr Virus: Diseases Linked to Infection and Transformation

“…However, only EBNA3A and EBNA3C are essential for viral transformation of B-lymphocytes, and all appear to significantly contribute to maintaining the viability of transformed cells, suggesting an important role in oncogenesis (Tomkinson et al, 1993). EBNA3C has been reported to interact with many cellular factors (Robertson et al, 1995; Choudhuri et al, 2007; Saha et al, 2011, 2015; Banerjee et al, 2013, 2014; Jha et al, 2013a, 2014, 2015a,b,c). One of these cellular antigens is RBP-JK, which binds to all the EBNA3 proteins (Robertson et al, 1995, 1996).…”

“…Cyclins, specifically Cyclin A, D1, E are also a crucial regulators in B-cell transformation through EBV (Saha et al, 2011). Several studies highlighted the importance of the DDR in EBV induced B-cell transformation (Choudhuri et al, 2007; Jha et al, 2013b, 2014). Briefly, ChK1, ChK2, and H2AX are critical components of EBV-derived B-cell transformation and are activated during early infection as well as provide a bridge to bypass the host immune system for virus propagation (Choudhuri et al, 2007; Jha et al, 2014).…”