Three Epstein–Barr virus latency proteins independently promote genomic instability by inducing DNA damage, inhibiting DNA repair and inactivating cell cycle checkpoints

ABSTRACTNasopharyngeal carcinoma (NPC) is closely associated with latent Epstein-Barr virus (EBV) infection. Although EBV infection of preneoplastic epithelial cells is not immortalizing, EBV can modulate oncogenic and cell death mechanisms. The viral latent membrane proteins 1 (LMP1) and LMP2A are consistently expressed in NPC and can cooperate in bitransgenic mice expressed from the keratin-14 promoter to enhance carcinoma development in an epithelial chemical carcinogenesis model. In this study, LMP1 and LMP2A were coexpressed in the EBV-negative NPC cell line HK1 and examined for combined effects in response to genotoxic treatments. In response to DNA damage activation, LMP1 and LMP2A coexpression reduced γH2AX (S139) phosphorylation and caspase cleavage induced by a lower dose (5 μM) of the topoisomerase II inhibitor etoposide. Regulation of γH2AX occurred before the onset of caspase activation without modulation of other DNA damage signaling mediators, including ATM, Chk1, or Chk2, and additionally was suppressed by inducers of DNA single-strand breaks (SSBs) and replication stress. Despite reduced DNA damage repair signaling, LMP1-2A coexpressing cells recovered from cytotoxic doses of etoposide; however, LMP1 expression was sufficient for this effect. LMP1 and LMP2A coexpression did not enhance cell growth, with a moderate increase of cell motility to fibronectin. This study supports that LMP1 and LMP2A jointly regulate DNA repair signaling and cell death activation with no further enhancement in the growth properties of neoplastic cells.IMPORTANCENPC is characterized by clonal EBV infection and accounts for >78,000 annual cancer cases with increased incidence in regions where EBV is endemic, such as southeast Asia. The latent proteins LMP1 and LMP2A coexpressed in NPC can individually enhance growth or survival properties in epithelial cells, but their combined effects and potential regulation of DNA repair and checkpoint mechanisms are relatively undetermined. In this study, LMP1-2A coexpression suppressed activation of the DNA damage response (DDR) protein γH2AX induced by selective genotoxins that promote DNA replication stress or SSBs. Expression of LMP1 was sufficient to recover cells, resulting in outgrowth of LMP1 and LMP1-2A-coexpressing cells and indicating distinct LMP1-dependent effects in the restoration of replicative potential. These findings demonstrate novel properties for LMP1 and LMP2A in the cooperative modulation of DDR and apoptotic signaling pathways, further implicating both proteins in the progression of NPC and epithelial malignancies.

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Correction to: miR-18a reactivates the Epstein-Barr virus through defective DNA damage response and promotes genomic instability in EBV-associated lymphomas

BMC Cancer ◽

10.1186/s12885-019-5378-x ◽

2019 ◽

Vol 19 (1) ◽

Author(s):

Pengfei Cao ◽

Meili Zhang ◽

Lujuan Wang ◽

Buqing Sai ◽

Jiuqi Tang ◽

...

Keyword(s):

Dna Damage ◽

Genomic Instability ◽

Dna Damage Response ◽

Epstein Barr Virus ◽

Barr Virus ◽

Damage Response ◽

Epstein Barr

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p16INK4A-independence of Epstein–Barr virus-induced cell proliferation and virus latency

Journal of General Virology ◽

10.1099/vir.0.79831-0 ◽

2004 ◽

Vol 85 (6) ◽

pp. 1381-1386 ◽

Cited By ~ 5

Author(s):

Michelle J. Hayes ◽

Anna Koundouris ◽

Nelleke Gruis ◽

Wilma Bergman ◽

Gordon G. Peters ◽

...

Keyword(s):

Cell Cycle ◽

Epstein Barr Virus ◽

Cell Cycle Progression ◽

Restriction Point ◽

Barr Virus ◽

Virus Latency ◽

Cycle Arrest ◽

Direct Binding ◽

Promote Cell Cycle Progression ◽

Epstein Barr

Epstein–Barr virus (EBV) has the ability to promote cell cycle progression following the initial infection of primary resting B-lymphocytes and to cause cell cycle arrest at the onset of the viral replicative cycle. Various mechanisms have been proposed for the proliferative effects, including the up-regulation of cyclin D2 by the viral EBNA-2 and EBNA-LP proteins, direct binding of EBNA3C to the retinoblastoma protein (pRb), and down-regulation of the p16INK4A tumour suppressor by the viral LMP1 product. To try to gain insight into the relative importance of these mechanisms, the ability of EBV to immortalize lymphocytes from an individual who is genetically deficient for p16INK4A was examined. From detailed analyses of the resultant lymphoblastoid cell lines it is concluded that p16INK4A status has little bearing on EBV's ability to manipulate the cell cycle machinery and a model to accommodate the previously proposed routes taken by EBV to bypass the restriction point is presented.

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Epstein-Barr Virus Nuclear Antigen 3C Interacts with and Enhances the Stability of the c-Myc Oncoprotein

Journal of Virology ◽

10.1128/jvi.02500-07 ◽

2008 ◽

Vol 82 (8) ◽

pp. 4082-4090 ◽

Cited By ~ 49

Author(s):

Bharat G. Bajaj ◽

Masanao Murakami ◽

Qiliang Cai ◽

Subhash C. Verma ◽

Ke Lan ◽

...

Keyword(s):

Cell Cycle ◽

B Lymphocytes ◽

Epstein Barr Virus ◽

Target Genes ◽

Cellular Transformation ◽

Cell Cycle Checkpoints ◽

Nuclear Antigen ◽

Small Subset ◽

Barr Virus ◽

Epstein Barr

ABSTRACT Epstein-Barr virus (EBV) was the first human DNA virus to be associated with cancer. Its oncogenic potential was further demonstrated by its ability to transform primary B lymphocytes in vitro. EBV nuclear antigen 3C (EBNA3C) is one of a small subset of latent antigens critical for the transformation of human primary B lymphocytes. Although EBNA3C has been shown to modulate several cellular functions, additional targets involved in cellular transformation remain to be explored. EBNA3C can recruit key components of the SCFSkp2 ubiquitin ligase complex. In this report, we show that EBNA3C residues 130 to 190, previously shown to bind to the SCFSkp2 complex, also can strongly associate with the c-Myc oncoprotein. Additionally, the interaction of EBNA3C with c-Myc was mapped to the region of c-Myc that includes the highly conserved Skp2 binding domain. Skp2 has been shown to regulate c-Myc stability and also has been shown to function as a coactivator of transcription for c-Myc target genes. We now show that the EBV latent oncoprotein EBNA3C can stabilize c-Myc and that the recruitment of both c-Myc and its cofactor Skp2 to c-Myc-dependent promoters can enhance c-Myc-dependent transcription. This same region of EBNA3C also recruits and modulates the activity of retinoblastoma and p27, both major regulators of the mammalian cell cycle. The inclusion of c-Myc in the group of cellular targets modulated by this domain further accentuates the importance of these critical residues of EBNA3C in bypassing the cell cycle checkpoints.

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The Translesion Polymerase Pol η Is Required for Efficient Epstein-Barr Virus Infectivity and Is Regulated by the Viral Deubiquitinating Enzyme BPLF1

Journal of Virology ◽

10.1128/jvi.00600-17 ◽

2017 ◽

Vol 91 (19) ◽

Cited By ~ 6

Author(s):

Ossie F. Dyson ◽

Joseph S. Pagano ◽

Christopher B. Whitehurst

Keyword(s):

Dna Damage ◽

Dna Repair ◽

Epstein Barr Virus ◽

Infectious Virus ◽

Lytic Cycle ◽

Viral Dna ◽

Barr Virus ◽

Polymerase Eta ◽

Epstein Barr ◽

Cellular Dna

ABSTRACT Epstein-Barr virus (EBV) infection and lytic replication are known to induce a cellular DNA damage response. We previously showed that the virally encoded BPLF1 protein interacts with and regulates several members of the translesion synthesis (TLS) pathway, a DNA damage tolerance pathway, and that these cellular factors enhance viral infectivity. BPLF1 is a late lytic cycle gene, but the protein is also packaged in the viral tegument, indicating that BPLF1 may function both early and late during infection. The BPLF1 protein expresses deubiquitinating activity that is strictly conserved across the Herpesviridae; mutation of the active site cysteine results in a loss of enzymatic activity. Infection with an EBV BPLF1 knockout virus results in decreased EBV infectivity. Polymerase eta (Pol η), a specialized DNA repair polymerase, functions in TLS and allows for DNA replication complexes to bypass lesions in DNA. Here we report that BPLF1 interacts with Pol η and that Pol η protein levels are increased in the presence of functional BPLF1. BPLF1 promotes a nuclear relocalization of Pol η molecules which are focus-like in appearance, consistent with the localization observed when Pol η is recruited to sites of DNA damage. Knockdown of Pol η resulted in decreased production of infectious virus, and further, Pol η was found to bind to EBV DNA, suggesting that it may allow for bypass of damaged viral DNA during its replication. The results suggest a mechanism by which EBV recruits cellular repair factors, such as Pol η, to sites of viral DNA damage via BPLF1, thereby allowing for efficient viral DNA replication. IMPORTANCE Epstein-Barr virus is the causative agent of infectious mononucleosis and infects approximately 90% of the world's population. It causes lymphomas in individuals with acquired and innate immune disorders and is strongly associated with Hodgkin's lymphoma, Burkitt's lymphoma, diffuse large B-cell lymphomas, nasopharyngeal carcinoma (NPC), and lymphomas that develop in organ transplant recipients. Cellular DNA damage is a major determinant in the establishment of oncogenic processes and is well studied, but there are few studies of endogenous repair of viral DNA. This work evaluates how EBV's BPLF1 protein and its conserved deubiquitinating activity regulate the cellular DNA repair enzyme polymerase eta and recruit it to potential sites of viral damage and replication, resulting in enhanced production of infectious virus. These findings help to establish how EBV enlists and manipulates cellular DNA repair factors during the viral lytic cycle, contributing to efficient infectious virion production.

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