Efficient replication of Epstein–Barr virus in stratified epithelium in vitro

Epstein-Barr virus (EBV) latently infects normal B cells and contributes to the development of certain human lymphomas. Newly infected B cells support a highly transforming form (type III) of viral latency; however, long-term EBV infection in immunocompetent hosts is limited to B cells with a more restricted form of latency (type I) in which most viral gene expression is silenced by promoter DNA methylation. How EBV converts latency type is unclear, although it is known that type I latency is associated with a germinal center (GC) B cell phenotype, and type III latency with an activated B cell (ABC) phenotype. In this study, we have examined whether expression of TET2, a cellular enzyme that initiates DNA demethylation by converting 5-methylcytosine (5mC) into 5-hydroxymethylcytosine (5hmC), regulates EBV latency type in B cells. We found that TET2 expression is inhibited in normal GC cells and GC type lymphomas. In contrast, TET2 is expressed in normal naive B cells and ABC type lymphomas. We also demonstrate that GC type cell lines have increased 5mC levels and reduced 5hmC levels in comparison to those of ABC type lines. Finally, we show that TET2 promotes the ability of the EBV transcription factor EBNA2 to convert EBV-infected cells from type I to type III latency. These findings demonstrate that TET2 expression is repressed in GC cells independent of EBV infection and suggest that TET2 promotes type III EBV latency in B cells with an ABC or naive phenotype by enhancing EBNA2 activation of methylated EBV promoters.IMPORTANCE EBV establishes several different types of viral latency in B cells. However, cellular factors that determine whether EBV enters the highly transforming type III latency, versus the more restricted type I latency, have not been well characterized. Here we show that TET2, a cellular enzyme that initiates DNA demethylation by converting 5-methylcytosine (5mC) into 5-hydroxymethylcytosine (5hmC), regulates EBV latency type in B cells by enhancing the ability of the viral transcription factor EBNA2 to activate methylated viral promoters that are expressed in type III (but not type I) latency. Furthermore, we demonstrate that (independent of EBV) TET2 is turned off in normal and malignant germinal center (GC) B cells but expressed in other B cell types. Thus, restricted TET2 expression in GC cells may promote type I EBV latency. KEYWORDS 5hmC, EBNA2, EBV, Epstein-Barr virus, TET2, germinal center B cell, latency E pstein-Barr virus (EBV) is human gammaherpesvirus that infects over 90% of the world population. EBV is the causative agent of infectious mononucleosis and is also associated with several malignancies of B cell and epithelial cell origin, including Burkitt lymphoma (BL), Hodgkin's disease, posttransplant lymphoproliferative disorder, diffuse large B cell lymphoma (DLBCL), nasopharyngeal carcinoma (NPC), and gastric carcinoma (1, 2). EBV establishes latent infection in normal B cells, in which the virus

Section: Tet2 Promotes Ebv Type III Latency Gene Expressionmentioning

confidence: 99%

Restricted TET2 Expression in Germinal Center Type B Cells Promotes Stringent Epstein-Barr Virus Latency

Wille

et al. 2017

“…More than 90% of the adult population is latently infected, and a subset can develop EBV-associated malignancies, including nasopharyngeal carcinoma (NPC), gastric cancer, Burkitt lymphoma, Hodgkin lymphoma, and lymphomas in the immunocompromised, including AIDS-associated lymphoma and posttransplant lymphoproliferative disease (2,3). Epithelial cell infection in vitro frequently results in productive replication, and latently infected oral epithelial cells are rare in persistently infected healthy individuals (4,5). However, epithelial tumors such as NPC consistently express a type II latency program, which includes latent membrane protein 1 (LMP1), LMP2A, and LMP2B (1,5).…”

Section: Importancementioning

confidence: 99%

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

Wasil

Wei

Chang

et al. 2015

Nasopharyngeal carcinoma (NPC) is closely associated with latent Epstein-Barr virus (EBV) infection. Although EBV infection of preneoplastic epithelial cells is not immortalizing, EBVcan 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 ␥H2AX (S139) phosphorylation and caspase cleavage induced by a lower dose (5 M) of the topoisomerase II inhibitor etoposide. Regulation of ␥H2AX 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. E pstein-Barr virus (EBV) is a human gammaherpesvirus that establishes lifelong latency in memory B cells, with sporadic reactivation and transmission from oral epithelia (1). More than 90% of the adult population is latently infected, and a subset can develop EBV-associated malignancies, including nasopharyngeal carcinoma (NPC), gastric cancer, Burkitt lymphoma, Hodgkin lymphoma, and lymphomas in the immunocompromised, including AIDS-associated lymphoma and posttransplant lymphoproliferative disease (2, 3). Epithelial cell infection in vitro frequently results in productive replication, and latently infected oral epithelial cells are rare in persistently infected healthy individuals (4, 5). However, epithelial tumors such as NPC consistently express a type II latency program, which includes latent membrane protein 1 (LMP1), LMP2A, and LMP2B (1, 5). Additionally, monoclonal EBV episomes are detected in NPC, suggesting that NPC tumors are the clonal outgrowth of an initially infected cell likely predisposed to oncogenic transformation from additional genetic and environmental cofactors, such as the loss of p16 and exposure to dietary nitrosamines (2, 3). In contrast to the immortalizing properties of EBV to primary B cells, the contribution of EBV infection to epithelial cell oncogenesis is less understood, as infection alone is insufficient to immortalize or induce oncogenic potential in preneoplastic cell lines from the nasopharynx (5, 6).LMP1 and LMP2A transcripts are ...

“…Normal human epithelial cells, when grown in air-interface organotypic raft cultures, differentiate to form a stratified epithelium. Such cells infected at the apical surface with EBV support complete lytic replication and produce new virus particles (19). In B cells, the viral lytic cascade is induced by the two immediate early genes BZLF1 and BRLF1, which cooperate to induce expression of all downstream lytic genes (20).…”

Section: Now and The Futurementioning

confidence: 99%

“…For example, overexpression of cyclin D1 has been reported as being required for the persistence of latent EBV in the absence of a drug resistance marker in telomerase-immortalized nasopharyngeal epithelial cells, and the loss of the tumor suppressor p16, which often occurs in nasopharyngeal tumors or premalignant lesions, negatively regulates cyclin D1 signaling (24). In addition, no evidence of latent EBV was found in the organotypic raft cultures infected from the apical surface (19).…”

Section: Now and The Futurementioning

confidence: 99%

The Long and Complicated Relationship between Epstein-Barr Virus and Epithelial Cells

Hutt-Fletcher

2017

The roles of epithelial cells in infection and persistence of the EpsteinBarr virus (EBV) have long been difficult to resolve. However, recent developments have reinforced the conclusion that these cells are a major site of virus replication and raised the possibility that, like papillomaviruses, EBV has evolved to take advantage of epithelial differentiation to ensure survival, persistence, and spread.KEYWORDS Epstein-Barr virus, epithelial cells, tropism E pstein-Barr virus (EBV) is well known as a human lymphotropic herpesvirus that was isolated from a Burkitt's lymphoma in the early 1960s (1). It is a well-described causal agent of infectious mononucleosis and an important player in the development of lymphoid tumors. It is well used as a tool for immortalizing human B cells. Soon after its discovery, an association between EBV and epithelial malignancies was revealed (2) and the presence of the virus was confirmed within epithelial cells of nasopharyngeal carcinoma (3). Yet, as late as 2005, a distinguished speaker at an international herpesvirus meeting declared that epithelial cells were not relevant to the general biology of EBV. How was that possible, and where are we today? EARLY WORKEarly works described EBV DNA and RNA in squamous epithelial cells shed in the oral cavity during acute infectious mononucleosis (4, 5). Thus, the model with B cells as the reservoir of latent EBV and epithelial cells as the site of productive lytic replication in vivo was developed. However, additional support for the routine involvement of epithelial cells in primary and persistent infections was, for many years, quite elusive. In the absence of any obvious lesions, such as those seen in herpes simplex infections, the finding of an infected cell in the oral cavity was much like looking for the proverbial needle in the haystack. Compounding the problem was the difficulty in infecting epithelial cells in vitro. With the advent of the AIDS epidemic, oral hairy leukoplakia appeared, which was the first and remains the only disease caused entirely by productive replication of EBV. Oral hairy leukoplakia, which is an epithelial hyperplasia typically found on the lateral tongue (6), is full of and driven by EBV lytic replication (7,8). This bolstered the case for epithelial cells in the oral cavity being responsible for the production of cell-free virus in saliva, but still, normal epithelial cells were readily found to contain virus only in individuals coinfected with the human immunodeficiency virus (9). For a while, the model of persistence pivoted toward the idea that in the absence of malignancy or other underlying disease, B cells alone are involved in infection (10).Three sets of observations subsequently helped spur a return to the yin-and-yang description of epithelial and B cell infection. Despite the uncertainty about the role of epithelial cells in EBV biology, its presence in epithelial cells of patients with nasopharyngeal carcinoma and gastric cancer indicates that EBV remains important for understanding...