Epstein‐Barr Virus (EBV) Infection in Epithelial Cells In Vivo: Rare Detection of EBV Replication in Tongue Mucosa but Not in Salivary Glands

ABSTRACT Epstein-Barr virus (EBV) is found frequently in certain epithelial pathologies, such as nasopharyngeal carcinoma and oral hairy leukoplakia, indicating that the virus can infect epithelial cells in vivo. Recent studies of cell lines imply that epithelial cells may also play a role in persistent EBV infection in vivo. In this report, we show the establishment and characterization of an ex vivo culture model of tonsil epithelial cells, a likely site for EBV infection in vivo. Primary epithelial-cell cultures, generated from tonsil explants, contained a heterogeneous mixture of cells with an ongoing process of differentiation. Keratin expression profiles were consistent with the presence of cells from both surface and crypt epithelia. A small subset of cells could be latently infected by coculture with EBV-releasing cell lines, but not with cell-free virus. We also detected viral-DNA, -mRNA, and -protein expression in cultures from EBV-positive tonsil donors prior to in vitro infection. We conclude that these cells were either already infected at the time of explantation or soon after through cell-to-cell contact with B cells replicating EBV in the explant. Taken together, these findings suggest that the tonsil epithelium of asymptomatic virus carriers is able to sustain EBV infection in vivo. This provides an explanation for the presence of EBV in naso- and oropharyngeal pathologies and is consistent with epithelial cells playing a role in the egress of EBV during persistent infection.

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Hippo signaling effectors YAP and TAZ induce Epstein-Barr Virus (EBV) lytic reactivation through TEADs in epithelial cells

PLoS Pathogens ◽

10.1371/journal.ppat.1009783 ◽

2021 ◽

Vol 17 (8) ◽

pp. e1009783

Author(s):

Nicholas Van Sciver ◽

Makoto Ohashi ◽

Nicholas P. Pauly ◽

Jillian A. Bristol ◽

Scott E. Nelson ◽

...

Keyword(s):

Epithelial Cell ◽

Epithelial Cells ◽

B Cell ◽

Epstein Barr Virus ◽

Hippo Signaling ◽

Oral Keratinocytes ◽

Barr Virus ◽

Ebv Infection ◽

Lytic Reactivation ◽

Epstein Barr

The Epstein-Barr virus (EBV) human herpesvirus is associated with B-cell and epithelial-cell malignancies, and both the latent and lytic forms of viral infection contribute to the development of EBV-associated tumors. Here we show that the Hippo signaling effectors, YAP and TAZ, promote lytic EBV reactivation in epithelial cells. The transcriptional co-activators YAP/TAZ (which are inhibited by Hippo signaling) interact with DNA-binding proteins, particularly TEADs, to induce transcription. We demonstrate that depletion of either YAP or TAZ inhibits the ability of phorbol ester (TPA) treatment, cellular differentiation or the EBV BRLF1 immediate-early (IE) protein to induce lytic EBV reactivation in oral keratinocytes, and show that over-expression of constitutively active forms of YAP and TAZ reactivate lytic EBV infection in conjunction with TEAD family members. Mechanistically, we find that YAP and TAZ interact with, and activate, the EBV BZLF1 immediate-early promoter. Furthermore, we demonstrate that YAP, TAZ, and TEAD family members are expressed at much higher levels in epithelial cell lines in comparison to B-cell lines, and find that EBV infection of oral keratinocytes increases the level of activated (dephosphorylated) YAP and TAZ. Finally, we have discovered that lysophosphatidic acid (LPA), a known YAP/TAZ activator that plays an important role in inflammation, induces EBV lytic reactivation in epithelial cells through a YAP/TAZ dependent mechanism. Together these results establish that YAP/TAZ are powerful inducers of the lytic form of EBV infection and suggest that the ability of EBV to enter latency in B cells at least partially reflects the extremely low levels of YAP/TAZ and TEADs in this cell type.

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Infection of Epstein–Barr Virus in Type III Latency Modulates Biogenesis of Exosomes and the Expression Profile of Exosomal miRNAs in the Burkitt Lymphoma Mutu Cell Lines

Cancers ◽

10.3390/cancers10070237 ◽

2018 ◽

Vol 10 (7) ◽

pp. 237 ◽

Cited By ~ 13

Author(s):

Asuka Nanbo ◽

Harutaka Katano ◽

Michiyo Kataoka ◽

Shiho Hoshina ◽

Tsuyoshi Sekizuka ◽

...

Keyword(s):

Epithelial Cells ◽

Burkitt Lymphoma ◽

Epstein Barr Virus ◽

Type I ◽

Type Iii ◽

Barr Virus ◽

Ebv Infection ◽

Latently Infected ◽

Infected Cells ◽

Epstein Barr

Infection of Epstein–Barr virus (EBV), a ubiquitous human gamma herpesvirus, is associated with various malignancies in B lymphocytes and epithelial cells. EBV encodes 49 microRNAs in two separated regions, termed the BART and BHRF1 loci. Although accumulating evidence demonstrates that EBV infection regulates the profile of microRNAs in the cells, little is known about the microRNAs in exosomes released from infected cells. Here, we characterized the expression profile of intracellular and exosomal microRNAs in EBV-negative, and two related EBV-infected Burkitt lymphoma cell lines having type I and type III latency by next-generation sequencing. We found that the biogenesis of exosomes is upregulated in type III latently infected cells compared with EBV-negative and type I latently infected cells. We also observed that viral and several specific host microRNAs were predominantly incorporated in the exosomes released from the cells in type III latency. We confirmed that multiple viral microRNAs were transferred to the epithelial cells cocultured with EBV-infected B cells. Our findings indicate that EBV infection, in particular in type III latency, modulates the biogenesis of exosomes and the profile of exosomal microRNAs, potentially contributing to phenotypic changes in cells receiving these exosomes.

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Transient Immunodeficiency During Asymptomatic Epstein-Barr Virus Infection

PEDIATRICS ◽

10.1542/peds.71.6.964 ◽

1983 ◽

Vol 71 (6) ◽

pp. 964-967

Author(s):

THOMAS J. BOWEN ◽

RALPH J. WEDGWOOD ◽

HANS D. OCHS ◽

WERNER HENLE

Keyword(s):

Epstein Barr Virus ◽

Mononuclear Cells ◽

Epstein Barr Virus Infection ◽

Peripheral Blood Mononuclear ◽

Immunoglobulin Synthesis ◽

Barr Virus ◽

Ebv Infection ◽

Epstein Barr

In vivo and in vitro humoral and cellular immune responses were studied in a 2½-year-old girl immediately before, during, and after an asymptomatic infection with Epstein-Barr virus. During the acute EBV infection, the patient's peripheral blood mononuclear cells were deficient in immunoglobulin synthesis and suppressed the in vitro immunoglobulin synthesis of normal allogeneic cells. In vitro mitogen transformation of lymphocytes was reduced. In vivo antibody responses to the T cell-dependent antigens bacteriophage φX 174 and Keyhole limpet hemocyanin were markedly depressed. These studies suggest that suppressor cells induced during acute EBV infection not only suppress immunoglobulin synthesis in vitro, but also interfere with in vivo antibody synthesis.

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Common Epstein-Barr virus (EBV) type-1 variant strains in both malignant and benign EBV-associated disorders

Blood ◽

10.1182/blood.v87.4.1579.bloodjournal8741579 ◽

1996 ◽

Vol 87 (4) ◽

pp. 1579-1585 ◽

Cited By ~ 24

Author(s):

V Schuster ◽

G Ott ◽

S Seidenspinner ◽

HW Kreth

Keyword(s):

Gastric Carcinoma ◽

Epstein Barr Virus ◽

Hodgkin's Lymphoma ◽

Sequence Pattern ◽

Barr Virus ◽

Ebv Infection ◽

Epstein Barr

In the present study, Epstein-Barr virus (EBV) isolates from 18 malignant tumors (angioimmunoblastic lymphadenopathy [AILD], n = 4; Hodgkin's disease [HD], n = 3; pleomorphic T-cell non-Hodgkin's lymphoma [T-NHL], n = 1; B-cell non-Hodgkin's lymphoma [B-NHL], n = 8; gastric carcinoma, n = 2) as well as from 10 tonsils of EBV- seropositive children and from peripheral blood mononuclear cells of 12 children with uncomplicated infectious mononucleosis (IM) and of a boy with severe chronic active EBV infection were genotyped in the EBV nuclear antigen-2 (EBNA-2) gene. A total of 40 of 41 isolates harbored EBV type 1; in 1 specimen (tonsil), only EBV type 2 was found. Further molecular characterization of EBV type-1 wild-type isolates in the EBNA- 2 gene and in the 40-kb distant EBV-encoded small RNAs (EBER) region showed that different groups of stable EBV type-1 variant strains exist in vivo both in benign and malignant lymphatic tissue. Group 1 is composed of EBV type-1 isolates (B-NHL, n = 3; T-NHL, n = 1; HD, n = 1; IM, n = 4) that showed a B95–8-like DNA sequence pattern in both viral genes. Group 2 isolates (HD, n = 1; AILD, n = B-NHL, n = 1; tonsils of EBV-seropositive children, n = 9; IM, n = 20 showed a nucleotide change at position 49095 in the EBNA-2 gene, leading to an amino acid substitution (Pro-->Ser), and EBV type-2 sequences in the EBER region. EBV type-1 isolates that fall into group 3 (AILD, n = 3; HD, n = 1; B- NHL, n = 4; gastric carcinoma, n = 2; IM, n = 6; severe chronic active EBV infection, n = 1) were characterized by typical nucleotide changes and a 3-bp insertion (CTC; extra Leu residue) in the EBNA-2 gene and an EBV type-2-specific sequence pattern in the EBER region. These EBV type- 1 variant strains may represent the most prevalent circulating EBV type- 1 strains in the exposed population and seem not to be restricted to a certain EBV-associated disease or tumor type. However, analysis of more EBV isolates from benign and malignant lesions must show whether more EBV type-1 substrains exist in vivo.

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Epstein–Barr Virus Infection of Pseudostratified Nasopharyngeal Epithelium Disrupts Epithelial Integrity

Cancers ◽

10.3390/cancers12092722 ◽

2020 ◽

Vol 12 (9) ◽

pp. 2722

Author(s):

Fenggang Yu ◽

Yanan Lu ◽

Yingying Li ◽

Yuji Uchio ◽

Utomo Andi Pangnguriseng ◽

...

Keyword(s):

Epithelial Cells ◽

Epstein Barr Virus ◽

Basal Layer ◽

Infection Model ◽

Hybridization Technique ◽

Epstein Barr Virus Infection ◽

Barr Virus ◽

Ebv Infection ◽

Epstein Barr ◽

Nasopharyngeal Epithelial

Epstein–Barr virus (EBV) is a human oncogenic virus that causes several types of tumor, such as Burkitt’s lymphoma and nasopharyngeal carcinoma (NPC). NPC tumor cells are clonal expansions of latently EBV-infected epithelial cells. However, the mechanisms by which EBV transforms the nasopharyngeal epithelium is hampered, because of the lack of good in vitro model to pursue oncogenic process. Our primary nasopharyngeal epithelial cell cultures developed pseudostratified epithelium at the air-liquid interface, which was susceptible to EBV infection. Using the highly sensitive RNA in situ hybridization technique, we detected viral infection in diverse cell types, including ciliated cells, goblet cells, and basal cells. EBV-encoded small RNA-positive cells were more frequently detected in the suprabasal layer than in the basal layer. We established the most physiologically relevant EBV infection model of nasopharyngeal epithelial cells. This model will advance our understanding of EBV pathogenesis in the development of NPC.

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Antibodies to gp350/220 Enhance the Ability of Epstein-Barr Virus To Infect Epithelial Cells

Journal of Virology ◽

10.1128/jvi.00622-06 ◽

2006 ◽

Vol 80 (19) ◽

pp. 9628-9633 ◽

Cited By ~ 41

Author(s):

Susan M. Turk ◽

Ru Jiang ◽

Liudmila S. Chesnokova ◽

Lindsey M. Hutt-Fletcher

Keyword(s):

B Cells ◽

Nasopharyngeal Carcinoma ◽

Epithelial Cells ◽

Epstein Barr Virus ◽

Lytic Cycle ◽

Virus Envelope ◽

Barr Virus ◽

High Risk Populations ◽

Epstein Barr

ABSTRACT Epstein-Barr virus (EBV) is a persistent, orally transmitted herpesvirus that replicates in B cells and epithelial cells and is associated with lymphoid and epithelial malignancies. The virus binds to CD21 on B cells via glycoprotein gp350/220 and infects efficiently. Infection of cultured epithelial cells has not typically been efficient but can occur in the absence of gp350/220 and CD21 and in vivo is thought to be important to the development of nasopharyngeal carcinoma. We report here that antibodies to gp350/220, which inhibit EBV infection of B cells, enhance infection of epithelial cells. The effect is not mediated by Fc receptor binding but is further enhanced by antibody cross-linking, which may patch gp350/220 in the virus envelope. Saliva from EBV-seropositive individuals has similar effects that can be reversed by depletion of antibody. The results are consistent with a model in which gp350/220 interferes with the access of other important players to the epithelial cell surface. The results may have implications for the development of nasopharyngeal carcinoma in high-risk populations in which elevated titers of antibody to EBV lytic cycle proteins are prognostic.

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A Positive Autoregulatory Loop of LMP1 Expression and STAT Activation in Epithelial Cells Latently Infected with Epstein-Barr Virus

Journal of Virology ◽

10.1128/jvi.77.7.4139-4148.2003 ◽

2003 ◽

Vol 77 (7) ◽

pp. 4139-4148 ◽

Cited By ~ 116

Author(s):

Honglin Chen ◽

Lindsey Hutt-Fletcher ◽

Liang Cao ◽

S. Diane Hayward

Keyword(s):

Epithelial Cells ◽

Cell Line ◽

Cell Lines ◽

Epstein Barr Virus ◽

Barr Virus ◽

Ebv Infection ◽

Lmp1 Expression ◽

Cell Line Hela ◽

Epstein Barr ◽

Phosphorylated Stat3

ABSTRACT STAT3 and STAT5 are constitutively activated and nuclear in nasopharyngeal carcinoma (NPC) cells. In normal signaling, STATs are only transiently activated. To investigate whether Epstein-Barr virus (EBV), and in particular the protein LMP1, contributes to sustained STAT phosphorylation and activation in epithelial cells, we examined STAT activity in two sets of paired cell lines, HeLa, an EBV-converted HeLa cell line, HeLa-Bx1, the NPC-derived cell line CNE2-LNSX, and an LMP1-expressing derivative, CNE2-LMP1. EBV infection was associated with a significant increase in the tyrosine-phosphorylated forms of STAT3 and STAT5 in HeLa-Bx1 cells. This effect correlated with LMP1 expression, since phosphorylated STAT3 and STAT5 levels were also increased in CNE2-LMP1 cells relative to the control CNE2-LNSX cells. No change was observed in STAT1 or STAT6 phosphorylation in these cell lines, nor was there a significant change in the levels of total STAT3, STAT5, STAT1, or STAT6 protein. Tyrosine phosphorylation allows the normally cytoplasmic STAT proteins to enter the nucleus and bind to their recognition sequences in responsive promoters. The ability of LMP1 to activate STAT3 was further established by immunofluorescence assays in which coexpression of LMP1 in transfected cells was sufficient to mediate nuclear relocalization of Flag-STAT3 and by an electrophoretic mobility shift assay which showed that LMP1 expression in CNE2-LNSX cells was associated with increased endogenous STAT3 DNA binding activity. In addition, the activity of a downstream target of STAT3, c-Myc, was upregulated in HeLa-Bx1 and CNE2-LMP1 cells. A linkage was established between interleukin-6 (IL-6)- and LMP1-mediated STAT3 activation. Treatment with IL-6 increased phosphorylated STAT3 levels in CNE2-LNSX cells, and conversely, treatment of CNE2-LMP1 cells with IL-6 neutralizing antibody ablated STAT3 activation and c-Myc upregulation. The previous observation that STAT3 activated the LMP1 terminal repeat promoter in reporter assays was extended to show upregulated expression of endogenous LMP1 mRNA and protein in HeLa-Bx1 cells transfected with a constitutively activated STAT3. A model is proposed in which EBV infection of an epithelial cell containing activated STATs would permit LMP1 expression. This in turn would establish a positive feedback loop of IL-6-induced STAT activation, LMP1 and Qp-EBNA1 expression, and viral genome persistence.

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Epstein-Barr-Virus-Encoded LMP2A Induces Primary Epithelial Cell Migration and Invasion: Possible Role in Nasopharyngeal Carcinoma Metastasis

Journal of Virology ◽

10.1128/jvi.79.24.15430-15442.2005 ◽

2005 ◽

Vol 79 (24) ◽

pp. 15430-15442 ◽

Cited By ~ 66

Author(s):

Dirk M. Pegtel ◽

Aravind Subramanian ◽

Tzung-Shiahn Sheen ◽

Ching-Hwa Tsai ◽

Todd R. Golub ◽

...

Keyword(s):

Cell Migration ◽

Epithelial Cell ◽

Epithelial Cells ◽

Epstein Barr Virus ◽

Cellular Migration ◽

Migration And Invasion ◽

Barr Virus ◽

Ebv Infection ◽

Epithelial Cell Migration ◽

Epstein Barr

ABSTRACT Nonkeratinizing nasopharyngeal carcinomas (NPC) are >95% associated with the expression of the Epstein-Barr virus (EBV) LMP2A latent protein. However, the role of EBV, in particular, LMP2A, in tumor progression is not well understood. Using Affymetrix chips and a pattern-matching computational technique (neighborhood analysis), we show that the level of LMP2A expression in NPC biopsy samples correlates with that of a cellular protein, integrin-alpha-6 (ITGα6), that is associated with cellular migration in vitro and metastasis in vivo. We have recently developed a primary epithelial model from tonsil tissue to study EBV infection in epithelial cells. Here we report that LMP2A expression in primary tonsil epithelial cells causes them to become migratory and invasive, that ITGα6 RNA levels are up-regulated in epithelial cells expressing LMP2, and that ITGα6 protein levels are increased in the migrating cells. Blocking antibodies against ITGα6 abrogated LMP2-induced invasion through Matrigel by primary epithelial cells. Our results provide a link between LMP2A expression, ITGα6 expression, epithelial cell migration, and NPC metastasis and suggest that EBV infection may contribute to the high incidence of metastasis in NPC progression.

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Epstein-Barr Virus Infection Promotes Epithelial Cell Growth by Attenuating Differentiation-Dependent Exit from the Cell Cycle

mBio ◽

10.1128/mbio.01332-19 ◽

2019 ◽

Vol 10 (4) ◽

Cited By ~ 5

Author(s):

Mark R. Eichelberg ◽

Rene Welch ◽

J. Tod Guidry ◽

Ahmed Ali ◽

Makoto Ohashi ◽

...

Keyword(s):

Gene Expression ◽

Cell Cycle ◽

Epithelial Cell ◽

Epithelial Cells ◽

Epstein Barr Virus ◽

Latent Infection ◽

Barr Virus ◽

Ebv Infection ◽

Epstein Barr ◽

Oral Epithelial

ABSTRACT Epstein-Barr virus (EBV) is a human herpesvirus that is associated with lymphomas as well as nasopharyngeal and gastric carcinomas. Although carcinomas account for almost 90% of EBV-associated cancers, progress in examining EBV’s role in their pathogenesis has been limited by difficulty in establishing latent infection in nontransformed epithelial cells. Recently, EBV infection of human telomerase reverse transcriptase (hTERT)-immortalized normal oral keratinocytes (NOKs) has emerged as a model that recapitulates aspects of EBV infection in vivo, such as differentiation-associated viral replication. Using uninfected NOKs and NOKs infected with the Akata strain of EBV (NOKs-Akata), we examined changes in gene expression due to EBV infection and differentiation. Latent EBV infection produced very few significant gene expression changes in undifferentiated NOKs but significantly reduced the extent of differentiation-induced gene expression changes. Gene set enrichment analysis revealed that differentiation-induced downregulation of the cell cycle and metabolism pathways was markedly attenuated in NOKs-Akata relative to that in uninfected NOKs. We also observed that pathways induced by differentiation were less upregulated in NOKs-Akata. We observed decreased differentiation markers and increased suprabasal MCM7 expression in NOKs-Akata versus NOKs when both were grown in raft cultures, consistent with our transcriptome sequencing (RNA-seq) results. These effects were also observed in NOKs infected with a replication-defective EBV mutant (AkataΔRZ), implicating mechanisms other than lytic-gene-induced host shutoff. Our results help to define the mechanisms by which EBV infection alters keratinocyte differentiation and provide a basis for understanding the role of EBV in epithelial cancers. IMPORTANCE Latent infection by Epstein-Barr virus (EBV) is an early event in the development of EBV-associated carcinomas. In oral epithelial tissues, EBV establishes a lytic infection of differentiated epithelial cells to facilitate the spread of the virus to new hosts. Because of limitations in existing model systems, the effects of latent EBV infection on undifferentiated and differentiating epithelial cells are poorly understood. Here, we characterize latent infection of an hTERT-immortalized oral epithelial cell line (NOKs). We find that although EBV expresses a latency pattern similar to that seen in EBV-associated carcinomas, infection of undifferentiated NOKs results in differential expression of a small number of host genes. In differentiating NOKs, however, EBV has a more substantial effect, reducing the extent of differentiation and delaying the exit from the cell cycle. This effect may synergize with preexisting cellular abnormalities to prevent exit from the cell cycle, representing a critical step in the development of cancer.

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