scholarly journals ΔNp63α promotes Epstein-Barr virus latency in undifferentiated epithelial cells

2021 ◽  
Vol 17 (11) ◽  
pp. e1010045
Author(s):  
Nicholas Van Sciver ◽  
Makoto Ohashi ◽  
Dhananjay M. Nawandar ◽  
Nicholas P. Pauly ◽  
Denis Lee ◽  
...  
Keyword(s):  
Transcription Factors ◽  
Nasopharyngeal Carcinoma ◽  
Epithelial Cell ◽  
Epithelial Cells ◽  
B Cell ◽  
Epstein Barr Virus ◽  
Viral Latency ◽  
Human Herpesvirus ◽  
Barr Virus ◽  
Epstein Barr

Epstein-Barr virus (EBV) is a human herpesvirus that causes infectious mononucleosis and contributes to both B-cell and epithelial-cell malignancies. EBV-infected epithelial cell tumors, including nasopharyngeal carcinoma (NPC), are largely composed of latently infected cells, but the mechanism(s) maintaining viral latency are poorly understood. Expression of the EBV BZLF1 (Z) and BRLF1 (R) encoded immediate-early (IE) proteins induces lytic infection, and these IE proteins activate each other’s promoters. ΔNp63α (a p53 family member) is required for proliferation and survival of basal epithelial cells and is over-expressed in NPC tumors. Here we show that ΔNp63α promotes EBV latency by inhibiting activation of the BZLF1 IE promoter (Zp). Furthermore, we find that another p63 gene splice variant, TAp63α, which is expressed in some Burkitt and diffuse large B cell lymphomas, also represses EBV lytic reactivation. We demonstrate that ΔNp63α inhibits the Zp promoter indirectly by preventing the ability of other transcription factors, including the viral IE R protein and the cellular KLF4 protein, to activate Zp. Mechanistically, we show that ΔNp63α promotes viral latency in undifferentiated epithelial cells both by enhancing expression of a known Zp repressor protein, c-myc, and by decreasing cellular p38 kinase activity. Furthermore, we find that the ability of cis-platinum chemotherapy to degrade ΔNp63α contributes to the lytic-inducing effect of this agent in EBV-infected epithelial cells. Together these findings demonstrate that the loss of ΔNp63α expression, in conjunction with enhanced expression of differentiation-dependent transcription factors such as BLIMP1 and KLF4, induces lytic EBV reactivation during normal epithelial cell differentiation. Conversely, expression of ΔNp63α in undifferentiated nasopharyngeal carcinoma cells and TAp63α in Burkitt lymphoma promotes EBV latency in these malignancies.

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

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.

scholarly journals Soluble Epstein-Barr Virus Glycoproteins gH, gL, and gp42 Form a 1:1:1 Stable Complex That Acts Like Soluble gp42 in B-Cell Fusion but Not in Epithelial Cell Fusion

2006 ◽  
Vol 80 (19) ◽  
pp. 9444-9454 ◽  
Author(s):  
Austin N. Kirschner ◽  
Jasmina Omerović ◽  
Boris Popov ◽  
Richard Longnecker ◽  
Theodore S. Jardetzky
Keyword(s):  
B Cells ◽  
Epithelial Cell ◽  
Epithelial Cells ◽  
B Cell ◽  
Membrane Fusion ◽  
Cell Fusion ◽  
Epstein Barr Virus ◽  
Stable Complex ◽  
Barr Virus ◽  
Epstein Barr

ABSTRACT Epstein-Barr virus (EBV) is a herpesvirus that infects cells by fusing its lipid envelope with the target cell membrane. The fusion process requires the actions of viral glycoproteins gH, gL, and gB for entry into epithelial cells and additionally requires gp42 for entry into B cells. To further study the roles of these membrane-associated glycoproteins, purified soluble forms of gp42, gH, and gL were expressed that lack the membrane-spanning regions. The soluble gH/gL protein complex binds to soluble gp42 with high affinity, forming a stable heterotrimer with 1:1:1 stoichiometry, and this complex is not formed by an N-terminally truncated variant of gp42. The effects of adding soluble gp42, gH/gL, and gH/gL/gp42 were examined with a virus-free cell-cell fusion assay. The results demonstrate that, in contrast to gp42, membrane fusion does not proceed with secreted gH/gL. The addition of soluble gH/gL does not inhibit or enhance B-cell or epithelial cell fusion when membrane-bound gH/gL, gB, and gp42 are present. However, the soluble gH/gL/gp42 complex does activate membrane fusion with B cells, similarly to soluble gp42, but it does not inhibit fusion with epithelial cells, as observed for gp42 alone. A gp42 peptide, derived from an N-terminal segment involved in gH/gL interactions, binds to soluble gH/gL and inhibits EBV-mediated epithelial cell fusion, mimicking gp42. These observations reveal distinct functional requirements for gH/gL and gp42 complexes in EBV-mediated membrane fusion.

scholarly journals Mutations of Epstein-Barr Virus gH That Are Differentially Able To Support Fusion with B Cells or Epithelial Cells

2005 ◽  
Vol 79 (17) ◽  
pp. 10923-10930 ◽  
Author(s):  
Liguo Wu ◽  
Corina M. Borza ◽  
Lindsey M. Hutt-Fletcher
Keyword(s):  
B Cells ◽  
Epithelial Cell ◽  
Epithelial Cells ◽  
B Cell ◽  
Cell Fusion ◽  
Epstein Barr Virus ◽  
Common Mechanism ◽  
Barr Virus ◽  
The Core ◽  
Epstein Barr

ABSTRACT The core fusion machinery of all herpesviruses consists of three conserved glycoproteins, gB and gHgL, suggesting a common mechanism for virus cell fusion, but fusion of Epstein-Barr virus (EBV) with B cells and epithelial cells is initiated differently. Fusion with B cells requires a fourth protein, gp42, which complexes with gHgL and interacts with HLA class II, the B-cell coreceptor. Fusion with an epithelial cell does not require gp42 but requires interaction of gHgL with a novel epithelial cell coreceptor. Epithelial cell fusion can be inhibited by gp42 binding to gHgL and by antibodies to gH that fail to block B-cell fusion. This suggests that regions of gHgL initiating fusion with each cell are separable from each other and from regions involved in fusion itself. To address this possibility we mapped the region of gH recognized by a monoclonal antibody to gH that blocks EBV fusion with epithelial cells but not B cells by making a series of chimeras with the gH homolog of rhesus lymphocryptovirus. Proteins with mutations engineered within this region included those that preferentially mediate fusion with B cells, those that preferentially mediate fusion with epithelial cells, and those that mediate fusion with neither cell type. These results support the hypothesis that the core fusion function of gH is the same for B cells and epithelial cells and that it differs only in the way in which it is triggered into a functionally active state.

scholarly journals Epstein-Barr Virus Shed in Saliva Is High in B-Cell-Tropic Glycoprotein gp42

2006 ◽  
Vol 80 (14) ◽  
pp. 7281-7283 ◽  
Author(s):  
R. Jiang ◽  
R. S. Scott ◽  
L. M. Hutt-Fletcher
Keyword(s):  
Epithelial Cells ◽  
B Cell ◽  
Epstein Barr Virus ◽  
Human Herpesvirus ◽  
Cell Types ◽  
Class Ii ◽  
Hla Class Ii ◽  
Barr Virus ◽  
Epstein Barr ◽  
Differential Ability

ABSTRACT Epstein-Barr virus is an orally transmitted human herpesvirus that infects epithelial cells and establishes latency in memory B lymphocytes. Movement of virus between the two cell types is facilitated by changes in amounts of an envelope glycoprotein, gp42, which are effected by interaction of gp42 with HLA class II in a B cell. Here we used the differential ability of virus to bind to CD21-positive B cells and CD21-negative epithelial cells, which is also influenced by levels of gp42, to determine that the majority of virus shed in saliva is derived from an HLA class II-negative cell.

scholarly journals Epstein-Barr Virus Induces Adhesion Receptor CD226 (DNAM-1) Expression during Primary B-Cell Transformation into Lymphoblastoid Cell Lines

mSphere ◽  
2017 ◽  
Vol 2 (6) ◽  
Author(s):  
Lisa Grossman ◽  
Chris Chang ◽  
Joanne Dai ◽  
Pavel A. Nikitin ◽  
Dereje D. Jima ◽  
...  
Keyword(s):  
B Cells ◽  
B Cell ◽  
Cell Lines ◽  
Epstein Barr Virus ◽  
Human Herpesvirus ◽  
Barr Virus ◽  
Ebv Infection ◽  
Cellular Aggregation ◽  
Epstein Barr

ABSTRACT Epstein-Barr virus (EBV) is a common human herpesvirus that establishes latency in B cells. While EBV infection is asymptomatic for most individuals, immune-suppressed individuals are at significantly higher risk of a form of EBV latent infection in which infected B cells are reactivated, grow unchecked, and generate lymphomas. This form of latency is modeled in the laboratory by infecting B cells from the blood of normal human donors in vitro. In this model, we identified a protein called CD226 that is induced by EBV but is not normally expressed on B cells. Rather, it is known to play a role in aggregation and survival signaling of non-B cells in the immune system. Cultures of EBV-infected cells adhere to one another in “clumps,” and while the proteins that are responsible for this cellular aggregation are not fully understood, we hypothesized that this form of cellular aggregation may provide a survival advantage. In this article, we characterize the mechanism by which EBV induces this protein and its expression on lymphoma tissue and cell lines and characterize EBV-infected cell lines in which CD226 has been knocked out. Epstein-Barr virus (EBV), an oncogenic herpesvirus, infects and transforms primary B cells into immortal lymphoblastoid cell lines (LCLs), providing a model for EBV-mediated tumorigenesis. EBV transformation stimulates robust homotypic aggregation, indicating that EBV induces molecules that mediate cell-cell adhesion. We report that EBV potently induced expression of the adhesion molecule CD226, which is not normally expressed on B cells. We found that early after infection of primary B cells, EBV promoted an increase in CD226 mRNA and protein expression. CD226 levels increased further from early proliferating EBV-positive B cells to LCLs. We found that CD226 expression on B cells was independent of B-cell activation as CpG DNA failed to induce CD226 to the extent of EBV infection. CD226 expression was high in EBV-infected B cells expressing the latency III growth program, but low in EBV-negative and EBV latency I-infected B-lymphoma cell lines. We validated this correlation by demonstrating that the latency III characteristic EBV NF-κB activator, latent membrane protein 1 (LMP1), was sufficient for CD226 upregulation and that CD226 was more highly expressed in lymphomas with increased NF-κB activity. Finally, we found that CD226 was not important for LCL steady-state growth, survival in response to apoptotic stress, homotypic aggregation, or adhesion to activated endothelial cells. These findings collectively suggest that EBV induces expression of a cell adhesion molecule on primary B cells that may play a role in the tumor microenvironment of EBV-associated B-cell malignancies or facilitate adhesion in the establishment of latency in vivo. IMPORTANCE Epstein-Barr virus (EBV) is a common human herpesvirus that establishes latency in B cells. While EBV infection is asymptomatic for most individuals, immune-suppressed individuals are at significantly higher risk of a form of EBV latent infection in which infected B cells are reactivated, grow unchecked, and generate lymphomas. This form of latency is modeled in the laboratory by infecting B cells from the blood of normal human donors in vitro. In this model, we identified a protein called CD226 that is induced by EBV but is not normally expressed on B cells. Rather, it is known to play a role in aggregation and survival signaling of non-B cells in the immune system. Cultures of EBV-infected cells adhere to one another in “clumps,” and while the proteins that are responsible for this cellular aggregation are not fully understood, we hypothesized that this form of cellular aggregation may provide a survival advantage. In this article, we characterize the mechanism by which EBV induces this protein and its expression on lymphoma tissue and cell lines and characterize EBV-infected cell lines in which CD226 has been knocked out.

scholarly journals Epstein-Barr Virus Nuclear Antigen Leader Protein Coactivates EP300

2018 ◽  
Vol 92 (9) ◽  
Author(s):  
Chong Wang ◽  
Hufeng Zhou ◽  
Yong Xue ◽  
Jun Liang ◽  
Yohei Narita ◽  
...  
Keyword(s):  
Transcription Factors ◽  
B Cells ◽  
B Cell ◽  
Epstein Barr Virus ◽  
Nuclear Antigen ◽  
Transcription Activator ◽  
Lymphoproliferative Diseases ◽  
Barr Virus ◽  
Leader Protein ◽  
Epstein Barr

ABSTRACTEpstein-Barr virus nuclear antigen (EBNA) leader protein (EBNALP) is one of the first viral genes expressed upon B-cell infection. EBNALP is essential for EBV-mediated B-cell immortalization. EBNALP is thought to function primarily by coactivating EBNA2-mediated transcription. Chromatin immune precipitation followed by deep sequencing (ChIP-seq) studies highlight that EBNALP frequently cooccupies DNA sites with host cell transcription factors (TFs), in particular, EP300, implicating a broader role in transcription regulation. In this study, we investigated the mechanisms of EBNALP transcription coactivation through EP300. EBNALP greatly enhanced EP300 transcription activation when EP300 was tethered to a promoter. EBNALP coimmunoprecipitated endogenous EP300 from lymphoblastoid cell lines (LCLs). EBNALP W repeat serine residues 34, 36, and 63 were required for EP300 association and coactivation. Deletion of the EP300 histone acetyltransferase (HAT) domain greatly reduced EBNALP coactivation and abolished the EBNALP association. An EP300 bromodomain inhibitor also abolished EBNALP coactivation and blocked the EP300 association with EBNALP. EBNALP sites cooccupied by EP300 had significantly higher ChIP-seq signals for sequence-specific TFs, including SPI1, RelA, EBF1, IRF4, BATF, and PAX5. EBNALP- and EP300-cooccurring sites also had much higher H3K4me1 and H3K27ac signals, indicative of activated enhancers. EBNALP-only sites had much higher signals for DNA looping factors, including CTCF and RAD21. EBNALP coactivated reporters under the control of NF-κB or SPI1. EP300 inhibition abolished EBNALP coactivation of these reporters. Clustered regularly interspaced short palindromic repeat interference targeting of EBNALP enhancer sites significantly reduced target gene expression, including that of EP300 itself. These data suggest a previously unrecognized mechanism by which EBNALP coactivates transcription through subverting of EP300 and thus affects the expression of LCL genes regulated by a broad range of host TFs.IMPORTANCEEpstein-Barr virus was the first human DNA tumor virus discovered over 50 years ago. EBV is causally linked to ∼200,000 human malignancies annually. These cancers include endemic Burkitt lymphoma, Hodgkin lymphoma, lymphoma/lymphoproliferative disease in transplant recipients or HIV-infected people, nasopharyngeal carcinoma, and ∼10% of gastric carcinoma cases. EBV-immortalized human B cells faithfully model key aspects of EBV lymphoproliferative diseases and are useful models of EBV oncogenesis. EBNALP is essential for EBV to transform B cells and transcriptionally coactivates EBNA2 by removing repressors from EBNA2-bound DNA sites. Here, we found that EBNALP can also modulate the activity of the key transcription activator EP300, an acetyltransferase that activates a broad range of transcription factors. Our data suggest that EBNALP regulates a much broader range of host genes than was previously appreciated. A small-molecule inhibitor of EP300 abolished EBNALP coactivation of multiple target genes. These findings suggest novel therapeutic approaches to control EBV-associated lymphoproliferative diseases.

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

2006 ◽  
Vol 80 (19) ◽  
pp. 9628-9633 ◽  
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.

scholarly journals Compatibility of the gH homologues of Epstein–Barr virus and related lymphocryptoviruses

2007 ◽  
Vol 88 (8) ◽  
pp. 2129-2136 ◽  
Author(s):  
Liguo Wu ◽  
Lindsey M. Hutt-Fletcher
Keyword(s):  
B Cells ◽  
Epithelial Cells ◽  
B Cell ◽  
Cell Fusion ◽  
Epstein Barr Virus ◽  
Common Marmoset ◽  
Barr Virus ◽  
Human B Cells ◽  
The Common ◽  
Epstein Barr

Glycoprotein gH, together with its chaperone gL and a third glycoprotein gB, is essential for cell–cell fusion and virus–cell fusion mediated by herpesviruses. Epstein–Barr virus (EBV), the prototype human lymphocryptovirus, requires a fourth glycoprotein gp42 to support fusion with B cells in addition to epithelial cells. Two other lymphocryptoviruses, the rhesus lymphocryptovirus (Rh-LCV) and the common marmoset lymphocryptovirus (CalHV3), have been sequenced in their entirety and each has a gp42 homologue. Combinations of proteins from EBV, Rh-LCV and CalHV3 were able to mediate fusion of epithelial cells, but, even when complexed with EBV gp42, only Rh-LCV and not CalHV3 proteins were able to mediate fusion with human B cells. CalHV3 gL was also unable to function effectively as a chaperone for EBV or Rh-LCV gH. The Rh-LCV gH homologue supported more fusion than EBV gH with an epithelial cell and supported the highest levels of fusion with a B cell. Chimeric constructs made from Rh-LCV gH and EBV gH that have 85.4 % sequence identity should prove useful for mapping the regions of gH that are of importance to fusion as a whole and to B-cell fusion in particular.

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

2005 ◽  
Vol 79 (24) ◽  
pp. 15430-15442 ◽  
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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