scholarly journals The EBNA2 Polyproline Region Is Dispensable for Epstein-Barr Virus-Mediated Immortalization Maintenance

2002 ◽  
Vol 76 (14) ◽  
pp. 7349-7355 ◽  
Author(s):  
Alexey V. Gordadze ◽  
David Poston ◽  
Paul D. Ling
Keyword(s):  
B Cells ◽  
Cell Lines ◽  
Epstein Barr Virus ◽  
Gene Dosage ◽  
Nuclear Antigen ◽  
Striking Difference ◽  
Loss Of Function ◽  
Wild Type ◽  
Barr Virus ◽  
Epstein Barr

ABSTRACT Epstein-Barr virus nuclear antigen 2 (EBNA2) is required for EBV-mediated immortalization of primary human B cells and is a direct transcriptional activator of viral and cellular genes. The prototype EBNA2 protein contains a unique motif in which 43 out of 45 amino acids are prolines (polyproline region [PPR]). Previous genetic analysis has shown that deletion of the PPR resulted in viruses unable to immortalize B cells, although the protein did appear transcriptionally functional (R. Yalamanchili, S. Harada, and E. Kieff, J. Virol. 70:2468-2473, 1996). The PPR's uniqueness and requirement for immortalization make it an attractive therapeutic target. However, the role of this highly unusual motif for immortalization remains enigmatic. We have recently developed a transcomplementation assay that allows both genetic and functional analyses of EBNA2 in EBV-mediated immortalization maintenance (A. V. Gordadze, R. Peng, J. Tan, G. Liu, R. Sutton, B. Kempkes, G. W. Bornkamm, and P. D. Ling, J. Virol. 75:5899-5912, 2001). Surprisingly, we found that ΔPPR-EBNA2 was able to support B-cell proliferation similar to that of wild-type EBNA2 in this assay, indicating that deletion of the PPR from EBNA2 does not result in a loss of function required for immortalization maintenance. Further analysis of this mutant EBNA2 revealed that it consistently activated the viral LMP1 and LMP2A promoters severalfold better than wild-type EBNA2 in transient cotransfection assays. In addition, one striking difference between lymphoblastoid cell lines expressing wild-type EBNA2 from those expressing ΔPPR-EBNA2 is that the latter cells have significantly reduced EBV genomic levels. The data are consistent with a model in which lower EBNA2 target gene dosage may be selected for in ΔPPR-EBNA2-dependent cell lines to compensate for hyperactive stimulation of viral genes, such as LMP-1, which is cytostatic for B cells when overexpressed. It is conceivable that the hyperactivity rather than the loss of function, as hypothesized previously, could be responsible for the inability of recombinant ΔPPR-EBNA2 EBVs to immortalize B cells.

scholarly journals Epstein-Barr Virus Nuclear Antigen Leader Protein Induces Expression of Thymus- and Activation-Regulated Chemokine in B Cells

2004 ◽  
Vol 78 (8) ◽  
pp. 3984-3993 ◽  
Author(s):  
Mikiko Kanamori ◽  
Shinya Watanabe ◽  
Reiko Honma ◽  
Masayuki Kuroda ◽  
Shosuke Imai ◽  
...  
Keyword(s):  
B Cells ◽  
B Cell ◽  
Cell Lines ◽  
Epstein Barr Virus ◽  
Critical Role ◽  
Nuclear Antigen ◽  
Viral Gene ◽  
Barr Virus ◽  
Leader Protein ◽  
Epstein Barr

ABSTRACT Epstein-Barr virus (EBV) nuclear antigen leader protein (EBNA-LP) plays a critical role in transformation of primary B lymphocytes to continuously proliferating lymphoblastoid cell lines (LCLs). To identify cellular genes in B cells whose expression is regulated by EBNA-LP, we performed microarray expression profiling on an EBV-negative human B-cell line, BJAB cells, that were transduced by a retroviral vector expressing the EBV EBNA-LP (BJAB-LP cells) and on BJAB cells that were transduced with a control vector (BJAB-vec cells). Microarray analysis led to the identification of a cellular gene encoding the CC chemokine TARC as a novel target gene that was induced by EBNA-LP. The levels of TARC mRNA expression and TARC secretion were significantly up-regulated in BJAB-LP compared with BJAB-vec cells. Induction of TARC was also observed when a subline of BJAB cells was converted by a recombinant EBV. Among the EBV-infected B-cell lines with the latency III phenotype that were tested, the LCLs especially secreted significantly high levels of TARC. The level of TARC secretion appeared to correlate with the level of full-length EBNA-LP expression. These results indicate that EBV infection induces TARC expression in B cells and that EBNA-LP is one of the viral gene products responsible for the induction.

scholarly journals Determining the Role of the Epstein-Barr Virus Cp EBNA2-Dependent Enhancer during the Establishment of Latency by Using Mutant and Wild-Type Viruses Recovered from Cottontop Marmoset Lymphoblastoid Cell Lines

2000 ◽  
Vol 74 (23) ◽  
pp. 11115-11120 ◽  
Author(s):  
Lina Yoo ◽  
Samuel H. Speck
Keyword(s):  
Steady State ◽  
B Cells ◽  
Cell Lines ◽  
Epstein Barr Virus ◽  
Lymphoblastoid Cell Lines ◽  
Wild Type ◽  
Barr Virus ◽  
Human B Cells ◽  
Steady State Levels ◽  
Epstein Barr

ABSTRACT Epstein-Barr virus (EBV) nuclear antigen (EBNA) 2 (EBNA2) is involved in upregulating the expression of both EBNAs and latency-associated membrane proteins. Transcription of the six EBNA genes, which are expressed in EBV-immortalized primary B cells, arises from one of two promoters, Cp and Wp, located near the left end of the viral genome. Wp is exclusively used to drive EBNA gene transcription during the initial stages of infection in primary B cells; induction of transcription from Cp follows. We previously have mapped an EBNA2-dependent enhancer upstream of Cp (M. Woisetschlaeger et al., Proc. Natl. Acad. Sci. USA 88:3942–3946, 1991) and, more recently, have demonstrated that deletion of this enhancer results in EBV-immortalized lymphoblastoid cell lines (LCLs) that are heavily biased toward the use of Wp to drive transcription of the EBNA genes (L. Yoo et al., J. Virol. 71:9134–9142, 1997). To assess the immortalizing capacity of this mutant EBV and to monitor the early events after infection of primary B cells, B cells isolated from cottontop marmosets were used to generate LCLs immortalized with the Cp EBNA2 enhancer deletion mutant virus. As previously reported, all EBV-infected marmoset LCLs examined could be triggered to produce significant levels of virus. Infection of human B cells with wild-type or Cp EBNA2 enhancer mutant viruses recovered from marmoset B-cell lines demonstrated that (i) the Cp EBNA2 enhancer mutant virus immortalizes primary human B cells nearly as efficiently as wild-type virus and (ii) the Cp EBNA2-dependent enhancer plays an important role in the induction of Cp activity during the early stages of infection. The latter is consistent with the phenotype of LCLs immortalized with the Cp EBNA2 enhancer mutant EBV. Finally, using an established LCL in which EBNA2 function is regulated by β-estradiol, we showed that the loss of EBNA2 function results in an ∼4-fold decrease in the steady-state levels of Cp-initiated transcripts and a concomitant increase in the steady-state levels of Wp-initiated transcripts. Taken together, these results provide strong evidence that EBNA2 plays an important role in regulating Cp activity. These results also demonstrate that diminished induction of Cp activity does not appear to affect the ability of EBV to immortalize primary B cells in cultures. Finally, as shown here, infection of marmoset B cells with immortalization-competent mutants of EBV provides a convenient reservoir for the production of mutant viruses.

scholarly journals Epstein-Barr Virus (EBV) Latent Protein EBNA3A Directly Targets and Silences the STK39 Gene in B Cells Infected by EBV

2018 ◽  
Vol 92 (7) ◽  
Author(s):  
Quentin Bazot ◽  
Kostas Paschos ◽  
Martin J. Allday
Keyword(s):  
B Cells ◽  
B Cell ◽  
Cell Lines ◽  
Epstein Barr Virus ◽  
Nuclear Antigen ◽  
Lymphoblastoid Cell Lines ◽  
Barr Virus ◽  
Cellular Genes ◽  
Wide Range ◽  
Epstein Barr

ABSTRACT Epstein-Barr virus (EBV) establishes latent infection in human B cells and is associated with a wide range of cancers. The EBV nuclear antigen 3 (EBNA3) family proteins are critical for B cell transformation and function as transcriptional regulators. It is well established that EBNA3A and EBNA3C cooperate in the regulation of cellular genes. Here, we demonstrate that the gene STK39 is repressed only by EBNA3A. This is the first example of a gene regulated only by EBNA3A in EBV-transformed lymphoblastoid cell lines (LCLs) without the help of EBNA3C. This was demonstrated using a variety of LCLs carrying either knockout, revertant, or conditional EBNA3 recombinants. Investigating the kinetics of EBNA3A-mediated changes in STK39 expression showed that STK39 becomes derepressed quickly after EBNA3A inactivation. This derepression is reversible as EBNA3A reactivation represses STK39 in the same cells expressing a conditional EBNA3A. STK39 is silenced shortly after primary B cell infection by EBV, and no STK39 -encoded protein (SPAK) is detected 3 weeks postinfection. Chromatin immunoprecipitation (ChIP) analysis indicates that EBNA3A directly binds to a regulatory region downstream of the STK39 transcription start site. For the first time, we demonstrated that the polycomb repressive complex 2 with the deposition of the repressive mark H3K27me3 is not only important for the maintenance of an EBNA3A target gene ( STK39 ) but is also essential for the initial establishment of its silencing. Finally, we showed that DNA methyltransferases are involved in the EBNA3A-mediated repression of STK39 . IMPORTANCE EBV is well known for its ability to transform B lymphocytes to continuously proliferating lymphoblastoid cell lines. This is achieved in part by the reprogramming of cellular gene transcription by EBV transcription factors, including the EBNA3 proteins that play a crucial role in this process. In the present study, we found that EBNA3A epigenetically silences STK39 . This is the first gene where EBNA3A has been found to exert its repressive role by itself, without needing its coregulators EBNA3B and EBNA3C. Furthermore, we demonstrated that the polycomb repressor complex is essential for EBNA3A-mediated repression of STK39 . Findings in this study provide new insights into the regulation of cellular genes by the transcription factor EBNA3A.

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 The Epstein-Barr virus (EBV) nuclear antigen 1 BamHI F promoter is activated on entry of EBV-transformed B cells into the lytic cycle.

1992 ◽  
Vol 66 (12) ◽  
pp. 7461-7468 ◽  
Author(s):  
A L Lear ◽  
M Rowe ◽  
M G Kurilla ◽  
S Lee ◽  
S Henderson ◽  
...  
Keyword(s):  
B Cells ◽  
Epstein Barr Virus ◽  
Lytic Cycle ◽  
Nuclear Antigen ◽  
Barr Virus ◽  
Epstein Barr

scholarly journals Characterization of naturally Epstein–Barr virus-infected gastric carcinoma cell line YCCEL1

2013 ◽  
Vol 94 (3) ◽  
pp. 497-506 ◽  
Author(s):  
Do Nyun Kim ◽  
Min Koo Seo ◽  
Hoyun Choi ◽  
Su Yeon Kim ◽  
Hee Jong Shin ◽  
...  
Keyword(s):  
Cell Line ◽  
Cell Lines ◽  
Blot Analysis ◽  
Epstein Barr Virus ◽  
Model Systems ◽  
Nuclear Antigen ◽  
Gastric Carcinoma Cell ◽  
Barr Virus ◽  
Epstein Barr

Epstein–Barr virus (EBV) is a herpesvirus associated with lymphomas and carcinomas. While EBV-associated epithelial cell lines are good model systems to investigate the role of EBV in carcinoma, only a few cell lines are available as they are hard to acquire. A greater variety of naturally EBV-infected cell lines which are derived from tumour patients are needed to represent various features of EBVaGC. We characterized cell line YCCEL1, established from a Korean EBVaGC patient, to ascertain whether it can be used to study the roles of EBV in EBVaGC. The expression of EBV genes and cell surface markers was examined by in situ hybridization, RT-PCR, Western blot analysis, immunofluorescence assay and Northern blot analysis. EBV episomal status was analysed by Southern blotting and real-time PCR. This cell line expressed EBV nuclear antigen 1 (EBNA1) and latent membrane protein 2A (LMP2A), but not EBNA2, LMP2B nor LMP1. The majority of the lytic proteins were not detected in YCCEL1 cells either before or after treatment with 12-O-tetradecanoylphorbol-13-acetate. YCCEL1 cells expressed BART microRNAs (miRNAs) at high level but did not express BHRF1 miRNAs. YCCEL1 cells expressed cytokeratin, but not CD21 and CD19, suggesting CD21-independent EBV infection. The latent EBV gene and EBV miRNA expression pattern of YCCEL1 cells closely resembled that of general EBVaGC cases. Our results support the value of YCCEL1 cells as a good model system to study the role of EBV in gastric carcinogenesis.

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.

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