Methylation of Transcription Factor Binding Sites in the Epstein-Barr Virus Latent Cycle Promoter Wp Coincides with Promoter Down-Regulation during Virus-Induced B-Cell Transformation

ABSTRACT Two Epstein-Barr virus latent cycle promoters for nuclear antigen expression, Wp and Cp, are activated sequentially during virus-induced transformation of B cells to B lymphoblastoid cell lines (LCLs) in vitro. Previously published restriction enzyme studies have indicated hypomethylation of CpG dinucleotides in the Wp and Cp regions of the viral genome in established LCLs, whereas these same regions appeared to be hypermethylated in Burkitt's lymphoma cells, where Wp and Cp are inactive. Here, using the more sensitive technique of bisulfite genomic sequencing, we reexamined the situation in established LCLs with the typical pattern of dominant Cp usage; surprisingly, this showed substantial methylation in the 400-bp regulatory region upstream of the Wp start site. This was not an artifact of long-term in vitro passage, since, in cultures of recently infected B cells, we found progressive methylation of Wp (but not Cp) regulatory sequences occurring between 7 and 21 days postinfection, coincident with the period in which dominant nuclear antigen promoter usage switches from Wp to Cp. Furthermore, in the equivalent in vivo situation, i.e., in the circulating B cells of acute infectious mononucleosis patients undergoing primary EBV infection, we again frequently observed selective methylation of Wp but not Cp sequences. An effector role for methylation in Wp silencing was supported by methylation cassette assays of Wp reporter constructs and by bandshift assays, where the binding of two sets of transcription factors important for Wp activation in B cells, BSAP/Pax5 and CREB/ATF proteins, was shown to be blocked by methylation of their binding sites.

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Epstein-Barr Virus LMP2A Alters In Vivo and In Vitro Models of B-Cell Anergy, but Not Deletion, in Response to Autoantigen

Journal of Virology ◽

10.1128/jvi.79.12.7355-7362.2005 ◽

2005 ◽

Vol 79 (12) ◽

pp. 7355-7362 ◽

Cited By ~ 39

Author(s):

Michelle A. Swanson-Mungerson ◽

Robert G. Caldwell ◽

Rebecca Bultema ◽

Richard Longnecker

Keyword(s):

B Cells ◽

B Cell ◽

Epstein Barr Virus ◽

Nuclear Translocation ◽

Cell Receptor ◽

Barr Virus ◽

Low Levels ◽

Epstein Barr

ABSTRACT A significant percentage of the population latently harbors Epstein-Barr virus (EBV) in B cells. One EBV-encoded protein, latent membrane protein 2A (LMP2A), is expressed in tissue culture models of EBV latent infection, in human infections, and in many of the EBV-associated proliferative disorders. LMP2A constitutively activates proteins involved in the B-cell receptor (BCR) signal transduction cascade and inhibits the antigen-induced activation of these proteins. In the present study, we investigated whether LMP2A alters B-cell receptor signaling in primary B cells in vivo and in vitro. LMP2A does not inhibit antigen-induced tolerance in response to strong stimuli in an in vivo tolerance model in which B cells are reactive to self-antigen. In contrast, LMP2A bypasses anergy induction in response to low levels of soluble hen egg lysozyme (HEL) both in vivo and in vitro as determined by the ability of LMP2A-expressing HEL-specific B cells to proliferate and induce NF-κB nuclear translocation after exposure to low levels of antigen. Furthermore, LMP2A induces NF-κB nuclear translocation independent of BCR cross-linking. Since NF-κB is required to bypass tolerance induction, this LMP2A-dependent NF-κB activation may complete the tolerogenic signal induced by low levels of soluble HEL. Overall, the findings suggest that LMP2A may not inhibit BCR-induced signals under all conditions as previously suggested by studies with EBV immortalized B cells.

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Epstein-Barr Virus Nuclear Antigen 3C Activates the Latent Membrane Protein 1 Promoter in the Presence of Epstein-Barr Virus Nuclear Antigen 2 through Sequences Encompassing an Spi-1/Spi-B Binding Site

Journal of Virology ◽

10.1128/jvi.74.11.5151-5160.2000 ◽

2000 ◽

Vol 74 (11) ◽

pp. 5151-5160 ◽

Cited By ~ 81

Author(s):

Bo Zhao ◽

Clare E. Sample

Keyword(s):

Dna Binding ◽

Binding Site ◽

Binding Sites ◽

Epstein Barr Virus ◽

Latent Membrane Protein ◽

Nuclear Antigen ◽

Latent Membrane Protein 1 ◽

Barr Virus ◽

Epstein Barr

ABSTRACT The Epstein-Barr virus (EBV) nuclear antigen 3C (EBNA-3C) protein is a transcriptional regulator of viral and cellular genes that is essential for EBV-mediated immortalization of B lymphocytes in vitro. EBNA-3C can inhibit transcription through an association with the cellular DNA-binding protein Jκ, a function shared by EBNA-3A and EBNA-3B. Here, we report a mechanism by which EBNA-3C can activate transcription from the EBV latent membrane protein 1 (LMP-1) promoter in conjunction with EBNA-2. Jκ DNA-binding sites were not required for this activation, and a mutant EBNA-3C protein unable to bind Jκ activated transcription as efficiently as wild-type EBNA-3C, indicating that EBNA-3C can regulate transcription through a mechanism that is independent of Jκ. Furthermore, activation of the LMP-1 promoter is a unique function of EBNA-3C, not shared by EBNA-3A and EBNA-3B. The DNA element through which EBNA-3C activates the LMP-1 promoter includes a Spi-1/Spi-B binding site, previously characterized as an important EBNA-2 response element. Although this element has considerable homology to mouse immunoglobulin light chain promoter sequences to which the mouse homologue of Spi-1 binds with its dimerization partner IRF4, we demonstrate that the IRF4-like binding sites in the LMP-1 promoter do not play a role in EBNA-3C-mediated activation. Both EBNA-2 and EBNA-3C were required for transcription mediated through a 41-bp region of the LMP-1 promoter encompassing the Spi binding site. However, EBNA-3C had no effect on transcription mediated in conjunction with the EBNA-2 activation domain fused to the GAL4 DNA-binding domain, suggesting that it does not function as an adapter between EBNA-2 and the cellular transcriptional machinery. Like EBNA-2, EBNA-3C bound directly to both Spi-1 and Spi-B in vitro. This interaction was mediated by a region of EBNA-3C encompassing a likely basic leucine zipper (bZIP) domain and the ets domain of Spi-1 or Spi-B, reminiscent of interactions between bZIP and ets domains of other transcription factors that result in their targeting to DNA. There are many examples of regulation of the hematopoietic-specific Spi transcription factors through protein-protein interactions, and a similar regulation by EBNA-3C, in conjunction with EBNA-2, is likely to be an important and unique contribution of EBNA-3C to EBV-mediated immortalization.

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The Epstein-Barr virus nuclear protein 2 acidic domain forms a complex with a novel cellular coactivator that can interact with TFIIE.

Molecular and Cellular Biology ◽

10.1128/mcb.15.9.4735 ◽

1995 ◽

Vol 15 (9) ◽

pp. 4735-4744 ◽

Cited By ~ 179

Author(s):

X Tong ◽

R Drapkin ◽

R Yalamanchili ◽

G Mosialos ◽

E Kieff

Keyword(s):

Epstein Barr Virus ◽

Nuclear Protein ◽

Lymphocyte Transformation ◽

Nuclear Antigen ◽

Cdna Clones ◽

Barr Virus ◽

Acidic Domain ◽

Epstein Barr

Epstein-Barr virus nuclear antigen 2 (EBNA 2) activates transcription of specific genes and is essential for B-lymphocyte transformation. EBNA 2 has an acidic activation domain which interacts with general transcription factors TFIIB, TFIIH, and TAF40. We now show that EBNA 2 is specifically bound to a novel nuclear protein, p100, and that p100 can coactivate gene expression mediated by the EBNA 2 acidic domain. The EBNA 2 acidic domain was used to affinity purify p100. cDNA clones encoding the p100 open reading frame were identified on the basis of peptide sequences of the purified protein. Antibody against p100 coimmunoprecipitated p100 and EBNA 2 from Epstein-Barr virus-transformed lymphocyte extracts, indicating that EBNA 2 and p100 are complexed in vivo. p100 overexpression in cells specifically augmented EBNA 2 acidic domain-mediated activation. The coactivating effect is probably mediated by p100 interaction with TFIIE. Bacterially expressed p100 specifically adsorbs TFIIE from nuclear extracts, and in vitro-translated p56 or p34 TFIIE subunit can independently bind to p100. p100 also appears to be essential for normal cell growth, since cell viability was reduced by antisense p100 RNA and restored by sense p100 RNA expression.

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Physical and Functional Interactions between the Corepressor CtBP and the Epstein-Barr Virus Nuclear Antigen EBNA3C

Journal of Virology ◽

10.1128/jvi.75.16.7749-7755.2001 ◽

2001 ◽

Vol 75 (16) ◽

pp. 7749-7755 ◽

Cited By ~ 75

Author(s):

Robert Touitou ◽

Mark Hickabottom ◽

Gillian Parker ◽

Tim Crook ◽

Martin J. Allday

Keyword(s):

Epstein Barr Virus ◽

Nuclear Antigen ◽

Rat Embryo ◽

Barr Virus ◽

Functional Interactions ◽

C Terminus ◽

Epstein Barr ◽

Repressor Activity

ABSTRACT CtBP has been shown to be a highly conserved corepressor of transcription. E1A and all the various transcription factors to which CtBP binds contain a conserved PLDLS CtBP-interacting domain, and EBNA3C includes a PLDLS motif (amino acids [aa] 728 to 732). Here we show that EBNA3C binds to CtBP both in vitro and in vivo and that the interaction requires an intact PLDLS. The C terminus of EBNA3C (aa 580 to 992) has modest trans-repressor activity when it is fused to the DNA-binding domain of Gal4, and deletion or mutation of the PLDLS sequence ablates this and unmasks a transactivation function within the fragment. However, loss of the CtBP interaction motif had little effect on the ability of full-length EBNA3C to repress transcription. A striking correlation between CtBP binding and the capacity of EBNA3C to cooperate with (Ha-)Ras in the immortalization and transformation of primary rat embryo fibroblasts was also revealed.

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Nuclear Factor-κB Binds to the Epstein-Barr Virus LMP1 Promoter and Upregulates Its Expression

Journal of Virology ◽

10.1128/jvi.01637-08 ◽

2008 ◽

Vol 83 (3) ◽

pp. 1393-1401 ◽

Cited By ~ 29

Author(s):

Pegah Johansson ◽

Ann Jansson ◽

Ulla Rüetschi ◽

Lars Rymo

Keyword(s):

B Cells ◽

Epstein Barr Virus ◽

Affinity Purification ◽

Critical Role ◽

Mobility Shift ◽

Barr Virus ◽

Exogenous Expression ◽

Epstein Barr

ABSTRACT The latent membrane protein 1 (LMP1) oncogene carried by Epstein-Barr virus (EBV) is essential for transformation and maintenance of EBV-immortalized B cells in vitro, and it is expressed in most EBV-associated tumor types. The activation of the NF-κB pathway by LMP1 plays a critical role in the upregulation of antiapoptotic proteins. The EBV-encoded EBNA2 transactivator is required for LMP1 activation in latency III, while LMP1 itself appears to be critical for its activation in the latency II gene expression program. In both cases, additional viral and cellular transcription factors are required in mediating transcription activation of the LMP1 promoter. Using DNA affinity purification and chromatin immunoprecipitation assay, we showed here that members of the NF-κB transcription factor family bound to the LMP1 promoter in vitro and in vivo. Electrophoretic mobility shift assay analyses indicated the binding of the p50-p50 homodimer and the p65-p50 heterodimer to an NF-κB site in the LMP1 promoter. Transient transfections and reporter assays showed that the LMP1 promoter is activated by exogenous expression of NF-κB factors in both B cells and epithelial cells. Exogenous expression of NF-κB factors in the EBNA2-deficient P3HR1 cell line induced LMP1 protein expression. Overall, our data are consistent with the presence of a positive regulatory circuit between NF-κB activation and LMP1 expression.

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Epstein-Barr Virus LMP2A Enhances B-Cell Responses In Vivo and In Vitro

Journal of Virology ◽

10.1128/jvi.00433-06 ◽

2006 ◽

Vol 80 (14) ◽

pp. 6764-6770 ◽

Cited By ~ 27

Author(s):

Michelle Swanson-Mungerson ◽

Rebecca Bultema ◽

Richard Longnecker

Keyword(s):

B Cells ◽

B Cell ◽

Epstein Barr Virus ◽

Plasma Cells ◽

Barr Virus ◽

Epstein Barr ◽

Hen Egg ◽

Hen Egg Lysozyme

ABSTRACT Epstein-Barr virus (EBV) establishes latent infections in a significant percentage of the population. Latent membrane protein 2A (LMP2A) is an EBV protein expressed during latency that inhibits B-cell receptor signaling in lymphoblastoid cell lines. In the present study, we have utilized a transgenic mouse system in which LMP2A is expressed in B cells that are specific for hen egg lysozyme (E/HEL-Tg). To determine if LMP2A allows B cells to respond to antigen, E/HEL-Tg mice were immunized with hen egg lysozyme. E/HEL-Tg mice produced antibody in response to antigen, indicating that LMP2A allows B cells to respond to antigen. In addition, E/HEL-Tg mice produced more antibody and an increased percentage of plasma cells after immunization compared to HEL-Tg littermates, suggesting that LMP2A increased the antibody response in vivo. Finally, in vitro studies determined that LMP2A acts directly on the B cell to increase antibody production by augmenting the expansion and survival of the activated B cells, as well as increasing the percentage of plasma cells generated. Taken together, these data suggest that LMP2A enhances, not diminishes, B-cell-specific antibody responses in vivo and in vitro in the E/HEL-Tg system.

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c-Myc and Rel/NF-κB Are the Two Master Transcriptional Systems Activated in the Latency III Program of Epstein-Barr Virus-Immortalized B Cells

Journal of Virology ◽

10.1128/jvi.02264-08 ◽

2009 ◽

Vol 83 (10) ◽

pp. 5014-5027 ◽

Cited By ~ 41

Author(s):

Nathalie Faumont ◽

Stéphanie Durand-Panteix ◽

Martin Schlee ◽

Sebastian Grömminger ◽

Marino Schuhmacher ◽

...

Keyword(s):

B Cells ◽

Cell Lines ◽

Epstein Barr Virus ◽

Expression Patterns ◽

Dominant Negative ◽

Barr Virus ◽

Conditional Expression ◽

Epstein Barr

ABSTRACT The Epstein-Barr virus (EBV) latency III program imposed by EBNA2 and LMP1 is directly responsible for immortalization of B cells in vitro and is thought to mediate most immunodeficiency-related posttransplant lymphoproliferative diseases in vivo. To answer the question whether and how this proliferation program is related to c-Myc, we have established the transcriptome of both c-Myc and EBV latency III proliferation programs using a Lymphochip specialized microarray. In addition to EBV-positive latency I Burkitt lymphoma lines and lymphoblastoid cell lines (LCLs), we used an LCL expressing an estrogen-regulatable EBNA2 fusion protein (EREB2-5) and derivative B-cell lines expressing a constitutively active or tetracycline-regulatable c-myc gene. A total of 897 genes were found to be fourfold or more up- or downregulated in either one or both proliferation programs compared to the expression profile of resting EREB2-5 cells. A total of 661 (74%) of these were regulated similarly in both programs. Numerous repressed genes were known targets of STAT1, and most induced genes were known to be upregulated by c-Myc and to be involved in cell proliferation. In keeping with the gene expression patterns, inactivation of c-Myc by a chemical inhibitor or by conditional expression of dominant-negative c-Myc and Max mutants led to proliferation arrest of LCLs. Most genes differently regulated in both proliferation programs corresponded to genes induced by NF-κB in LCLs, and many of them coded for immunoregulatory and/or antiapoptotic molecules. Thus, c-Myc and NF-κB are the two main transcription factors responsible for the phenotype, growth pattern, and biological properties of cells driven into proliferation by EBV.

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Faculty Opinions recommendation of The Epstein-Barr virus induces the expression of the LPAM-1 integrin in B-cells in vitro and in vivo.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.734628037.793554825 ◽

2019 ◽

Author(s):

Micah Luftig ◽

Brent Stanfield

Keyword(s):

B Cells ◽

Epstein Barr Virus ◽

Barr Virus ◽

Epstein Barr

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Novel Therapeutics for Epstein–Barr Virus

Molecules ◽

10.3390/molecules24050997 ◽

2019 ◽

Vol 24 (5) ◽

pp. 997 ◽

Cited By ~ 14

Author(s):

Graciela Andrei ◽

Erika Trompet ◽

Robert Snoeck

Keyword(s):

B Cells ◽

Epithelial Cells ◽

Epstein Barr Virus ◽

Adult Population ◽

Antiviral Drug ◽

Barr Virus ◽

Epstein Barr ◽

New Strategies

Epstein–Barr virus (EBV) is a human γ-herpesvirus that infects up to 95% of the adult population. Primary EBV infection usually occurs during childhood and is generally asymptomatic, though the virus can cause infectious mononucleosis in 35–50% of the cases when infection occurs later in life. EBV infects mainly B-cells and epithelial cells, establishing latency in resting memory B-cells and possibly also in epithelial cells. EBV is recognized as an oncogenic virus but in immunocompetent hosts, EBV reactivation is controlled by the immune response preventing transformation in vivo. Under immunosuppression, regardless of the cause, the immune system can lose control of EBV replication, which may result in the appearance of neoplasms. The primary malignancies related to EBV are B-cell lymphomas and nasopharyngeal carcinoma, which reflects the primary cell targets of viral infection in vivo. Although a number of antivirals were proven to inhibit EBV replication in vitro, they had limited success in the clinic and to date no antiviral drug has been approved for the treatment of EBV infections. We review here the antiviral drugs that have been evaluated in the clinic to treat EBV infections and discuss novel molecules with anti-EBV activity under investigation as well as new strategies to treat EBV-related diseases.

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