Top PDF Epstein-Barr Virus Nuclear Antigen Leader Protein Coactivates EP300

Epstein-Barr Virus Nuclear Antigen Leader Protein Coactivates EP300

Epstein-Barr Virus Nuclear Antigen Leader Protein Coactivates EP300

IMPORTANCE Epstein-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, nasopha- ryngeal carcinoma, and ⬃10% of gastric carcinoma cases. EBV-immortalized human B cells faithfully model key aspects of EBV lymphoproliferative diseases and are use- ful 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 tran- scription activator EP300, an acetyltransferase that activates a broad range of tran- scription 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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Epstein-Barr Virus Nuclear Antigen Leader Protein Induces Expression of Thymus- and Activation-Regulated Chemokine in B Cells

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

Consistent with the in vitro data, it has been reported that TARC is associated with Th2-type diseases, such as atopic dermatitis and bronchial asthma (18, 32, 55, 56, 71). TARC, therefore, plays a key role in regulating the trafficking and effector functions of Th2 cells. These features of TARC render it an ideal target for a viral protein such as EBNA-LP, since it is well known that some viruses, including EBV, have evolved mechanisms to evade detection and ultimately deregulate the host immune response.

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Epstein-Barr virus nuclear antigen 2 activates transcription of the terminal protein gene.

Epstein-Barr virus nuclear antigen 2 activates transcription of the terminal protein gene.

Stable transfection of Epstein-Barr virus (EBV) nuclear antigen 2 in lymphoma cells containing the EBV P3HR1 genome induces expression of B-cell activation molecules CD21 and CD23. Ident[r]

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Epstein-Barr virus nuclear antigen 2 transactivates latent membrane protein LMP1.

Epstein-Barr virus nuclear antigen 2 transactivates latent membrane protein LMP1.

Epstein-Barr virus latent membrane protein (LMP1) and nuclear proteins 2 and 3C are effectors of phenotypic changes in B lymphocytes: EBNA-2 and LMP1 cooperatively induce CD23. A bicistr[r]

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Identification and characterization of a cellular protein that cross-reacts with the Epstein-Barr virus nuclear antigen.

Identification and characterization of a cellular protein that cross-reacts with the Epstein-Barr virus nuclear antigen.

This serum detected both the 72K EBNA component and the 62K cellular protein in the genome-positive cell lines (Fig. 2, lanes B through F) and the 62K protein in genome-negative cell lin[r]

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Epstein-Barr virus nuclear antigen 3C is a transcriptional regulator.

Epstein-Barr virus nuclear antigen 3C is a transcriptional regulator.

However, the DNA sequences through which EBNA-3C ex- erts activation of the LMP-1 promoter are likely to be distinct from the RBP-J k binding sites. The fact that EBNA-3C re- presses EBNA-2-stimulated transcription from the isolated EBNA-2 response element, which is composed of RBP-J k binding sites, clearly demonstrates that EBNA-3C does not act in a manner analogous to that of EBNA-2 by binding to the promoter via RBP-J k and providing an activation domain, even though the experiments described here demonstrate that EBNA-3C contains sequences which can act as a transcrip- tional activation domain. Since RBP-J k protein has been char- acterized as an inhibitor, one possible explanation for EBNA- 3C-mediated activation is the relief of inhibition by RBP-J k , leading to increased transcription as a result of the binding of other transcription factors to the promoter. Although this may explain activation by EBNA-3C alone, it does not explain aug- mented activity by EBNA-3C in the presence of EBNA-2, since mutation of the RBP-Jk sites leads to inhibition rather than increased activation of the promoter by EBNA-2, relative to that of the wild-type promoter. The most convincing demon- stration that EBNA-3C mediates its activation via distinct se- quences is its ability to activate the RBP-J k -deleted LMP-1 promoter. The ability of EBNA-3C to activate expression from the LMP-1 promoter is consistent with the findings of Allday and Farrell (2), who demonstrated that stable expression of EBNA-3C in Raji cells, which normally express only a trun- cated EBNA-3C protein, prevents a decrease in LMP-1 which otherwise occurs in growth-saturated arrest.
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Epstein-Barr virus nuclear antigen 2 induces expression of the virus-encoded latent membrane protein.

Epstein-Barr virus nuclear antigen 2 induces expression of the virus-encoded latent membrane protein.

Infection of Epstein-Barr virus-negative human B-lymphoma cell lines with the fully transforming B95.8 Epstein-Barr virus strain was associated with complete virus latent gene expression[r]

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Solubilization of the Epstein-Barr virus-determined nuclear antigen and its characterization as a DNA-binding protein.

Solubilization of the Epstein-Barr virus-determined nuclear antigen and its characterization as a DNA-binding protein.

The Epstein-Barr virus (EBV)-determined nuclear antigen (EBNA) was solu- bilized from isolated nuclei of two EBV-transformed cell lines, Raji and AW- Ramos, by high-salt treatment.. Its [r]

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Cellular Target Genes of Epstein-Barr Virus Nuclear Antigen 2

Cellular Target Genes of Epstein-Barr Virus Nuclear Antigen 2

In summary, CBF1 is not a low-abundance protein and the intracellular CBF1 concentration in DG75 is unlikely to be rate limiting. We thus would like to suggest an alternative scenario for CBF1 action. EBNA-2 might not only use CBF1 as a simple adaptor but in addition may facilitate CBF1/DNA complex formation. If CBF1/DNA complex formation were facilitated by EBNA-2 or cellular factors in the context of specific pro- moters, we would anticipate that the corresponding genes are not actively repressed by CBF1. Recently, it has been demon- strated that CBF1 binding to the promoter of CD23 and to the FcRH5 promoter is enhanced by EBNA-2 (41). This finding might support the latter scenario, which implies that target genes actively repressed by CBF1 carry specific or high-affinity binding sites.
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Conserved Region CR2 of Epstein-Barr Virus Nuclear Antigen Leader Protein Is a Multifunctional Domain That Mediates Self-Association as well as Nuclear Localization and Nuclear Matrix Association

Conserved Region CR2 of Epstein-Barr Virus Nuclear Antigen Leader Protein Is a Multifunctional Domain That Mediates Self-Association as well as Nuclear Localization and Nuclear Matrix Association

In conclusion, homotypic associates form between molecules of EBNA-LP and CR2, which is responsible for the self-asso- ciation and is a multifunctional domain mediating several aspects of EBNA-LP function. A series of recent reports including this study indicate that key functional domains of EBNA-LP center on the W repeat domain (11, 19, 31, 33, 51, 52). The EBNA-LP mutant viruses examined to date lack the ability to express the Y1Y2 domain, and studies of such viruses showed a much reduced but not a complete loss of transform- ing activity, leading to the consensus that EBNA-LP is not essential for the EBV-induced transformation process. It is, therefore, of interest and importance to determine whether EBNA-LP is truly a nonessential transforming protein by using a mutant virus that is unable to express the W1W2 domain of EBNA-LP.
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The Epstein-Barr virus nuclear protein encoded by the leader of the EBNA RNAs is important in B-lymphocyte transformation.

The Epstein-Barr virus nuclear protein encoded by the leader of the EBNA RNAs is important in B-lymphocyte transformation.

Thus, the phenotype of the deletion of the last 45 amino acids of EBNA-LP was profoundly evident when primary B lymphocytes were infected in an endpoint dilution clonal transformation as[r]

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Interaction of Epstein-Barr Virus Nuclear Antigen Leader Protein (EBNA-LP) with HS1-Associated Protein X-1: Implication of Cytoplasmic Function of EBNA-LP

Interaction of Epstein-Barr Virus Nuclear Antigen Leader Protein (EBNA-LP) with HS1-Associated Protein X-1: Implication of Cytoplasmic Function of EBNA-LP

Received 12 June 2000/Accepted 11 August 2000 Epstein-Barr virus (EBV) nuclear antigen leader protein (EBNA-LP) consists of W1W2 repeats and a unique C-terminal Y1Y2 domain and has been suggested to play an important role in EBV-induced transformation. To identify the cellular factors interacting with EBNA-LP, we performed a yeast two-hybrid screen, using EBNA-LP cDNA containing four W1W2 repeats as bait and an EBV-transformed human peripheral blood lymphocyte cDNA library as the source of cellular genes. Our results were as follows. (i) All three cDNAs in positive yeast colonies were found to encode the same cellular protein, HS1-associated protein X-1 (HAX-1), which is localized mainly in the cytoplasm and has been suggested to be involved in the regulation of B-cell signal transduction and apoptosis. (ii) Mutational analysis of EBNA-LP revealed that the association with HAX-1 is mediated by the W1W2 repeat domain. (iii) A purified chimeric protein consisting of glutathione S-transferase fused to EBNA-LP specifically formed complexes with HAX-1 transiently expressed in COS-7 cells. (iv) When EBNA-LP and HAX-1 were coexpressed in COS-7 cells, EBNA-LP was specifically coimmu- noprecipitated with HAX-1. (v) Careful cell fractionation experiments of an EBV-infected lymphoblastoid cell line revealed that EBNA-LP is localized in the cytoplasm as well as in the nucleus. (vi) When EBNA-LP containing four W1W2 repeats was expressed in COS-7 cells, EBNA-LP was detected mainly in the nucleus by immunofluorescence assay. Interestingly, when EBNA-LP containing a single W1W2 repeat was expressed in COS-7 cells, EBNA-LP was localized predominantly in the cytoplasm and was colocalized with HAX-1. These results indicate that EBNA-LP is in fact present and may have a significant function in the cytoplasm, possibly by interacting with and affecting the function of HAX-1.
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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

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

IRF4 binding sites in the Ig ␬ enhancer, the Spi site in the LMP-1 promoter did not support the assembly of a similar complex between Spi proteins and IRF4. Instead, our data demonstrate that the ets domains of both Spi-1 and Spi-B proteins interact with a region of EBNA-3C containing a likely bZIP domain. Although we have thus far been unable to detect a protein-DNA complex containing EBNA-3C and Spi pro- teins by EMSA, there is ample precedent for interactions be- tween a bZIP domain and an ets domain mediating the for- mation of a protein-DNA complex (4, 47). Thus, although EBNA-3C has no known DNA-binding capability, by providing a strong activation domain, it may provide a function analo- gous to that of IRF4 in its interaction with Spi-1. The simplest model that incorporates both the previous findings with EBNA-2 and the data presented here, therefore, is that EBNA-2, EBNA-3C, and Spi proteins are each needed to form a transcriptionally active complex on DNA.
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EBP2, a Human Protein That Interacts with Sequences of the Epstein-Barr Virus Nuclear Antigen 1 Important for Plasmid Maintenance

EBP2, a Human Protein That Interacts with Sequences of the Epstein-Barr Virus Nuclear Antigen 1 Important for Plasmid Maintenance

The replication and stable maintenance of latent Epstein-Barr virus (EBV) DNA episomes in human cells requires only one viral protein, Epstein-Barr nuclear antigen 1 (EBNA1). To gain insight into the mechanisms by which EBNA1 functions, we used a yeast two-hybrid screen to detect human proteins that interact with EBNA1. We describe here the isolation of a protein, EBP2 (EBNA1 binding protein 2), that specifically interacts with EBNA1. EBP2 was also shown to bind to DNA-bound EBNA1 in a one-hybrid system, and the EBP2-EBNA1 interaction was confirmed by coimmunoprecipitation from insect cells expressing these two proteins. EBP2 is a 35-kDa protein that is conserved in a variety of organisms and is predicted to form coiled-coil interactions. We have mapped the region of EBNA1 that binds EBP2 and generated internal deletion mutants of EBNA1 that are deficient in EBP2 interactions. Functional analyses of these EBNA1 mutants show that the ability to bind EBP2 correlates with the ability of EBNA1 to support the long-term maintenance in human cells of a plasmid containing the EBV origin, oriP. An EBNA1 mutant lacking amino acids 325 to 376 was defective for EBP2 binding and long-term oriP plasmid maintenance but supported the transient repli- cation of oriP plasmids at wild-type levels. Thus, our results suggest that the EBNA1-EBP2 interaction is important for the stable segregation of EBV episomes during cell division but not for the replication of the episomes.
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Purification and biochemical characterization of the Epstein-Barr virus-determined nuclear antigen and an associated protein with a 53,000-dalton subunit.

Purification and biochemical characterization of the Epstein-Barr virus-determined nuclear antigen and an associated protein with a 53,000-dalton subunit.

The Epstein-Barr virus-determined nuclear antigen (EBNA) was purified 700- fold to apparent homogeneity from Raji and Namalwa cell extracts by a three-.. step procedure involving heat tr[r]

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Epstein-Barr Virus Nuclear Antigen 3C Interacts with Histone Deacetylase To Repress Transcription

Epstein-Barr Virus Nuclear Antigen 3C Interacts with Histone Deacetylase To Repress Transcription

Consistent with these functional assays, in vitro-translated HDAC1 bound to a glutathione S-transferase (GST) fusion protein including full-length EBNA3C, and in the reciprocal experiment EBNA3C bound to a GST fusion with the N terminus of HDAC1. Coimmunoprecipitations also revealed an EBNA3C-HDAC1 interaction in vivo, and GST-EBNA3C bound functional histone deacetylase enzyme activity from HeLa cell nuclear extracts. The region of EBNA3C involved in the interaction with HDAC1 appears to correspond to the region which is necessary for binding to CBF1/RBP-J k . A direct physical interaction between EBNA3C and HDAC1 was demonstrated with recombinant proteins purified from bacterial cells, and we therefore conclude that HDAC1 and CBF1/RBP-J k bind to the same or adjacent regions of EBNA3C. These data suggest that recruitment of histone deacetylase activity makes a significant contribution to the repression of transcription from Cp because EBNA3C bridges an interaction between CBF1/RBP-J k and HDAC1.
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Epstein Barr virus induced autoimmune responses  I  Immunoglobulin M autoantibodies to proteins mimicking and not mimicking Epstein Barr virus nuclear antigen 1

Epstein Barr virus induced autoimmune responses I Immunoglobulin M autoantibodies to proteins mimicking and not mimicking Epstein Barr virus nuclear antigen 1

We have cloned and expressed fragments of genes encoding two of these autoantigens. One gene (p542) encodes a protein containing a glycine-rich 28-mer, which is its chief autoantigenic epitope and which represents a newly identified class of evolutionarily conserved autoepitopes. The other gene (p554) encodes a protein that is not demonstrably cross-reactive with Epstein-Barr virus nuclear antigen-1 or with any other EBV protein, but forms complexes with other proteins. Immunoaffinity-purified anti-p542 and anti-p554 have relatively high binding affinities, as evidenced by inhibition at 10(6)-10(8) M-1, and neither autoantibody showed polyreactivity with other common antigens. The data thus suggest that neither autoantibody is simply an expression of polyclonal B cell activation. We conclude that the two autoantigens stimulate autoantibody synthesis by different mechanisms. One autoantigen shares homology to a viral protein which generates cross-reacting antibodies to the autoantigenic epitope. The other has no recognizable cross-reaction with the infecting pathogen and may become immunogenic through complexing with other proteins.
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Epstein-Barr Virus Nuclear Antigen 1 Hijacks the Host Kinase CK2 To Disrupt PML Nuclear Bodies

Epstein-Barr Virus Nuclear Antigen 1 Hijacks the Host Kinase CK2 To Disrupt PML Nuclear Bodies

Nirojini Sivachandran,† Jennifer Yinuo Cao,† and Lori Frappier* Department of Molecular Genetics, University of Toronto, 1 Kings College Circle, Toronto, Ontario M5S 1A8, Canada Received 2 June 2010/Accepted 12 August 2010 Latent Epstein-Barr virus (EBV) infection is an important causative factor in the development of several cancers, including nasopharyngeal carcinoma (NPC). The one EBV protein expressed in the nucleus of NPC cells, EBNA1, has been shown to disrupt promyelocitic leukemia (PML) nuclear bodies (NBs) by inducing the degradation of PML proteins, leading to impaired DNA repair and increased cell survival. Although EBNA1- mediated PML disruption is likely to be an important factor in the development of NPC, little is known about its mechanism. We now show that an interaction between EBNA1 and the host CK2 kinase is crucial for EBNA1 to disrupt PML bodies and degrade PML proteins. EBNA1 increases the association of CK2 with PML proteins, thereby increasing the phosphorylation of PML proteins by CK2, a modification that is known to trigger the polyubiquitylation and degradation of PML. The interaction between EBNA1 and CK2 is direct and occurs through the ␤ regulatory subunit of CK2 and EBNA1 amino acids 387 to 394. The binding of EBNA1 to the host ubiquitin specific protease USP7 has also been shown to be important for EBNA1-mediated PML disruption. We show that EBNA1 also increases the occupancy of USP7 at PML NBs and that CK2 and USP7 bind independently and simultaneously to EBNA1 to form a ternary complex. The combined results indicate that EBNA1 usurps two independent cellular pathways to trigger the loss of PML NBs.
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Epstein-Barr virus nuclear antigen 3C is a powerful repressor of transcription when tethered to DNA.

Epstein-Barr virus nuclear antigen 3C is a powerful repressor of transcription when tethered to DNA.

Virol. 69:3624–3630, 1995). Although EBNA3C binds DNA, a specific site for EBNA3C binding has not been identified; to test the ability of full-length EBNA3C to regulate transcription, EBNA3C (amino acids 11 to 992) was fused to the DNA-binding domain of GAL4. We show that this fusion protein does not transactivate but rather is a potent repressor of reporter gene expression. Repression is dependent on the dose of GAL4- EBNA3C and on the presence of GAL4-binding sites within reporter plasmids. Repression is not restricted to B cells nor is it species or promoter specific. Repression is independent of the location of the GAL4-binding sites relative to the transcription start site. A fragment of EBNA3C (amino acids 280 to 525) which represses expression in a manner which is nearly identical to that of the full-length protein has been identified; this fragment is rich in acidic and proline residues. A second, less potent repressor region located C terminal to amino acids 280 to 525 has also been identified; this domain is rich in proline and glutamine residues. We also show binding of EBNA3C, in vitro, to the TATA-binding protein component of TFIID, and this suggests a mechanism by which EBNA3C may communicate with the basal transcription complex.
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Epstein-Barr Virus Nuclear Antigen 3C Facilitates Cell Proliferation by Regulating Cyclin D2

Epstein-Barr Virus Nuclear Antigen 3C Facilitates Cell Proliferation by Regulating Cyclin D2

IMPORTANCE Epstein-Barr virus (EBV) is the first identified human tumor virus and is associated with a range of human cancers. During EBV-induced lymphomas, the essential viral latent proteins modify the expression of cell cycle-related proteins to disturb the cell cycle process, thereby facilitating the proliferative process. The es- sential EBV nuclear antigen 3C (EBNA3C) plays an important role in EBV-mediated B-cell transformation. Here we show that EBNA3C stabilizes cyclin D2 to regulate cell cycle progression. More specifically, EBNA3C directly binds to cyclin D2, and they co- localize together in nuclear compartments. EBNA3C enhances cyclin D2 stability by inhibiting its ubiquitin-dependent degradation and significantly promotes cell prolif- eration in the presence of cyclin D2. Our results provide novel insights into the func- tion of EBNA3C on cell progression by regulating the cyclin D2 protein and raise the possibility of the development of new anticancer therapies against EBV-associated cancers.
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