The Metastatic Suppressor Nm23-H1 Interacts with EBNA3C at Sequences Located between the Glutamine- and Proline-Rich Domains and Can Cooperate in Activation of Transcription

ABSTRACT Epstein-Barr virus (EBV) is a lymphotrophic herpesvirus infecting most of the world's population. It is associated with a number of human lymphoid and epithelial tumors and lymphoproliferative diseases in immunocompromised patients. Recent studies have shown an in vitro and in vivo interaction between the EBV nuclear antigen 3C (EBNA3C) and the metastatic suppressor Nm23-H1, known to be downregulated in human invasive breast carcinoma. In this study, we have identified the domain of EBNA3C that specifically binds to Nm23-H1. This domain lies within the region comprising amino acids 637 to 675 of EBNA3C flanked by the proline- and glutamine-rich domains. Furthermore, we show that Nm23-H1 activates transcription when fused to the Gal4 DNA-binding domain and is coexpressed with a luciferase reporter construct containing the Gal4 binding sites upstream of a basal promoter. Gal4-Nm23-H1, when tethered to the promoter by binding to the Gal4 DNA binding sequences, consistently activated transcription. The level of activation increased when increasing amounts of Gal4-Nm23-H1 were introduced into the system. Moreover, EBNA3C when cotransfected with Gal4-Nm23-H1 enhanced the transcriptional activity. These results suggest that Nm23-H1 may have intrinsic transcription activities in EBV-infected cells and that this activity can be modulated in the presence of the essential latent antigen EBNA3C.

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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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Epstein–Barr virus nuclear antigen (EBNA) 3A induces the expression of and interacts with a subset of chaperones and co-chaperones

Journal of General Virology ◽

10.1099/vir.0.83414-0 ◽

2008 ◽

Vol 89 (4) ◽

pp. 866-877 ◽

Cited By ~ 38

Author(s):

Paul Young ◽

Emma Anderton ◽

Kostas Paschos ◽

Rob White ◽

Martin J. Allday

Keyword(s):

Epstein Barr Virus ◽

Expression System ◽

Nuclear Antigen ◽

Human Diploid Fibroblasts ◽

Barr Virus ◽

Cellular Genes ◽

Infected Cells ◽

Epstein Barr ◽

Chaperone Complex

Viral nuclear oncoproteins EBNA3A and EBNA3C are essential for the efficient immortalization of B cells by Epstein–Barr virus (EBV) in vitro and it is assumed that they play an essential role in viral persistence in the human host. In order to identify cellular genes regulated by EBNA3A expression, cDNA encoding EBNA3A was incorporated into a recombinant adenoviral vector. Microarray analysis of human diploid fibroblasts infected with either adenovirus EBNA3A or an empty control adenovirus consistently showed an EBNA3A-specific induction of mRNA corresponding to the chaperones Hsp70 and Hsp70B/B′ and co-chaperones Bag3 and DNAJA1/Hsp40. Analysis of infected fibroblasts by real-time quantitative RT-PCR and Western blotting confirmed that EBNA3A, but not EBNA3C, induced expression of Hsp70, Hsp70B/B′, Bag3 and DNAJA1/Hsp40. This was also confirmed in a stable, inducible expression system. EBNA3A activated transcription from the Hsp70B promoter, but not multimerized heat-shock elements in transient transfection assays, consistent with specific chaperone and co-chaperone upregulation. Co-immunoprecipitation experiments suggest that EBNA3A can form a complex with the chaperone/co-chaperone proteins in both adenovirus-infected cells and EBV-immortalized lymphoblastoid cell lines. Consistent with this, induction of EBNA3A resulted in redistribution of Hsp70 from the cytoplasm to the nucleus. EBNA3A therefore specifically induces (and then interacts with) all of the factors necessary for an active Hsp70 chaperone complex.

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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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Epstein–Barr virus nuclear antigen 1 is a DNA-binding protein with strong RNA-binding activity

Journal of General Virology ◽

10.1099/vir.0.80239-0 ◽

2004 ◽

Vol 85 (10) ◽

pp. 2755-2765 ◽

Cited By ~ 19

Author(s):

Chih-Chung Lu ◽

Chia-Wei Wu ◽

Shin C. Chang ◽

Tzu-Yi Chen ◽

Chwan-Ren Hu ◽

...

Keyword(s):

Dna Binding ◽

Epstein Barr Virus ◽

Rna Binding ◽

Binding Protein ◽

Dna Binding Protein ◽

Binding Activity ◽

Nuclear Antigen ◽

Barr Virus ◽

Infected Cells ◽

Epstein Barr

Epstein–Barr virus (EBV) nuclear antigen 1 (EBNA-1) plays key roles in both the regulation of gene expression and the replication of the EBV genome in latently infected cells. To characterize the RNA-binding activity of EBNA-1, it was demonstrated that EBNA-1 binds efficiently to RNA homopolymers that are composed of poly(G) and weakly to those composed of poly(U). All three RGG boxes of EBNA-1 contributed additively to poly(G)-binding activity and could mediate RNA binding when attached to a heterologous protein in an RNA gel mobility-shift assay. In vitro-transcribed EBV and non-EBV RNA probes revealed that EBNA-1 bound to most RNAs examined and the affinity increased as the content of G and U increased, as demonstrated in competition assays. Among these probes, the 5′ non-coding region (NCR) (nt 131–278) of hepatitis C virus RNA appeared to be the strongest competitor for EBNA-1 binding to the EBV-encoded small nuclear RNA 1 (EBER1) probe, whereas a mutant 5′ NCR RNA with partially disrupted secondary structure was a weak competitor. Furthermore, the interaction of endogenous EBNA-1 and EBER1 in EBV-infected cells was demonstrated by a ribonucleoprotein immunoprecipitation assay. These results revealed that EBNA-1 is a DNA-binding protein with strong binding activity to a relatively broad spectrum of RNA and suggested an additional biological impact of EBNA-1 through its ability to bind to RNA.

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Epstein-Barr Virus Nuclear Antigen 3C Augments Mdm2-Mediated p53 Ubiquitination and Degradation by Deubiquitinating Mdm2

Journal of Virology ◽

10.1128/jvi.02408-08 ◽

2009 ◽

Vol 83 (9) ◽

pp. 4652-4669 ◽

Cited By ~ 81

Author(s):

Abhik Saha ◽

Masanao Murakami ◽

Pankaj Kumar ◽

Bharat Bajaj ◽

Karen Sims ◽

...

Keyword(s):

Ubiquitin Ligase ◽

Epstein Barr Virus ◽

Mutational Analysis ◽

Nuclear Antigen ◽

Cell Cycle Regulators ◽

Barr Virus ◽

Terminal Domain ◽

Epstein Barr

ABSTRACT Epstein-Barr virus (EBV) nuclear antigen 3C (EBNA3C) is one of the essential latent antigens for primary B-cell transformation. Previous studies established that EBNA3C facilitates degradation of several vital cell cycle regulators, including the retinoblastoma (pRb) and p27KIP proteins, by recruitment of the SCFSkp2 E3 ubiquitin ligase complex. EBNA3C was also shown to be ubiquitinated at its N-terminal residues. Furthermore, EBNA3C can bind to and be degraded in vitro by purified 20S proteasomes. Surprisingly, in lymphoblastoid cell lines, EBNA3C is extremely stable, and the mechanism for this stability is unknown. In this report we show that EBNA3C can function as a deubiquitination enzyme capable of deubiquitinating itself in vitro as well as in vivo. Functional mapping using deletion and point mutational analysis showed that both the N- and C-terminal domains of EBNA3C contribute to the deubiquitination activity. We also show that EBNA3C efficiently deubiquitinates Mdm2, an important cellular proto-oncogene, which is known to be overexpressed in several human cancers. The data presented here further demonstrate that the N-terminal domain of EBNA3C can bind to the acidic domain of Mdm2. Additionally, the N-terminal domain of EBNA3C strongly stabilizes Mdm2. Importantly, EBNA3C simultaneously binds to both Mdm2 and p53 and can form a stable ternary complex; however, in the presence of p53 the binding affinity of Mdm2 toward EBNA3C was significantly reduced, suggesting that p53 and Mdm2 might share a common overlapping domain of EBNA3C. We also showed that EBNA3C enhances the intrinsic ubiquitin ligase activity of Mdm2 toward p53, which in turn facilitated p53 ubiquitination and degradation. Thus, manipulation of the oncoprotein Mdm2 by EBNA3C potentially provides a favorable environment for transformation and proliferation of EBV-infected cells.

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The Cooperative Functions of the EBNA3 Proteins Are Central to EBV Persistence and Latency

Pathogens ◽

10.3390/pathogens7010031 ◽

2018 ◽

Vol 7 (1) ◽

pp. 31 ◽

Cited By ~ 7

Author(s):

Christine Styles ◽

Kostas Paschos ◽

Robert White ◽

Paul Farrell

Keyword(s):

Cell Transformation ◽

Nuclear Antigen ◽

Barr Virus ◽

Transcriptional Reprogramming ◽

Long Latency ◽

Cooperative Activity ◽

Epstein Barr ◽

Sequence Similarities

The Epstein–Barr nuclear antigen 3 (EBNA3) family of proteins, comprising EBNA3A, EBNA3B, and EBNA3C, play pivotal roles in the asymptomatic persistence and life-long latency of Epstein–Barr virus (EBV) in the worldwide human population. EBNA3-mediated transcriptional reprogramming of numerous host cell genes promotes in vitro B cell transformation and EBV persistence in vivo. Despite structural and sequence similarities, and evidence of substantial cooperative activity between the EBNA3 proteins, they perform quite different, often opposing functions. Both EBNA3A and EBNA3C are involved in the repression of important tumour suppressive pathways and are considered oncogenic. In contrast, EBNA3B exhibits tumour suppressive functions. This review focuses on how the EBNA3 proteins achieve the delicate balance required to support EBV persistence and latency, with emphasis on the contribution of the Allday laboratory to the field of EBNA3 biology.

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Epstein-Barr Virus Protein Can Upregulate Cyclo-Oxygenase-2 Expression through Association with the Suppressor of Metastasis Nm23-H1

Journal of Virology ◽

10.1128/jvi.80.3.1321-1331.2006 ◽

2006 ◽

Vol 80 (3) ◽

pp. 1321-1331 ◽

Cited By ~ 27

Author(s):

Rajeev Kaul ◽

Subhash C. Verma ◽

Masanao Murakami ◽

Ke Lan ◽

Tathagata Choudhuri ◽

...

Keyword(s):

Epstein Barr Virus ◽

Nuclear Antigen ◽

Luciferase Reporter ◽

Metastasis Suppressor ◽

Prostaglandin E ◽

Barr Virus ◽

Human Cancers ◽

Cox 2 ◽

Epstein Barr

ABSTRACT Previous studies have demonstrated the interaction between the Epstein-Barr virus (EBV) nuclear antigen 3C (EBNA3C) and the metastatic suppressor Nm23-H1 both in vitro and in vivo (C. Subramanian, M. A. Cotter II, and E. S. Robertson, Nat. Med. 7:350-355, 2001). EBNA3C can reverse the ability of Nm23-H1 to suppress migration of Burkitt's lymphoma and breast carcinoma cell lines in vitro. EBNA3C contributes to EBV-associated human cancers by regulating transcription of a number of cellular and viral promoters and by targeting and altering the transcription activities of the metastasis suppressor Nm23-H1. Cyclo-oxygenase-2 (COX-2), an inducible enzyme important in inflammation, is overexpressed in a variety of cancers and can influence cell migration. In this report we show that Nm23-H1 and EBNA3C can modulate expression of COX-2 in the context of EBV infection and transformation. The levels of COX-2 were consistently higher in EBV-positive cells than in EBV-negative cells. Additionally, we show that Nm23-H1 can upregulate the COX-2 promoter element in luciferase reporter assays, whereas EBNA3C alone did not affect the level of response but clearly contributed to an additive increase when coexpressed with Nm23-H1. The downstream effect of COX-2 expression was also evaluated and showed that prostaglandin E2 levels increased with Nm23-H1 and that there was some level of cooperativity in the presence of EBNA3C. The majority of this response was mediated through the cyclic AMP response element and NF-κB sites. These studies suggest a potential role for COX-2 in EBV-associated human cancers.

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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

Journal of Virology ◽

10.1128/jvi.74.22.10468-10479.2000 ◽

2000 ◽

Vol 74 (22) ◽

pp. 10468-10479 ◽

Cited By ~ 51

Author(s):

R. J. Tierney ◽

H. E. Kirby ◽

J. K. Nagra ◽

J. Desmond ◽

A. I. Bell ◽

...

Keyword(s):

B Cells ◽

Binding Sites ◽

Epstein Barr Virus ◽

Regulatory Region ◽

Nuclear Antigen ◽

Regulatory Sequences ◽

Barr Virus ◽

Epstein Barr

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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EBNA3C Can Modulate the Activities of the Transcription Factor Necdin in Association with Metastasis Suppressor Protein Nm23-H1

Journal of Virology ◽

10.1128/jvi.02286-08 ◽

2009 ◽

Vol 83 (10) ◽

pp. 4871-4883 ◽

Cited By ~ 21

Author(s):

Rajeev Kaul ◽

Masanao Murakami ◽

Ke Lan ◽

Tathagata Choudhuri ◽

Erle S. Robertson

Keyword(s):

Transcriptional Repression ◽

Cellular Protein ◽

Nuclear Antigen ◽

Metastasis Suppressor ◽

Barr Virus ◽

Human Cancers ◽

Differentiated Cells ◽

Epstein Barr

ABSTRACT Previous studies have demonstrated the interaction between the Epstein-Barr virus (EBV) nuclear antigen 3C (EBNA3C) and the metastatic suppressor Nm23-H1 both in vitro and in vivo (C. Subramanian, M. A. Cotter II, and E. S. Robertson, Nat. Med. 7:350-355, 2001). Importantly EBNA3C can reverse the ability of Nm23-H1 to suppress migration of human cells in vitro. EBNA3C contributes to EBV-associated human cancers by regulating transcription of a number of cellular and viral promoters as well as targeting and altering the transcription activities of the metastasis suppressor Nm23-H1. Furthermore, Necdin is a cellular protein which is highly induced in terminally differentiated cells; it contributes to the regulation of cell growth and is also known to interact with viral oncoproteins. In this report, we show that Nm23-H1 and EBNA3C can modulate the biological functions of Necdin in the context of EBV infection and transformation. The levels of Necdin were consistently lower in EBV-positive cells, and EBNA3C could change the subcellular localization of Necdin as well as rescue cells from the antiangiogenic and antiproliferative effects mediated by Necdin. We also show that Necdin directly interacts with Nm23-H1, resulting in modulation of the biochemical function of Nm23-H1 as well as the biological function of Necdin. Both EBNA3C and Nm23-H1 were able to rescue not only Necdin-mediated transcriptional repression of the downstream vascular endothelial growth factor promoter but also Necdin-mediated growth suppression and antiangiogenic effects on cancer cells. The majority of this response was mediated through amino acid residues 191 to 222 of Necdin, which are also known to be important for nuclear matrix targeting. These studies suggest a role for Necdin in the regulation of downstream cellular targets in a hypoxic environment in virus-associated human cancers.

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