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

Pathogens ◽  
2018 ◽  
Vol 7 (1) ◽  
pp. 31 ◽  
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.

scholarly journals Epstein-Barr Virus Nuclear Antigen 2 Binds via Its Methylated Arginine-Glycine Repeat to the Survival Motor Neuron Protein

2003 ◽  
Vol 77 (8) ◽  
pp. 5008-5013 ◽  
Author(s):  
Stephanie Barth ◽  
Michael Liss ◽  
Marc D. Voss ◽  
Thomas Dobner ◽  
Utz Fischer ◽  
...  
Keyword(s):  
Transcriptional Activation ◽  
Epstein Barr Virus ◽  
Cell Transformation ◽  
Survival Motor Neuron ◽  
Nuclear Antigen ◽  
Survival Motor Neuron Protein ◽  
Barr Virus ◽  
Epstein Barr

ABSTRACT Here we provide evidence that EBNA2 is methylated in vivo and that methylation of EBNA2 is a prerequisite for binding to SMN. We present SMN as a novel binding partner of EBNA2 by showing that EBNA2 colocalizes with SMN in nuclear gems and that both proteins can be coimmunoprecipitated from cellular extract. Furthermore, in vitro methylation of either wild-type EBNA2 or a glutathione S-transferase-EBNA2 fusion protein encompassing the arginine-glycine (RG) repeat element is necessary for in vitro binding to the Tudor domain of SMN. The recently shown functional cooperation of SMN and EBNA2 in transcriptional activation and the previous observation of a severely reduced transformation potential yet strongly enhanced transcriptional activity of an EBNA2 mutant lacking the RG repeat indicate that binding of SMN to EBNA2 is a critical step in B-cell transformation by Epstein-Barr virus.

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

1995 ◽  
Vol 15 (9) ◽  
pp. 4735-4744 ◽  
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.

scholarly journals 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

2002 ◽  
Vol 76 (17) ◽  
pp. 8702-8709 ◽  
Author(s):  
Chitra Subramanian ◽  
Erle S. Robertson
Keyword(s):  
Dna Binding ◽  
Nuclear Antigen ◽  
Luciferase Reporter ◽  
Luciferase Reporter Construct ◽  
Barr Virus ◽  
Activation Of Transcription ◽  
Infected Cells ◽  
Epstein Barr

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.

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

2001 ◽  
Vol 75 (16) ◽  
pp. 7749-7755 ◽  
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.

scholarly journals Epstein–Barr virus-induced B-cell transformation: quantitating events from virus binding to cell outgrowth

2005 ◽  
Vol 86 (11) ◽  
pp. 3009-3019 ◽  
Author(s):  
Claire Shannon-Lowe ◽  
Gouri Baldwin ◽  
Regina Feederle ◽  
Andrew Bell ◽  
Alan Rickinson ◽  
...  
Keyword(s):  
B Cell ◽  
Cell Nucleus ◽  
Epstein Barr Virus ◽  
Cell Activation ◽  
Cell Transformation ◽  
Nuclear Antigen ◽  
Virus Binding ◽  
Barr Virus ◽  
Epstein Barr

Epstein–Barr virus (EBV) infection and growth activation of human B cells is central to virus biology and disease pathogenesis, but is poorly understood in quantitative terms. Here, using virus at defined m.o.i., the different stages of this process at the single-cell level are followed in vitro. Virus binding to the B-cell surface, assayed by quantitative PCR, is highly efficient, particularly at the low m.o.i. values that most likely reflect physiologic events in vivo. However, only 10–15 % of bound virus genomes reach the cell nucleus, as visualized by sensitive fluorescence in situ hybridization (FISH) assay; viral genomes acquired per cell nucleus range from 1 to >10, depending on the m.o.i. Thereafter, despite differences in initial genome load, almost all nuclear genome-positive cells then go on to express the virus-encoded nuclear antigen EBNA2, upregulate the cell activation antigen CD23 and transit the cell cycle. EBNA2-positive cells in the first cycle post-infection then grow out to lymphoblastoid cell lines (LCLs) just as efficiently as do cells limiting-diluted from already established LCLs. This study therefore identifies EBV genome delivery to the nucleus as a key rate-limiting step in B-cell transformation, and highlights the remarkable efficiency with which a single virus genome, having reached the nucleus, then drives the transformation programme.

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

2009 ◽  
Vol 83 (9) ◽  
pp. 4652-4669 ◽  
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.

scholarly journals Epstein-Barr Virus Nuclear Antigen 3C Regulates Cyclin A/p27 Complexes and Enhances Cyclin A-Dependent Kinase Activity

2004 ◽  
Vol 78 (4) ◽  
pp. 1981-1991 ◽  
Author(s):  
Jason S. Knight ◽  
Erle S. Robertson
Keyword(s):  
Cell Cycle ◽  
Cell Cycle Progression ◽  
Kinase Activity ◽  
Cell Transformation ◽  
Cyclin A ◽  
Nuclear Antigen ◽  
Cycle Progression ◽  
Barr Virus ◽  
Epstein Barr

ABSTRACT Epstein-Barr virus (EBV) nuclear antigen 3C (EBNA3C) is essential for primary B-cell transformation. In this report we show that cyclin A, an activator of S phase progression, bound tightly to EBNA3C. EBNA3C interacted with cyclin A in vitro and associated with cyclin A complexes in EBV-transformed lymphoblastoid cell lines. Importantly, EBNA3C stimulated cyclin A-dependent kinase activity and rescued p27-mediated inhibition of cyclin A/Cdk2 kinase activity by decreasing the molecular association between cyclin A and p27 in cells. Additionally, phosphorylation of the retinoblastoma protein, a major regulator of cell cycle progression, was enhanced both in vitro and in vivo in the presence of EBNA3C. Cyclin A interacted with a region of the carboxy terminus of EBNA3C, shown to be important both for stimulation of cyclin A-dependent kinase activity and for cell cycle progression. This provides the first evidence of an essential EBV latent antigen's directly targeting a cell cycle regulatory protein and suggests a novel mechanism by which EBV deregulates the mammalian cell cycle, which is of critical importance in B-cell transformation.

scholarly journals 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

2000 ◽  
Vol 74 (22) ◽  
pp. 10468-10479 ◽  
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.

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

2009 ◽  
Vol 83 (10) ◽  
pp. 4871-4883 ◽  
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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