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 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 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 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.

Characterization of the Epstein–Barr virus BRRF1 gene, located between early genes BZLF1 and BRLF1

Microbiology ◽  
2000 ◽  
Vol 81 (7) ◽  
pp. 1791-1799 ◽  
Author(s):  
Carine Segouffin-Cariou ◽  
Géraldine Farjot ◽  
Alain Sergeant ◽  
Henri Gruffat
Keyword(s):  
Transcriptional Activation ◽  
Binding Sites ◽  
Epstein Barr Virus ◽  
Regulatory Elements ◽  
Barr Virus ◽  
Viral Genes ◽  
Epstein Barr

The switch from latency to a productive cycle in Epstein–Barr virus (EBV)-infected B cells proliferating in vitro is thought to be due to the transcriptional activation of two viral genes, BZLF1 and BRLF1, encoding two transcription factors called EB1 and R respectively. However, a third gene, BRRF1 is contained in the BZLF1/BRLF1 locus, overlapping with BRLF1 but in inverse orientation. We have characterized the 5′ end of the BRRF1 mRNA and the promoter, PNa, at which BRRF1 pre-mRNA is initiated. We show that although a single BRRF1 mRNA species is induced by 12-O-tetradecanoylphorbol 13-acetate/sodium butyrate in several EBV-infected B cell lines, in Akata cells treated with anti-IgG two BRRF1 mRNAs can be detected. Transcription initiated at the BRRF1 promoter was activated by EB1 but not by R, and EB1-binding sites which contribute to the EB1-activated transcription have been mapped to between positions −469 and +1. A 34 kDa protein could be translated from the BRRF1 mRNA both in vitro and in vivo, and was found predominantly in the nucleus of HeLa cells transfected with a BRRF1 expression vector. Thus there are three promoters in the region of the EBV chromatin containing the BZLF1/BRLF1 genes, two of which, PZ and PNa, potentially share regulatory elements.

scholarly journals Epstein-Barr Virus Nuclear Antigen 3C Recruits Histone Deacetylase Activity and Associates with the Corepressors mSin3A and NCoR in Human B-Cell Lines

2003 ◽  
Vol 77 (7) ◽  
pp. 4261-4272 ◽  
Author(s):  
Jason S. Knight ◽  
Ke Lan ◽  
Chitra Subramanian ◽  
Erle S. Robertson
Keyword(s):  
Histone Deacetylase ◽  
Cell Lines ◽  
Transcriptional Activation ◽  
Epstein Barr Virus ◽  
Trichostatin A ◽  
Nuclear Antigen ◽  
Histone Deacetylase 1 ◽  
Barr Virus ◽  
Epstein Barr

ABSTRACT Epstein-Barr virus (EBV) nuclear antigen 3C (EBNA3C) is a known regulatory transcription factor that has been shown to interact with histone deacetylase 1 (HDAC1) when cotransfected in human cell lines and by in vitro binding experiments. Previous studies have shown that EBNA3C interacts with p300 and prothymosin alpha (ProTα) in EBV-infected cells and may be involved in recruiting acetyltransferases to the chromatin for acetylation of histones and transcriptional activation. EBNA3C has also been shown to function as a repressor of transcription when directed to promoters. In this report, we show that EBNA3C complexed with ProTα can also recruit deacetylase activity and associates in a complex that includes HDAC1 and HDAC2 in human B cells. A complex of EBNA3C and ProTα coimmunoprecipitated with HDAC1 and HDAC2 in cell lines stably expressing EBNA3C. Additionally, this complex associated with the mSin3A and NCoR corepressors in EBNA3C-expressing cell lines and may function in a complex with additional transcription factors known to be repressors of transcription. EBNA3C in complex with ProTα recruited deacetylase activity in cell lines stably expressing EBNA3C, and this activity was shown to be partially sensitive to trichostatin A (TSA). This suggests an association with other deacetylases that are insensitive to the general inhibitory effects of TSA, as the entire activity was not abolished in multiple assays. The association between EBNA3C and the corepressors as well as HDACs is likely to depend on the presence of ProTα in the complex. Immunoprecipitation with anti-ProTα antibody immunoprecipitated EBNA3C and the other repressors, whereas immunoprecipitation with anti-EBNA3C antibody resulted in little or no association with these molecules associated with transcription repression. Clearly, EBNA3C functions as a component of a number of dynamic complexes which function in repression and activation of transcription.

scholarly journals Nucleosome Assembly Proteins Bind to Epstein-Barr Virus Nuclear Antigen 1 and Affect Its Functions in DNA Replication and Transcriptional Activation

2009 ◽  
Vol 83 (22) ◽  
pp. 11704-11714 ◽  
Author(s):  
Shan Wang ◽  
Lori Frappier
Keyword(s):  
Dna Replication ◽  
Transcriptional Activation ◽  
Epstein Barr Virus ◽  
Nuclear Antigen ◽  
Nucleosome Assembly ◽  
Barr Virus ◽  
Ds Element ◽  
Epstein Barr ◽  
Assembly Proteins

ABSTRACT The EBNA1 protein of Epstein-Barr virus (EBV) plays several important roles in EBV latent infection, including activating DNA replication from the latent origin of replication (oriP) and activating the transcription of other latency genes within the EBV chromatin. These functions require EBNA1 binding to the DS and FR elements within oriP, respectively, although how these interactions activate these processes is not clear. We previously identified interactions of EBNA1 with the related nucleosome assembly proteins NAP1 and TAF-I, known to affect the replication and transcription of other chromatinized templates. We have further investigated these interactions, showing that EBNA1 binds directly to NAP1 and to the β isoform of TAF-I (also called SET) and that these interactions greatly increase the solubility of EBNA1 in vitro. These interactions were confirmed in EBV-infected cells, and chromatin immunoprecipitation with these cells showed that NAP1 and TAF-I both localized with EBNA1 to the FR element, while only TAF-I was detected with EBNA1 at the DS element. In keeping with these observations, alteration of the NAP1 or TAF-Iβ level by RNA interference and overexpression inhibited transcriptional activation by EBNA1 in FR reporter assays. In addition, EBNA1-mediated DNA replication was stimulated when TAF-I (but not NAP1) was downregulated and was inhibited by TAF-Iβ overexpression. The results indicate that the interaction of EBNA1 with NAP1 and TAF-I is important for transcriptional activation and that EBNA1 recruits TAF-I to the DS element, where it negatively regulates DNA replication.

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