scholarly journals An ATF/CRE Element Mediates both EBNA2-Dependent and EBNA2-Independent Activation of the Epstein-Barr Virus LMP1 Gene Promoter

1998 ◽  
Vol 72 (2) ◽  
pp. 1365-1376 ◽  
Author(s):  
Anna Sjöblom ◽  
Weiwen Yang ◽  
Lars Palmqvist ◽  
Ann Jansson ◽  
Lars Rymo
Keyword(s):  
Epstein Barr Virus ◽  
Mutational Analysis ◽  
Expression Vectors ◽  
Nuclear Antigen ◽  
Regulatory Sequence ◽  
Mobility Shift ◽  
Independent Manner ◽  
Barr Virus ◽  
Heterodimeric Complex ◽  
Epstein Barr

ABSTRACT The Epstein-Barr virus (EBV) latent membrane protein 1 (LMP1) is a viral oncogene whose expression is regulated by both viral and cellular factors. EBV nuclear antigen 2 (EBNA2) is a potent transactivator of LMP1 expression in human B cells, and several EBNA2 response elements have been identified in the promoter regulatory sequence (LRS). We have previously shown that an activating transcription factor/cyclic AMP response element (ATF/CRE) site in LRS is involved in EBNA2 responsiveness. We now establish the importance of the ATF/CRE element by mutational analysis and show that both EBNA2-dependent activation and EBNA2-independent activation of the promoter occur via this site but are mediated by separate sets of factors. An electrophoretic mobility shift assay (EMSA) with specific antibodies showed that the ATF-1, CREB-1, ATF-2 and c-Jun factors bind to the site as ATF-1/CREB-1 and ATF-2/c-Jun heterodimers whereas the Sp1 and Sp3 factors bind to an adjacent Sp site. Overexpression of ATF-1 and CREB-1 in the cells by expression vectors demonstrated that homodimeric as well as heterodimeric forms of the factors transactivate the LMP1 promoter in an EBNA2-independent manner. The homodimers of ATF-2 and c-Jun did not significantly stimulate promoter activity. In contrast, the ATF-2/c-Jun heterodimer had only a minor stimulatory effect in the absence of EBNA2 but induced a strong transactivation of the LMP1 promoter when coexpressed with this protein. Evidence for a direct interaction between the ATF-2/c-Jun heterodimeric complex and EBNA2 was obtained by EMSA and coimmunoprecipitation experiments. Thus, our results suggest that EBNA2-induced transactivation via the ATF/CRE site occurs through a direct contact between EBNA2 and an ATF-2/c-Jun heterodimer. EBNA2-independent promoter activation via this site, on the other hand, is mediated by a heterodimeric complex between the ATF-1 and CREB-1 factors.

scholarly journals Mutational analysis of the Epstein--Barr virus nuclear antigen 2 by far-Western blotting and DNA-binding studies

1996 ◽  
Vol 77 (5) ◽  
pp. 991-996 ◽  
Author(s):  
C. Sauder ◽  
N. Gotzinger ◽  
W. H. Schubach ◽  
G. C. Horvath ◽  
E. Kremmer ◽  
...  
Keyword(s):  
Dna Binding ◽  
Western Blotting ◽  
Epstein Barr Virus ◽  
Mutational Analysis ◽  
Nuclear Antigen ◽  
Binding Studies ◽  
Barr Virus ◽  
Dna Binding Studies ◽  
Epstein Barr

scholarly journals Mutational analysis of Epstein-Barr virus nuclear antigen 1 (EBNA 1)

1988 ◽  
Vol 16 (8) ◽  
pp. 3415-3435 ◽  
Author(s):  
Maryellen Polvino-Bodnar ◽  
Janette Kiso ◽  
Priscilla A. Schaffer
Keyword(s):  
Epstein Barr Virus ◽  
Mutational Analysis ◽  
Nuclear Antigen ◽  
Barr Virus ◽  
Epstein Barr

scholarly journals Mechanism for Specificity by HMG-1 in Enhanceosome Assembly

2000 ◽  
Vol 20 (12) ◽  
pp. 4359-4370 ◽  
Author(s):  
Katharine B. Ellwood ◽  
Yi-Meng Yen ◽  
Reid C. Johnson ◽  
Michael Carey
Keyword(s):  
Conformational Changes ◽  
Regulatory Sequence ◽  
Mobility Shift ◽  
Independent Manner ◽  
Barr Virus ◽  
Sequence Recognition ◽  
Epstein Barr ◽  
Mobility Shift Assay ◽  
Architectural Protein

ABSTRACT Assembly of enhanceosomes requires architectural proteins to facilitate the DNA conformational changes accompanying cooperative binding of activators to a regulatory sequence. The architectural protein HMG-1 has been proposed to bind DNA in a sequence-independent manner, yet, paradoxically, it facilitates specific DNA binding reactions in vitro. To investigate the mechanism of specificity we explored the effect of HMG-1 on binding of the Epstein-Barr virus activator ZEBRA to a natural responsive promoter in vitro. DNase I footprinting, mutagenesis, and electrophoretic mobility shift assay reveal that HMG-1 binds cooperatively with ZEBRA to a specific DNA sequence between two adjacent ZEBRA recognition sites. This binding requires a strict alignment between two adjacent ZEBRA sites and both HMG boxes of HMG-1. Our study provides the first demonstration of sequence-dependent binding by a nonspecific HMG-box protein. We hypothesize how a ubiquitous, nonspecific architectural protein can function in a specific context through the use of rudimentary sequence recognition coupled with cooperativity. The observation that an abundant architectural protein can bind DNA cooperatively and specifically has implications towards understanding HMG-1's role in mediating DNA transactions in a variety of enzymological systems.

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 Processing of a Multiple Membrane Spanning Epstein-Barr Virus Protein for Cd8+T Cell Recognition Reveals a Proteasome-Dependent, Transporter Associated with Antigen Processing–Independent Pathway

2001 ◽  
Vol 194 (8) ◽  
pp. 1053-1068 ◽  
Author(s):  
Georg Lautscham ◽  
Sabine Mayrhofer ◽  
Graham Taylor ◽  
Tracey Haigh ◽  
Alison Leese ◽  
...  
Keyword(s):  
Antigen Processing ◽  
Epstein Barr Virus ◽  
Hla Class I ◽  
Nuclear Antigen ◽  
Virus Protein ◽  
Clear Correlation ◽  
Independent Manner ◽  
Barr Virus ◽  
Epstein Barr ◽  
Membrane Spanning

Epstein-Barr virus (EBV) latent membrane protein (LMP)2 is a multiple membrane spanning molecule which lacks ectodomains projecting into the lumen of the endoplasmic reticulum (ER). Human CD8+ cytotoxic T lymphocytes (CTL)s recognize a number of epitopes within LMP2. Assays with epitope-specific CTLs in two different cell backgrounds lacking the transporter associated with antigen processing (TAP) consistently show that some, but not all, LMP2 epitopes are presented in a TAP-independent manner. However, unlike published examples of TAP-independent processing from endogenously expressed antigens, presentation of TAP-independent LMP2 epitopes was abrogated by inhibition of proteasomal activity. We found a clear correlation between hydrophobicity of the LMP2 epitope sequence and TAP independence, and experiments with vaccinia minigene constructs expressing cytosolic epitope peptides confirmed that these more hydrophobic peptides were selectively able to access the HLA class I pathway in TAP-negative cells. Furthermore, the TAP-independent phenotype of particular epitope sequences did not require membrane location of the source antigen since (i) TAP-independent LMP2 epitopes inserted into an EBV nuclear antigen and (ii) hydrophobic epitope sequences native to EBV nuclear antigens were both presented in TAP-negative cells. We infer that there is a proteasome-dependent, TAP-independent pathway of antigen presentation which hydrophobic epitopes can selectively access.

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

2000 ◽  
Vol 74 (21) ◽  
pp. 10104-10111 ◽  
Author(s):  
Yasushi Kawaguchi ◽  
Kaori Nakajima ◽  
Mie Igarashi ◽  
Tomoko Morita ◽  
Michiko Tanaka ◽  
...  
Keyword(s):  
Epstein Barr Virus ◽  
Mutational Analysis ◽  
Human Peripheral Blood ◽  
Cellular Protein ◽  
Nuclear Antigen ◽  
Cell Fractionation ◽  
Barr Virus ◽  
Leader Protein ◽  
Protein X ◽  
Epstein Barr

ABSTRACT 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 glutathioneS-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 coimmunoprecipitated 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.

scholarly journals Biophysical and Mutational Analysis of the Putative bZIP Domain of Epstein-Barr Virus EBNA 3C

2004 ◽  
Vol 78 (17) ◽  
pp. 9431-9445 ◽  
Author(s):  
Michelle J. West ◽  
Helen M. Webb ◽  
Alison J. Sinclair ◽  
Derek N. Woolfson
Keyword(s):  
Epstein Barr Virus ◽  
Leucine Zipper ◽  
Mutational Analysis ◽  
Nuclear Antigen ◽  
Barr Virus ◽  
Bzip Domain ◽  
Cell Immortalization ◽  
Epstein Barr ◽  
Cellular Transcription

ABSTRACT Epstein-Barr virus nuclear antigen 3C (EBNA 3C) is essential for B-cell immortalization and functions as a regulator of viral and cellular transcription. EBNA 3C contains glutamine-rich and proline-rich domains and a region in the N terminus consisting of a stretch of basic residues followed by a run of leucine residues spaced seven amino acids apart. This N-terminal domain is widely believed to represent a leucine zipper dimerization motif (bZIP). We have performed the first structural and functional analysis of this motif and demonstrated that this domain is not capable of forming stable homodimers. Peptides encompassing the EBNA 3C zipper domain are approximately 54 to 67% α-helical in solution but cannot form dimers at physiologically relevant concentrations. Moreover, the EBNA 3C leucine zipper cannot functionally substitute for another homodimerizing zipper domain in domain-swapping experiments. Our data indicate, however, that the EBNA 3C zipper domain behaves as an atypical bZIP domain and is capable of self-associating to form higher-order α-helical oligomers. Using directed mutagenesis, we also identified a new role for the bZIP domain in maintaining the interaction between EBNA 3C and RBP-Jκ in vivo. Disruption of the helical nature of the zipper domain by the introduction of proline residues reduces the ability of EBNA 3C to inhibit EBNA 2 activation and interact with RBP-Jκ in vivo by 50%, and perturbation of the charge on the basic region completely abolishes this function of EBNA 3C.

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