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 Overexpression, Purification, and Crystallization of the DNA Binding and Dimerization Domains of the Epstein-Barr Virus Nuclear Antigen 1

1995 ◽  
Vol 270 (35) ◽  
pp. 20556-20559 ◽  
Author(s):  
Jean A. Barwell ◽  
Alexey Bochkarev ◽  
Richard A. Pfuetzner ◽  
Harry Tong ◽  
Daniel S. C. Yang ◽  
...  
Keyword(s):  
Dna Binding ◽  
Epstein Barr Virus ◽  
Nuclear Antigen ◽  
Barr Virus ◽  
Epstein Barr

scholarly journals 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

2000 ◽  
Vol 74 (11) ◽  
pp. 5151-5160 ◽  
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.

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.

Sequence-specific DNA binding of the Epstein-Barr virus nuclear antigen (EBNA-1) to clustered sites in the plasmid maintenance region

Cell ◽  
1985 ◽  
Vol 42 (3) ◽  
pp. 859-868 ◽  
Author(s):  
Dan R. Rawlins ◽  
Gregory Milman ◽  
S.Diane Hayward ◽  
Gary S. Hayward
Keyword(s):  
Dna Binding ◽  
Epstein Barr Virus ◽  
Nuclear Antigen ◽  
Plasmid Maintenance ◽  
Barr Virus ◽  
Epstein Barr

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

2004 ◽  
Vol 85 (10) ◽  
pp. 2755-2765 ◽  
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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