scholarly journals Replication from oriP of Epstein-Barr Virus Requires Exact Spacing of Two Bound Dimers of EBNA1 Which Bend DNA

2001 ◽  
Vol 75 (22) ◽  
pp. 10603-10611 ◽  
Author(s):  
Jacqueline M. Bashaw ◽  
John L. Yates
Keyword(s):  
Electrophoretic Mobility ◽  
Binding Sites ◽  
Epstein Barr Virus ◽  
Mobility Shift ◽  
Barr Virus ◽  
Cellular Factors ◽  
Specific Nucleotide Sequence ◽  
Epstein Barr ◽  
Stable Maintenance ◽  
Electrophoretic Mobility Shift Assays

ABSTRACT oriP is a 1.7-kb region of the Epstein-Barr virus (EBV) chromosome that supports replication and stable maintenance of plasmids in human cells that contain EBV-encoded protein EBNA1. Plasmids that depend on oriP are replicated once per cell cycle by cellular factors. The replicator of oriPis an ∼120-bp region called DS which depends on either of two pairs of closely spaced EBNA1 binding sites. Here we report that changing the distance between the EBNA1 sites of a functional pair by inserting or deleting 1 or 2 bp abolished replication activity. The results indicated that, while the distance separating the binding sites is critical, the specific nucleotide sequence between them is unlikely to be important. The use of electrophoretic mobility shift assays to investigate binding by EBNA1 to the sites with normal or altered spacing revealed that EBNA1 induces DNA to bend significantly when it binds, with the center of bending coinciding with the center of binding. EBNA1 binding to a functional pair of sites which are spaced 21 bp apart center to center and which thus are in helical phase induces a larger symmetrical bend, which based on electrophoretic mobility approximates the sum of two separate EBNA1-induced DNA bends. The results imply that replication from oriP requires a precise structure in which DNA forms a large bend around two EBNA1 dimers.

scholarly journals HCF1 and OCT2 Cooperate with EBNA1 To Enhance OriP-Dependent Transcription and Episome Maintenance of Latent Epstein-Barr Virus

2016 ◽  
Vol 90 (11) ◽  
pp. 5353-5367 ◽  
Author(s):  
Jayaraju Dheekollu ◽  
Andreas Wiedmer ◽  
Daniel Sentana-Lledo ◽  
Joel Cassel ◽  
Troy Messick ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Herpes Simplex Virus ◽  
Herpes Simplex ◽  
Epstein Barr Virus ◽  
Histone H3 ◽  
Type I ◽  
Barr Virus ◽  
Cellular Factors ◽  
Epstein Barr ◽  
Simplex Virus

ABSTRACTEpstein-Barr virus (EBV) establishes latent infections as multicopy episomes with complex patterns of viral gene transcription and chromatin structure. The EBV origin of plasmid replication (OriP) has been implicated as a critical control element for viral transcription, as well as viral DNA replication and episome maintenance. Here, we examine cellular factors that bind OriP and regulate histone modification, transcription regulation, and episome maintenance. We found that OriP is enriched for histone H3 lysine 4 (H3K4) methylation in multiple cell types and latency types. Host cell factor 1 (HCF1), a component of the mixed-lineage leukemia (MLL) histone methyltransferase complex, and transcription factor OCT2 (octamer-binding transcription factor 2) bound cooperatively with EBNA1 (Epstein-Barr virus nuclear antigen 1) at OriP. Depletion of OCT2 or HCF1 deregulated latency transcription and histone modifications at OriP, as well as the OriP-regulated latency type-dependent C promoter (Cp) and Q promoter (Qp). HCF1 depletion led to a loss of histone H3K4me3 (trimethylation of histone H3 at lysine 4) and H3 acetylation at Cp in type III latency and Qp in type I latency, as well as an increase in heterochromatic H3K9me3 at these sites. HCF1 depletion resulted in the loss of EBV episomes from Burkitt's lymphoma cells with type I latency and reactivation from lymphoblastoid cells (LCLs) with type III latency. These findings indicate that HCF1 and OCT2 function at OriP to regulate viral transcription, histone modifications, and episome maintenance. As HCF1 is best known for its function in herpes simplex virus 1 (HSV-1) immediate early gene transcription, our findings suggest that EBV latency transcription shares unexpected features with HSV gene regulation.IMPORTANCEEBV latency is associated with several human cancers. Viral latent cycle gene expression is regulated by the epigenetic control of the OriP enhancer region. Here, we show that cellular factors OCT2 and HCF1 bind OriP in association with EBNA1 to maintain elevated histone H3K4me3 and transcriptional enhancer function. HCF1 is known as a transcriptional coactivator of herpes simplex virus (HSV) immediate early (IE) transcription, suggesting that OriP enhancer shares aspects of HSV IE transcription control.

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 Characterization of the Epstein–Barr virus BALF2 promoter

1999 ◽  
Vol 80 (10) ◽  
pp. 2747-2750 ◽  
Author(s):  
Chien-Hui Hung ◽  
Shih-Tung Liu
Keyword(s):  
Transcription Factors ◽  
Epstein Barr Virus ◽  
Early Stage ◽  
Lytic Cycle ◽  
Mobility Shift ◽  
Response Elements ◽  
Barr Virus ◽  
Epstein Barr ◽  
Mobility Shift Assay

BALF2, which encodes the major DNA-binding protein of Epstein–Barr virus (EBV), is expressed during the early stage of the lytic cycle. The location of the BALF2 promoter was identified by primer extension, which indicated that the transcription start is located at nucleotide 164,782 of the EBV genome. Transfection analyses revealed that, similar to other EBV early promoters, the BALF2 promoter is activated by the EBV-encoded transcription factors Rta and Zta. The promoter is also synergistically activated if both transcription factors are present in B lymphocytes and in epithelial cells. Deletion analysis and electrophoretic mobility-shift assay revealed that the region between nucleotides −134 and −64 contains Zta- response elements and the region between nucleotides −287 and −254 contains Rta-response elements. This study demonstrates the importance of Rta and Zta in regulating the transcription of EBV early genes.

scholarly journals Functional interaction of Oct transcription factors with the family of repeats in Epstein–Barr virus oriP

2005 ◽  
Vol 86 (5) ◽  
pp. 1261-1267 ◽  
Author(s):  
J. Almqvist ◽  
J. Zou ◽  
Y. Linderson ◽  
C. Borestrom ◽  
E. Altiok ◽  
...  
Keyword(s):  
Transcription Factors ◽  
Epstein Barr Virus ◽  
Mobility Shift ◽  
Barr Virus ◽  
Binding Factor ◽  
The Family ◽  
Epstein Barr ◽  
Mobility Shift Assay ◽  
Specific Regulation ◽  
Cellular Transcription

The family of repeats (FR) is a major upstream enhancer of the Epstein–Barr virus (EBV) latent C promoter (Cp) that controls transcription of six different latent nuclear proteins following interaction with the EBV nuclear protein EBNA1. Here, it was shown that Cp could also be activated by octamer-binding factor (Oct) proteins. Physical binding to the FR by the cellular transcription factors Oct-1 and Oct-2 was demonstrated by using an electrophoretic mobility-shift assay. Furthermore, Oct-1 in combination with co-regulator Bob.1, or Oct-2 alone, could drive transcription of a heterologous thymidine kinase promoter linked to the FR in both B cells and epithelial cells. Cp controlled by the FR was also activated by binding of Oct-2 to the FR. This may have direct implications for B cell-specific regulation of Cp.

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 Binding of CCCTC-binding factor in vivo to the region located between Rep* and the C promoter of Epstein–Barr virus is unaffected by CpG methylation and does not correlate with Cp activity

2009 ◽  
Vol 90 (5) ◽  
pp. 1183-1189 ◽  
Author(s):  
Daniel Salamon ◽  
Ferenc Banati ◽  
Anita Koroknai ◽  
Mate Ravasz ◽  
Kalman Szenthe ◽  
...  
Keyword(s):  
Cell Lines ◽  
Epstein Barr Virus ◽  
Cpg Methylation ◽  
Ctcf Binding ◽  
Mobility Shift ◽  
Barr Virus ◽  
Binding Factor ◽  
Epstein Barr ◽  
Mobility Shift Assay

In this study, the binding of the insulator protein CCCTC-binding factor (CTCF) to the region located between Rep* and the C promoter (Cp) of Epstein–Barr virus (EBV) was analysed using chromatin immunoprecipitation and in vivo footprinting. CTCF binding was found to be independent of Cp usage in cell lines corresponding to the major EBV latency types. Bisulfite sequencing and an electrophoretic mobility-shift assay (using methylated and unmethylated probes) revealed that CTCF binding was insufficient to induce local CpG demethylation in certain cell lines and was unaffected by CpG methylation in the region between Rep* and Cp. In addition, CTCF binding to the latency promoter, Qp, did not correlate with Qp activity.

scholarly journals Nuclear Factor-κB Binds to the Epstein-Barr Virus LMP1 Promoter and Upregulates Its Expression

2008 ◽  
Vol 83 (3) ◽  
pp. 1393-1401 ◽  
Author(s):  
Pegah Johansson ◽  
Ann Jansson ◽  
Ulla Rüetschi ◽  
Lars Rymo
Keyword(s):  
B Cells ◽  
Epstein Barr Virus ◽  
Affinity Purification ◽  
Critical Role ◽  
Mobility Shift ◽  
Barr Virus ◽  
Exogenous Expression ◽  
Epstein Barr

ABSTRACT The latent membrane protein 1 (LMP1) oncogene carried by Epstein-Barr virus (EBV) is essential for transformation and maintenance of EBV-immortalized B cells in vitro, and it is expressed in most EBV-associated tumor types. The activation of the NF-κB pathway by LMP1 plays a critical role in the upregulation of antiapoptotic proteins. The EBV-encoded EBNA2 transactivator is required for LMP1 activation in latency III, while LMP1 itself appears to be critical for its activation in the latency II gene expression program. In both cases, additional viral and cellular transcription factors are required in mediating transcription activation of the LMP1 promoter. Using DNA affinity purification and chromatin immunoprecipitation assay, we showed here that members of the NF-κB transcription factor family bound to the LMP1 promoter in vitro and in vivo. Electrophoretic mobility shift assay analyses indicated the binding of the p50-p50 homodimer and the p65-p50 heterodimer to an NF-κB site in the LMP1 promoter. Transient transfections and reporter assays showed that the LMP1 promoter is activated by exogenous expression of NF-κB factors in both B cells and epithelial cells. Exogenous expression of NF-κB factors in the EBNA2-deficient P3HR1 cell line induced LMP1 protein expression. Overall, our data are consistent with the presence of a positive regulatory circuit between NF-κB activation and LMP1 expression.

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