scholarly journals The Replicator of the Epstein-Barr Virus Latent Cycle Origin of DNA Replication, oriP, Is Composed of Multiple Functional Elements

2001 ◽  
Vol 75 (22) ◽  
pp. 10582-10592 ◽  
Author(s):  
Michelle D. Koons ◽  
Sarah Van Scoy ◽  
Janet Hearing
Keyword(s):  
Binding Sites ◽  
Epstein Barr Virus ◽  
Protein S ◽  
Virus Genome ◽  
Nuclear Antigen ◽  
Cell Protein ◽  
Present Evidence ◽  
Replication Assay ◽  
Barr Virus ◽  
Epstein Barr

ABSTRACT Replication of the Epstein-Barr virus genome initiates at one of several sites in latently infected, dividing cells. One of these replication origins is close to the viral DNA maintenance element, and, together, this replication origin and the maintenance element are referred to as oriP. The replicator oforiP contains four binding sites for Epstein-Barr virus nuclear antigen 1 (EBNA-1), the sole viral protein required for the replication and maintenance of oriP plasmids. We showed previously that these EBNA-1 sites function in pairs and that mutational inactivation of one pair does not eliminate replicator function. In this study we characterized the contribution of each EBNA-1 site within the replicator and flanking sequences through the use of an internally controlled replication assay. We present evidence that shows that all four EBNA-1 sites are required for anoriP plasmid to be replicated in every cell cycle. Results from these experiments also show that the paired EBNA-1 binding sites are not functionally equivalent and that the low affinity of sites 2 and 3 compared to that of sites 1 and 4 is not essential for replicator function. Our results suggest that a host cell protein(s) binds sequences flanking the EBNA-1 sites and that interactions between EBNA-1 and this protein(s) are critical for replicator function. Finally, we present evidence that shows that the minimal replicator oforiP consists of EBNA-1 sites 3 and 4 and two copies of a 14-bp repeat that is present in inverse orientation flanking these EBNA-1 sites. EBNA-1 sites 1 and 2, together with an element(s) within nucleotides 9138 to 9516, are ancillary elements required for full replicator activity.

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 Initiation of latent DNA replication in the Epstein-Barr virus genome can occur at sites other than the genetically defined origin.

1995 ◽  
Vol 15 (5) ◽  
pp. 2893-2903 ◽  
Author(s):  
R D Little ◽  
C L Schildkraut
Keyword(s):  
Mammalian Cells ◽  
Epstein Barr Virus ◽  
Virus Genome ◽  
Replication Initiation ◽  
Nuclear Antigen ◽  
Replication Forks ◽  
Small Plasmid ◽  
Barr Virus ◽  
Latently Infected ◽  
Epstein Barr

Our laboratory has previously shown that replication of a small plasmid, p174, containing the genetically defined Epstein-Barr virus (EBV) latent origin of replication, oriP, initiates within oriP at or near a dyad symmetry (DS) element and terminates specifically at a family of repeated sequences (FR), also located within oriP. We describe here an analysis of the replication of intact approximately 170-kb EBV genomes in four latently infected cell lines that uses two-dimensional gel replicon mapping. Initiation was detected at oriP in all EBV genomes examined; however, some replication forks appear to originate from alternative initiation sites. In addition, pausing of replication forks was observed at the two clusters of EBV nuclear antigen 1 binding sites within oriP and at or near two highly expressed viral genes 0.5 to 1 kb upstream of oriP, the EBV-encoded RNA (EBER) genes. In the Raji EBV genome, the relative abundance of these stalled forks and the direction in which they are stalled indicate that most replication forks originate upstream of oriP. We thus searched for additional initiation sites in the Raji EBV and found that the majority of initiation events were distributed over a broad region to the left of oriP. This delocalized pattern of initiation resembles initiation of replication in several well-characterized mammalian chromosomal loci and is the first described for any viral genome. EBV thus provides a unique model system with which to investigate factors influencing the selection of replication initiation and termination sites in mammalian cells.

scholarly journals Identifying Sites Bound by Epstein-Barr Virus Nuclear Antigen 1 (EBNA1) in the Human Genome: Defining a Position-Weighted Matrix To Predict Sites Bound by EBNA1 in Viral Genomes

2009 ◽  
Vol 83 (7) ◽  
pp. 2930-2940 ◽  
Author(s):  
Lindsay R. Dresang ◽  
David T. Vereide ◽  
Bill Sugden
Keyword(s):  
Dna Binding ◽  
Human Genome ◽  
Binding Sites ◽  
Epstein Barr Virus ◽  
Consensus Sequence ◽  
Nuclear Antigen ◽  
Barr Virus ◽  
Dna Binding Sites ◽  
Weighted Matrix ◽  
Epstein Barr

ABSTRACT We identified binding sites for Epstein-Barr virus (EBV) nuclear antigen 1 (EBNA1) in the human genome using chromatin immunoprecipitation and microarrays. The sequences for these newly identified sites were used to generate a position-weighted matrix (PWM) for EBNA1's DNA-binding sites. This PWM helped identify additional DNA-binding sites for EBNA1 in the genomes of EBV, Kaposi's sarcoma-associated herpesvirus, and cercopithecine herpesvirus 15 (CeHV-15) (also called herpesvirus papio 15). In particular, a homologue of the Rep* locus in EBV was predicted in the genome of CeHV-15, which is notable because Rep* of EBV was not predicted by the previously developed consensus sequence for EBNA1's binding DNA. The Rep* of CeHV-15 functions as an origin of DNA synthesis in the EBV-positive cell line Raji; this finding thus builds on a set of DNA-binding sites for EBNA1 predicted in silico.

scholarly journals The Latency-Associated Nuclear Antigen Encoded by Kaposi's Sarcoma-Associated Herpesvirus Activates Two Major Essential Epstein-Barr Virus Latent Promoters

2001 ◽  
Vol 75 (19) ◽  
pp. 9446-9457 ◽  
Author(s):  
Angela K. Groves ◽  
Murray A. Cotter ◽  
Chitra Subramanian ◽  
Erle S. Robertson
Keyword(s):  
Transcription Factors ◽  
Cell Line ◽  
Binding Sites ◽  
Kaposi's Sarcoma ◽  
Epstein Barr Virus ◽  
Kaposi’S Sarcoma ◽  
Nuclear Antigen ◽  
Barr Virus ◽  
Epstein Barr ◽  
Kaposi’S Sarcoma Associated Herpesvirus

ABSTRACT The latency-associated nuclear antigen (LANA) encoded by the Kaposi's sarcoma-associated herpesvirus (KSHV) is expressed in the majority of KSHV-infected cells and in cells coinfected with Epstein-Barr virus (EBV). In coinfected body cavity-based lymphomas (BCBLs), EBV latent membrane protein 1 (LMP1), which is essential for B-lymphocyte transformation, is expressed. EBNA2 upregulates the expression of LMP1 and other cellular genes through specific interactions with cellular transcription factors tethering EBNA2 to its responsive promoters. In coinfected BCBL cells, EBNA2 is not detected but LANA, which is constitutively expressed, contains motifs suggestive of potential transcriptional activity. Additionally, recent studies have shown that LANA is capable of activating cellular promoters. Therefore, we investigated whether LANA can affect transcription from two major EBV latent promoters. In this study, we demonstrated that LANA can efficiently transactivate both the LMP1 and C promoters in the human B-cell line BJAB as well as in the human embryonic kidney 293 cell line. Moreover, we demonstrated that specific domains of LANA containing the putative leucine zipper and the glutamic acid-rich region are highly effective in upregulating these viral promoters, while the amino-terminal region (435 amino acids) exhibited little or no transactivation activity in our assays. We also specifically tested truncations of the LMP1 promoter element and showed that the −204 to +40 region had increased levels of activation compared with a larger region, −512 to +40, which contains two recombination signal-binding protein Jκ binding sites. The smaller, −204 to +40 promoter region contains specific binding sites for the Ets family transcription factor PU.1, transcription activating factor/cyclic AMP response element, and Sp1, all of which are known to function as activators of transcription. Our data therefore suggest a potential role for LANA in regulation of the major EBV latent promoters in KSHV- and EBV-coinfected cells. Furthermore, LANA may be able to activate transcription of viral and cellular promoters in the absence of EBNA2, potentially through association with transcription factors bound to their cognate sequences within the −204 to +40 region. This regulation of viral gene expression is critical for persistence of these DNA tumor viruses and most likely involved in mediating the oncogenic process in these coinfected cells.

Epstein Barr virus nuclear antigen 2 binding sites distribution overlap with MS-associated regions

2014 ◽  
Vol 275 (1-2) ◽  
pp. 51
Author(s):  
Vito Ag Ricigliano ◽  
Adam Handel ◽  
Viviana Annibali ◽  
Claudia Policano ◽  
Giovanni Ristori ◽  
...  
Keyword(s):  
Binding Sites ◽  
Epstein Barr Virus ◽  
Nuclear Antigen ◽  
Barr Virus ◽  
Epstein Barr
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