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 Variant Chromatin Structure of theoriP Region of Epstein-Barr Virus and Regulation of EBER1 Expression by Upstream Sequences andoriP

2001 ◽  
Vol 75 (13) ◽  
pp. 6235-6241 ◽  
Author(s):  
Barbara Wensing ◽  
Albert Stühler ◽  
Peter Jenkins ◽  
Martine Hollyoake ◽  
Claudio Elgueta Karstegl ◽  
...  
Keyword(s):  
Epstein Barr Virus ◽  
Virus Genome ◽  
Micrococcal Nuclease ◽  
Matrix Attachment Region ◽  
Barr Virus ◽  
Latently Infected ◽  
Attachment Region ◽  
Nuclear Matrix Attachment Region ◽  
Epstein Barr ◽  
Nucleosomal Structure

ABSTRACT Most of the Epstein-Barr virus genome in latently infected cells is in a standard nucleosomal structure, but the region encompassingoriP and the Epstein-Barr virus-encoded small RNA (EBER) genes shows a distinctive pattern when digested with micrococcal nuclease. This pattern corresponds to a previously mapped nuclear matrix attachment region. Although the EBER genes are adjacent to oriP, there is only a two- to fourfold effect oforiP on EBER expression. However, sequences containing a consensus ATF site upstream of EBER1 are important for EBER1 expression.

Role of EBNA-1 in arresting replication forks at the Epstein-Barr virus oriP family of tandem repeats

1991 ◽  
Vol 11 (12) ◽  
pp. 6268-6278
Author(s):  
V Dhar ◽  
C L Schildkraut
Keyword(s):  
Replication Fork ◽  
Epstein Barr Virus ◽  
Tandem Repeats ◽  
Simian Virus 40 ◽  
T Antigen ◽  
Nuclear Antigen ◽  
Replication Forks ◽  
Barr Virus ◽  
Epstein Barr ◽  
Sv40 Dna

The 20-member family of 30-bp tandem repeats located within the oriP region of Epstein-Barr virus (EBV) can act as a transcriptional enhancer in the presence of EBV nuclear antigen 1 (EBNA-1). A replication fork barrier and a termination site of plasmid replication in human B cells is also found within or near the EBV tandem repeats. Within each tandem repeat is a consensus binding sequence for the EBNA-1 protein that is required for extrachromosomal maintenance of oriP-containing plasmids. To investigate the factors that contribute to the arrest of replication forks and termination in the region of the family of repeats, we have used an in vitro replication system in which replication of EBV recombinant plasmids is initiated from the simian virus 40 (SV40) DNA replication origin in the presence of SV40 T antigen and soluble extracts prepared from human cells. The system can support bidirectional replication, initiating from the SV40 DNA origin with termination occurring in a region opposite the origin. Using two-dimensional agarose gel electrophoresis, we observed a barrier to replication forks in the presence of EBNA-1 in the region of the EBV repeats. Termination occurs at or near the tandem repeats in a manner similar to that observed in vivo (T.A. Gahn and C.L. Schildkraut, Cell 58:527-535, 1989). Reducing the number of repeats from 20 to 6 had little effect on the strength of the replication fork barrier. In the absence of EBNA-1, replication forks also arrested at the EBV repeats, but at a much lower efficiency. The addition of competitor DNA containing the EBV family of repeats can almost completely abolish the replication barrier produced in the presence of EBNA-1.

Calcium modulation activates Epstein-Barr virus genome in latently infected cells

Science ◽  
1986 ◽  
Vol 232 (4757) ◽  
pp. 1554-1556 ◽  
Author(s):  
A Faggioni ◽  
C Zompetta ◽  
S Grimaldi ◽  
G Barile ◽  
L Frati ◽  
...  
Keyword(s):  
Epstein Barr Virus ◽  
Virus Genome ◽  
Barr Virus ◽  
Calcium Modulation ◽  
Latently Infected ◽  
Infected Cells ◽  
Epstein Barr

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 Hyperphosphorylation of EBNA2 by Epstein-Barr Virus Protein Kinase Suppresses Transactivation of the LMP1 Promoter

2005 ◽  
Vol 79 (9) ◽  
pp. 5880-5885 ◽  
Author(s):  
Wei Yue ◽  
Edward Gershburg ◽  
Joseph S. Pagano
Keyword(s):  
Protein Kinase ◽  
B Lymphocytes ◽  
Epstein Barr Virus ◽  
Lytic Cycle ◽  
Nuclear Antigen ◽  
Virus Protein ◽  
Protein Levels ◽  
Barr Virus ◽  
Latently Infected ◽  
Epstein Barr

ABSTRACT The Epstein-Barr virus (EBV) BGLF4 gene encodes a serine/threonine protein kinase (PK) that is expressed in the cytolytic cycle. EBV nuclear antigen 2 (EBNA2) is a key latency gene essential for immortalization of B lymphocytes and transactivation of viral and cellular promoters. Here we report that EBV PK phosphorylates EBNA2 at Ser-243 and that these two proteins physically associate. PK suppresses EBNA2's ability to transactivate the LMP1 promoter, and Ser-243 of EBNA2 is involved in this suppression. Moreover, EBNA2 is hyperphosphorylated during EBV reactivation in latently infected B cells, which is associated with decreased LMP1 protein levels. This is the first report about the effect of EBV PK on the function of one of its target proteins and regulation of EBNA2 phosphorylation during the EBV lytic cycle.

scholarly journals Autorepression of Epstein-Barr Virus Nuclear Antigen 1 Expression by Inhibition of Pre-mRNA Processing

2007 ◽  
Vol 82 (4) ◽  
pp. 1679-1687 ◽  
Author(s):  
Mikio Yoshioka ◽  
Michelle M. Crum ◽  
Jeffery T. Sample
Keyword(s):  
Cell Lines ◽  
Epstein Barr Virus ◽  
Rna Binding ◽  
Cytotoxic T Cells ◽  
Nuclear Antigen ◽  
Genome Maintenance ◽  
Barr Virus ◽  
Latently Infected ◽  
Infected Cells ◽  
Epstein Barr

ABSTRACT Epstein-Barr virus (EBV) latent infection, and its associated oncogenic potential, is dependent on genome maintenance functions of EBV nuclear antigen 1 (EBNA-1), one of six EBNAs expressed from a common promoter (Wp and then Cp) upon infection of naive B cells. Subsequent host-mediated silencing, however, necessitates the expression of EBNA-1 from the EBNA-1-specific promoter Qp to ensure against genome loss during cell division, including EBV-associated malignancy. Here we addressed the mechanism by which EBNA-1 represses Qp through binding downstream of the transcription start site and the role of this autoregulatory function in EBV latency. Our results revealed that EBNA-1 does not inhibit transcription from Qp, as previously predicted, but acts post- or cotranscriptionally to block the processing of primary transcripts. This does not, however, require the RGG motifs responsible for strong but nonspecific RNA binding by EBNA-1. Within isogenic B-cell lines using either Cp/Wp or Qp, EBNA-1 occupancy of Qp is equivalent, suggesting that autoregulation occurs, albeit to different degrees, during full and restricted EBV latency programs. Finally, in cell lines using Cp or Wp for EBNA expression, unprocessed transcripts from Qp are detectable in the absence of corresponding mRNAs, providing further evidence that this novel mechanism of EBNA-1 action functions during latency. This posttranscriptional mechanism of regulation would provide an efficient means to monitor and regulate EBNA-1 expression from Qp, ensuring levels adequate for genome maintenance but, perhaps more importantly, below an immunogenic threshold above which latently infected cells may be at risk for elimination by EBNA-1-specific cytotoxic T cells.

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