scholarly journals Open chromatin structures regulate the efficiencies of pre-RC formation and replication initiation in Epstein-Barr virus

2012 ◽  
Vol 198 (4) ◽  
pp. 509-528 ◽  
Author(s):  
Peer Papior ◽  
José M. Arteaga-Salas ◽  
Thomas Günther ◽  
Adam Grundhoff ◽  
Aloys Schepers
Keyword(s):  
Cell Cycle ◽  
Internal Control ◽  
Epstein Barr Virus ◽  
Origin Recognition Complex ◽  
Replication Initiation ◽  
Micrococcal Nuclease ◽  
Open Chromatin ◽  
Barr Virus ◽  
A Genome ◽  
Epstein Barr

Whether or not metazoan replication initiates at random or specific but flexible sites is an unsolved question. The lack of sequence specificity in origin recognition complex (ORC) DNA binding complicates genome-scale chromatin immunoprecipitation (ChIP)-based studies. Epstein-Barr virus (EBV) persists as chromatinized minichromosomes that are replicated by the host replication machinery. We used EBV to investigate the link between zones of pre-replication complex (pre-RC) assembly, replication initiation, and micrococcal nuclease (MNase) sensitivity at different cell cycle stages in a genome-wide fashion. The dyad symmetry element (DS) of EBV’s latent origin, a well-established and very efficient pre-RC assembly region, served as an internal control. We identified 64 pre-RC zones that correlate spatially with 57 short nascent strand (SNS) zones. MNase experiments revealed that pre-RC and SNS zones were linked to regions of increased MNase sensitivity, which is a marker of origin strength. Interestingly, although spatially correlated, pre-RC and SNS zones were characterized by different features. We propose that pre-RCs are formed at flexible but distinct sites, from which only a few are activated per single genome and cell cycle.

scholarly journals Chromatin Profiling of Epstein-Barr Virus Latency Control Region

2007 ◽  
Vol 81 (12) ◽  
pp. 6389-6401 ◽  
Author(s):  
Latasha Day ◽  
Charles M. Chau ◽  
Michael Nebozhyn ◽  
Andrew J. Rennekamp ◽  
Michael Showe ◽  
...  
Keyword(s):  
Control Region ◽  
Histone Modifications ◽  
Transcription Initiation ◽  
Epstein Barr Virus ◽  
Origin Recognition Complex ◽  
Replication Initiation ◽  
Pcr Analysis ◽  
Chromatin Domain ◽  
Barr Virus ◽  
Epstein Barr

ABSTRACT Epstein-Barr virus (EBV) escapes host immunity by the reversible and epigenetic silencing of immunogenic viral genes. We previously presented evidence that a dynamic chromatin domain, which we have referred to as the latency control region (LCR), contributes to the reversible repression of EBNA2 and LMP1 gene transcription. We now explore the protein-DNA interaction profiles for a few known regulatory factors and histone modifications that regulate LCR structure and activity. A chromatin immunoprecipitation assay combined with real-time PCR analysis was used to analyze protein-DNA interactions at ∼500-bp intervals across the first 60,000 bp of the EBV genome. We compared the binding patterns of EBNA1 with those of the origin recognition complex protein ORC2, the chromatin boundary factor CTCF, the linker histone H1, and several histone modifications. We analyzed three EBV-positive cell lines (MutuI, Raji, and LCL3459) with distinct transcription patterns reflecting different latency types. Our findings suggest that histone modification patterns within the LCR are complex but reflect differences in each latency type. The most striking finding was the identification of CTCF sites immediately upstream of the Qp, Cp, and EBER transcription initiation regions in all three cell types. In transient assays, CTCF facilitated EBNA1-dependent transcription activation of Cp, suggesting that CTCF coordinates interactions between different chromatin domains. We also found that histone H3 methyl K4 clustered with CTCF and EBNA1 at sites of active transcription or DNA replication initiation. Our findings support a model where CTCF delineates multiple domains within the LCR and regulates interactions between these domains that correlate with changes in gene expression.

scholarly journals Regulation of Epstein-Barr Virus OriP Replication by Poly(ADP-Ribose) Polymerase 1

2010 ◽  
Vol 84 (10) ◽  
pp. 4988-4997 ◽  
Author(s):  
Italo Tempera ◽  
Zhong Deng ◽  
Constandache Atanasiu ◽  
Chi-Ju Chen ◽  
Maria D'Erme ◽  
...  
Keyword(s):  
Dna Replication ◽  
Dna Binding ◽  
Epstein Barr Virus ◽  
Origin Recognition Complex ◽  
Structural Changes ◽  
Replication Initiation ◽  
Barr Virus ◽  
Ds Element ◽  
Epstein Barr

ABSTRACT Poly(ADP-ribose) polymerase (PARP) is an abundant, chromatin-associated, NAD-dependent enzyme that functions in multiple chromosomal processes, including DNA replication and chromatin remodeling. The Epstein-Barr virus (EBV) origin of plasmid replication (OriP) is a dynamic genetic element that confers stable episome maintenance, DNA replication initiation, and chromatin organization functions. OriP function depends on the EBV-encoded origin binding protein EBNA1. We have previously shown that EBNA1 is subject to negative regulation by poly(ADP-ribosyl)ation (PARylation). We now show that PARP1 physically associates with OriP in latently EBV-infected B cells. Short hairpin RNA depletion of PARP1 enhances OriP replication activity and increases EBNA1, origin recognition complex 2 (ORC2), and minichromosome maintenance complex (MCM) association with OriP. Pharmacological inhibitors of PARP1 enhance OriP plasmid maintenance and increase EBNA1, ORC2, and MCM3 occupancy at OriP. PARylation in vitro inhibits ORC2 recruitment and remodels telomere repeat factor (TRF) binding at the dyad symmetry (DS) element of OriP. Purified PARP1 can ribosylate EBNA1 at multiple sites throughout its amino terminus but not in the carboxy-terminal DNA binding domain. We also show that EBNA1 linking regions (LR1 and LR2) can bind directly to oligomers of PAR. We propose that PARP1-dependent PARylation of EBNA1 and adjacently bound TRF2 induces structural changes at the DS element that reduce EBNA1 DNA binding affinity and functional recruitment of ORC.

scholarly journals Notch1, Notch2, and Epstein-Barr virus–encoded nuclear antigen 2 signaling differentially affects proliferation and survival of Epstein-Barr virus–infected B cells

Blood ◽  
2009 ◽  
Vol 113 (22) ◽  
pp. 5506-5515 ◽  
Author(s):  
Hella Kohlhof ◽  
Franziska Hampel ◽  
Reinhard Hoffmann ◽  
Helmut Burtscher ◽  
Ulrich H. Weidle ◽  
...  
Keyword(s):  
Cell Cycle ◽  
B Cells ◽  
B Cell ◽  
Expression Analysis ◽  
Epstein Barr Virus ◽  
Nuclear Antigen ◽  
Barr Virus ◽  
A Genome ◽  
Cell Immortalization ◽  
Epstein Barr

AbstractThe canonical mode of transcriptional activation by both the Epstein-Barr viral protein, Epstein-Barr virus–encoded nuclear antigen 2 (EBNA2), and an activated Notch receptor (Notch-IC) requires their recruitment to RBPJ, suggesting that EBNA2 uses the Notch pathway to achieve B-cell immortalization. To gain further insight into the biologic equivalence between Notch-IC and EBNA2, we performed a genome-wide expression analysis, revealing that Notch-IC and EBNA2 exhibit profound differences in the regulation of target genes. Whereas Notch-IC is more potent in regulating genes associated with differentiation and development, EBNA2 is more potent in inducing viral and cellular genes involved in proliferation, survival, and chemotaxis. Because both EBNA2 and Notch-IC induced the expression of cell cycle–associated genes, we analyzed whether Notch1-IC or Notch2-IC can replace EBNA2 in B-cell immortalization. Although Notch-IC could drive quiescent B cells into the cell cycle, B-cell immortalization was not maintained, partially due to an increased apoptosis rate in Notch-IC–expressing cells. Expression analysis revealed that both EBNA2 and Notch-IC induced the expression of proapoptotic genes, but only in EBNA2-expressing cells were antiapoptotic genes strongly up-regulated. These findings suggest that Notch signaling in B cells and B-cell lymphomas is only compatible with proliferation if pathways leading to antiapototic signals are active.

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.

scholarly journals Direct and Indirect Regulation of Cytokine and Cell Cycle Proteins by EBNA-2 during Epstein-Barr Virus Infection

2001 ◽  
Vol 75 (8) ◽  
pp. 3537-3546 ◽  
Author(s):  
Lindsay C. Spender ◽  
Georgina H. Cornish ◽  
Benjamin Rowland ◽  
Bettina Kempkes ◽  
Paul J. Farrell
Keyword(s):  
Cell Cycle ◽  
Epstein Barr Virus ◽  
Transient Transfection ◽  
Cyclin D2 ◽  
Transient Transfection Assay ◽  
Rnase Protection ◽  
Barr Virus ◽  
Tnf Α ◽  
Epstein Barr ◽  
Transfection Assay

ABSTRACT We have studied the pathways of regulation of cytokine and cell cycle control proteins during infection of human B lymphocytes by Epstein-Barr virus (EBV). Among 30 cytokine RNAs analyzed by the RNase protection assay, tumor necrosis factor alpha (TNF-α), granulocyte colony-stimulating factor, lymphotoxin (LT), and LTβ were found to be regulated within 20 h of EBV infection of primary B cells. Similar results were obtained using the estrogen-regulated EBNA-2 cell line EREB2.5, in which RNAs for LT and TNF-α were induced within 6 h of activation of EBNA-2. Expression of Notch also caused an induction of TNF-α RNA. The induction of TNF-α RNA by EBNA-2 was indirect, and constitutive expression of either LMP-1 or c-myc proteins did not substitute for EBNA-2 in induction of TNF-α RNA. Cyclin D2 is also an indirect target of EBNA-2-mediated transactivation. EBNA-2 was found to activate the cyclin D2 promoter in a transient-transfection assay. A mutant of EBNA-2 that does not bind RBP-Jκ retained some activity in this assay, and activation did not depend on the presence of B-cell-specific factors. Deletion analysis of the cyclin D2 promoter revealed that removal of sequences containing E-box c-myc consensus DNA binding sequences did not reduce EBNA-2-mediated activation of the cyclin D2 promoter in the transient-transfection assay. The results indicate that cytokines are an early target of EBNA-2 and that EBNA-2 can regulate cyclin D2 transcription in EBV-infected cells by mechanisms additional to the c-myc pathway.

scholarly journals Presence of Human Papillomavirus and Epstein–Barr Virus, but Absence of Merkel Cell Polyomavirus, in Head and Neck Cancer of Non-Smokers and Non-Drinkers

2021 ◽  
Vol 10 ◽  
Author(s):  
Frans J. Mulder ◽  
Faisal Klufah ◽  
Famke M. E. Janssen ◽  
Farzaneh Farshadpour ◽  
Stefan M. Willems ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Human Papillomavirus ◽  
Head And Neck ◽  
Epstein Barr Virus ◽  
Merkel Cell Polyomavirus ◽  
Merkel Cell ◽  
Cell Cycle Proteins ◽  
P53 Expression ◽  
Barr Virus ◽  
Epstein Barr

ObjectiveDetermine the presence and prognostic value of human papillomavirus (HPV), Epstein-Barr virus (EBV), Merkel cell polyomavirus (MCPyV), and cell cycle proteins in head and neck squamous cell carcinoma (HNSCC) of non-smokers and non-drinkers (NSND).MethodsClinical characteristics and tumors of 119 NSND with HNSCC were retrospectively collected and analyzed on tissue microarrays. RNAscope in situ hybridization (ISH) was used to screen for the presence of HPV and MCPyV mRNA. Immunohistochemistry was performed for expression of p16 as surrogate marker for HPV, Large T-antigen for MCPyV, and cell cycle proteins p53 and pRb. Positive virus results were confirmed with polymerase chain reaction. For EBV, EBV encoded RNA ISH was performed. Differences in 5-year survival between virus positive and negative tumors were determined by log rank analysis.ResultsAll oropharyngeal tumors (OPSCC) (n = 10) were HPV-positive, in addition to one oral (OSCC) and one nasopharyngeal tumor (NPSCC). The other three NPSCC were EBV-positive. MCPyV was not detected. Patients with HPV or EBV positive tumors did not have a significantly better 5-year disease free or overall survival. Over 70% of virus negative OSCC showed mutant-type p53 expression.ConclusionIn this cohort, all OPSCC and NPSCC showed HPV or EBV presence. Besides one OSCC, all other oral (n = 94), hypopharyngeal (n = 1), and laryngeal (n = 9) tumors were HPV, EBV, and MCPyV negative. This argues against a central role of these viruses in the ethiopathogenesis of tumors outside the oro- and nasopharynx in NSND. So, for the majority of NSND with virus negative OSCC, more research is needed to understand the carcinogenic mechanisms in order to consider targeted therapeutic options.

scholarly journals Radiosensitization Effect of Nedaplatin on Nasopharyngeal Carcinoma Cells in Different Status of Epstein-Barr Virus Infection

2014 ◽  
Vol 2014 ◽  
pp. 1-7 ◽  
Author(s):  
Li Yin ◽  
Jing Wu ◽  
Jianfeng Wu ◽  
Jinjun Ye ◽  
Xuesong Jiang ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Nasopharyngeal Carcinoma ◽  
Cell Cycle Arrest ◽  
Cell Lines ◽  
Epstein Barr Virus ◽  
Barr Virus ◽  
Cycle Arrest ◽  
M Phase ◽  
Epstein Barr ◽  
Mts Assay

This study aims to evaluate the radiosensitization effect of nedaplatin on nasopharyngeal carcinoma (NPC) cell lines with different Epstein-Barr virus (EBV) status. Human NPC cell lines CNE-2 (EBV-negative) and C666 (EBV-positive) were treated with 0–100 μg/mL nedaplatin, and inhibitory effects on cell viability and IC50were calculated by MTS assay. We assessed changes in radiosensitivity of cells by MTS and colony formation assays, and detected the apoptosis index and changes in cell cycle by flow cytometry. MTS assay showed that nedaplatin caused significant cytotoxicity in CNE-2 and C666 cells in a time- and dose-dependent manner. After 24 h, nedaplatin inhibited growth of CNE-2 and C666 cells with IC50values of 34.32 and 63.69 μg/mL, respectively. Compared with radiation alone, nedaplatin enhanced the radiation effect on both cell lines. Nedaplatin markedly increased apoptosis and cell cycle arrest in G2/M phase. Nedaplatin radiosensitized human NPC cells CNE-2 and C666, with a significantly greater effect on the former. The mechanisms of radiosensitization include induction of apoptosis and enhancement of cell cycle arrest in G2/M phase.

Sign in / Sign up

Export Citation Format

Share Document