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 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 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.

Semiquantitative PCR analysis of Epstein-Barr virus DNA in clinical samples of patients with EBV-associated diseases

2001 ◽  
Vol 65 (2) ◽  
pp. 348-357 ◽  
Author(s):  
Astrid Meerbach ◽  
Bernd Gruhn ◽  
Renate Egerer ◽  
Udo Reischl ◽  
Felix Zintl ◽  
...  
Keyword(s):  
Epstein Barr Virus ◽  
Clinical Samples ◽  
Pcr Analysis ◽  
Barr Virus ◽  
Associated Diseases ◽  
Epstein Barr

Modulation of Caspase-8 and FLICE-Inhibitory Protein Expression as a Potential Mechanism of Epstein-Barr Virus Tumorigenesis in Burkitt’s Lymphoma

Blood ◽  
1999 ◽  
Vol 94 (5) ◽  
pp. 1727-1737 ◽  
Author(s):  
Clifford G. Tepper ◽  
Michael F. Seldin
Keyword(s):  
Cell Lines ◽  
Epstein Barr Virus ◽  
Burkitt’S Lymphoma ◽  
Burkitt's Lymphoma ◽  
Pcr Analysis ◽  
Caspase 8 ◽  
Rt Pcr ◽  
Inhibitory Protein ◽  
Barr Virus ◽  
Epstein Barr

Abstract Ligation of the Fas receptor induces death-inducing signaling complex (DISC) formation, caspase activation, and subsequent apoptotic death of several cell types. Epstein-Barr virus (EBV)-positive group III Burkitt’s lymphoma (BL) cell lines have a marked resistance to Fas-mediated apoptosis, although expressing each of the DISC components, Fas/ APO-1–associated death domain protein (FADD), and caspase-8 (FLICE/MACH/Mch5). The apoptotic pathway distal to the DISC is intact because ceramide analogs, staurosporine, and granzyme B activate caspase-3 and induce apoptosis. Fas resistance was not explained by the putative death-attenuating caspase-8 isoforms. However, while Fas-activated cytosolic extracts from sensitive cells were capable of processing both procaspase-8 and procaspase-3 into active subunit forms, resistant cell extracts did not possess either of these activities. Accordingly, reverse transcriptase-polymerase chain reaction (RT-PCR) analysis showed higher transcript levels for the FLICE-inhibitory protein (FLIPL) in resistant cells and the ratio of caspase-8 to FLIPLmeasured by competition RT-PCR analysis directly correlated with susceptibility to Fas-mediated apoptosis of all cell lines. In addition, modification of the caspase-8/FLIPL ratio by caspase-8 or FLIPL overexpression was able to alter the susceptibility status of the cell lines tested. Our results imply that the relative levels of caspase-8 and FLIPL are an important determinant of susceptibility to Fas-mediated apoptosis.

scholarly journals Deletion of Epstein-Barr Virus Regulatory Sequences Upstream of the EBNA Gene Promoter Wp1 Is Unfavorable for B-Cell Immortalization

2002 ◽  
Vol 76 (22) ◽  
pp. 11763-11769 ◽  
Author(s):  
Lina I. Yoo ◽  
Josh Woloszynek ◽  
Steven Templeton ◽  
Samuel H. Speck
Keyword(s):  
B Cell ◽  
Transcription Initiation ◽  
Epstein Barr Virus ◽  
Regulatory Region ◽  
Regulatory Sequences ◽  
Recombinant Viruses ◽  
Barr Virus ◽  
Cell Immortalization ◽  
Epstein Barr ◽  
The Impact

ABSTRACT Transcription of the six Epstein-Barr virus (EBV) EBNA genes is coordinately regulated, being driven by either the Cp promoter, which is encoded within the unique region just upstream of the EBV major internal repeat (IR-1), or by the Wp promoter, which is encoded within the IR-1 repeat and thus present in multiple copies. Previous analyses of Cp- and Wp-initiated transcription have identified a shared cis-regulatory element mapping to the region extending from −169 to −369 bp upstream of the Wp transcription initiation site (M. T. Puglielli, N. Desai, and S. H. Speck, J. Virol. 71:120-128, 1997). To assess the impact of this regulatory region on Cp and Wp activity in the context of the viral genome, we attempted to delete this regulatory region upstream of the first copy of Wp (Wp1). While 10 recombinant viruses were obtained in which this deletion was incorporated in the interior of the IR-1 repeat, only a single lymphoblastoid cell line (LCL) immortalized by a recombinant EBV harboring the deletion upstream of Wp1 was recovered. In contrast, using a control targeting vector in which the Wp regulatory sequences were intact but which contained a sequence tag within the W0 exon, we demonstrated that of the five recombinant viruses analyzed in which the crossover event had occurred upstream of the Wp sequence tag, four had incorporated the tagged sequences into Wp1 of the virus. Taken together, these results indicate that deletion of the regulatory sequences from −369 to −169 bp upstream of Wp1 is unfavorable for EBV-driven B-cell immortalization but is tolerated within the interior of the IR-1 repeat. Analysis of promoter usage in the clone 9-60 LCL, in which the W enhancer sequences were deleted upstream of Wp1, revealed the following: (i) the level of Cp-initiated transcription was significantly diminished compared to that of wild-type LCLs; (ii) the decreased Cp-initiated transcription was not efficiently compensated by transcription initiation from Wp1; and (iii) transcription initiation from downstream Wp promoters was detectable. This is the first report of an LCL in which transcription initiation from a Wp downstream of Wp1 has been documented.

scholarly journals Cells Expressing the Epstein-Barr Virus Growth Program Are Present in and Restricted to the Naive B-Cell Subset of Healthy Tonsils

2000 ◽  
Vol 74 (21) ◽  
pp. 9964-9971 ◽  
Author(s):  
Alexandra M. Joseph ◽  
Gregory J. Babcock ◽  
David A. Thorley-Lawson
Keyword(s):  
B Cells ◽  
B Cell ◽  
Epstein Barr Virus ◽  
Cell Subset ◽  
Pcr Analysis ◽  
Virus Growth ◽  
Barr Virus ◽  
Latently Infected ◽  
Infected Cells ◽  
Epstein Barr

ABSTRACT In this paper we demonstrate, for the first time, that Epstein-Barr virus (EBV)-infected cells expressing the lymphoblastoid growth program are present in healthy carriers of the virus. Previously we observed that latently infected naive B cells are present in tonsils only when viral replication is detected, suggesting that these may represent newly infected B cells. We have tested this idea by performing a reverse transcription-PCR analysis for the expression of latent genes (EBNA2 and the EBNA3s) that are characteristically expressed only by newly infected cells expressing the growth latency program. EBNA2 expression is regularly detected in purified naive (IgD+) tonsillar B cells (13 of 16 tonsils tested) but was never found in the IgD− population (0 of 16). More detailed analysis revealed that the mRNAs for the latent genes EBNA1 (3 of 3 tonsils tested), EBNA3a (3 of 5), EBNA3b (3 of 5), EBNA3c (3 of 5), LMP1 (6 of 6), and LMP2 (5 of 6) were also present in the IgD+ population, but the EBNA1Q-K transcript, characteristic of nonlymphoblastoid forms of latency, was never detected (0 of 6). Finally, we demonstrate that the latently infected naive (IgD+) cells express CD80 (B7.1), a marker characteristically expressed on activated naive lymphoblasts but absent from resting naive B cells. The infected naive (IgD+) population in the tonsil therefore has the viral and cellular phenotype of a B-cell directly infected with EBV—an activated lymphoblast expressing the growth program.

scholarly journals Investigation of human papillomaviruses (HPV), mouse mammary tumour virus (MMTV), Epstein–Barr virus (EBV), and human polyomavirus entities in canine mammary tumours

2016 ◽  
Vol 60 (1) ◽  
pp. 49-53 ◽  
Author(s):  
Kivilcim Sonmez ◽  
Eda Altan ◽  
Funda Yildirim ◽  
Seçkin Serdar Arun ◽  
Nuri Turan ◽  
...  
Keyword(s):  
Epstein Barr Virus ◽  
Human Papillomaviruses ◽  
Human Polyomavirus ◽  
Mammary Tumour ◽  
Mammary Tissue ◽  
Pcr Analysis ◽  
Mouse Mammary Tumour Virus ◽  
Barr Virus ◽  
Mammary Tumours ◽  
Epstein Barr

Abstract Introduction: The aim of the study was to investigate the presence of human papillomaviruses (HPV), mouse mammary tumour virus (MMTV), Epstein-Barr virus (EBV), and human polyomavirus BK in canine mammary tumours (CMTs) and to correlate the results of histopathological classification with the results of virological examination. Material and Methods: Eighty CMTs and ten normal canine mammary gland samples were evaluated using histopathological methods and TaqMan real-time PCR analysis. Results: The results indicated that all mammary tumours and normal mammary tissue samples were negative for HPV16 and other HPV, EBV, human polyomavirus, and human mammary tumour virus strains. Conclusion: Further studies should be performed to investigate the existence of other strains of HPV, EBV, and human polyomavirus in CMTs.

scholarly journals Genetic Evidence that EBNA-1 Is Needed for Efficient, Stable Latent Infection by Epstein-Barr Virus

1999 ◽  
Vol 73 (4) ◽  
pp. 2974-2982 ◽  
Author(s):  
May-Ann Lee ◽  
Margaret E. Diamond ◽  
John L. Yates
Keyword(s):  
B Cells ◽  
Cell Lines ◽  
Epstein Barr Virus ◽  
Frameshift Mutation ◽  
Latent Infection ◽  
Replication Initiation ◽  
Viral Gene ◽  
Circular Chromosome ◽  
Barr Virus ◽  
Epstein Barr

ABSTRACT Replication and maintenance of the 170-kb circular chromosome of Epstein-Barr virus (EBV) during latent infection are generally believed to depend upon a single viral gene product, the nuclear protein EBNA-1. EBNA-1 binds to two clusters of sites at oriP, an 1,800-bp sequence on the EBV genome which can support replication and maintenance of artificial plasmids introduced into cell lines that contain EBNA-1. To investigate the importance of EBNA-1 to latent infection by EBV, we introduced a frameshift mutation into the EBNA-1 gene of EBV by recombination along with a flanking selectable marker. EBV genomes carrying the frameshift mutation could be isolated readily after superinfecting EBV-positive cell lines, but not if recombinant virus was used to infect EBV-negative B-cell lines or to immortalize peripheral blood B cells. EBV mutants lacking almost all of internal repeat 3, which encode a repetitive glycine and alanine domain of EBNA-1, were generated in the same way and found to immortalize B cells normally. An EBNA-1-deficient mutant of EBV was isolated and found to be incapable of establishing a latent infection of the cell line BL30 at a detectable frequency, indicating that the mutant was less than 1% as efficient as an isogenic, EBNA-1-positive strain in this assay. The data indicate that EBNA-1 is required for efficient and stable latent infection by EBV under the conditions tested. Evidence from other studies now indicates that autonomous maintenance of the EBV chromosome during latent infection does not depend on the replication initiation function of oriP. It is therefore likely that the viral chromosome maintenance (segregation) function of oriP and EBNA-1 is what is required.

scholarly journals Establishment and Characterization of a Human Epstein-Barr Virus-Associated Gastric Carcinoma in SCID Mice

1998 ◽  
Vol 72 (10) ◽  
pp. 8321-8326 ◽  
Author(s):  
Yoshiaki Iwasaki ◽  
Ja-Mun Chong ◽  
Yukiko Hayashi ◽  
Rie Ikeno ◽  
Kuniyoshi Arai ◽  
...  
Keyword(s):  
Gene Expression ◽  
Gastric Carcinoma ◽  
Epstein Barr Virus ◽  
Severe Combined Immunodeficiency ◽  
Scid Mice ◽  
Pcr Analysis ◽  
Epithelial Tissue ◽  
Unique Type ◽  
Barr Virus ◽  
Epstein Barr

ABSTRACT A transplantable human Epstein-Barr virus-associated gastric carcinoma (EBVaGC), designated KT, was propagated in severe combined immunodeficiency (SCID) mice for 12 passages. Mucin and cytokeratin expression and the Alu sequence in tumor DNA confirmed that the KT tumor was derived from human epithelial tissue. The identity of clonal EBV in the original and KT tumors was demonstrated by terminal repeat analysis of EBV DNA. The pattern of latency gene expression of EBV was the same in both tumors. EBER1 was presented similarly in tumor cell nuclei by in situ hybridization. Reverse transcription-PCR analysis also demonstrated Q-promoter-driven EBNA1 expression but not BZLF1, EBNA2, or LMP1 expression. Thus, the transplantable human EBVaGC KT retains the original EBV with the same latency gene expression and can serve as a model for this unique type of gastric carcinoma.

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