scholarly journals Epstein-Barr Virus Episome Stability Is Coupled to a Delay in Replication Timing

2008 ◽  
Vol 83 (5) ◽  
pp. 2154-2162 ◽  
Author(s):  
Jing Zhou ◽  
Andrew R. Snyder ◽  
Paul M. Lieberman
Keyword(s):  
Histone Deacetylases ◽  
Genome Stability ◽  
Epstein Barr Virus ◽  
Replication Timing ◽  
Replication Initiation ◽  
Stable Complex ◽  
Telomere Repeat ◽  
Temporal Regulation ◽  
Barr Virus ◽  
Epstein Barr

ABSTRACT The temporal regulation of DNA replication is thought to be important for chromosome organization and genome stability. We show here that Epstein-Barr virus (EBV) genomes replicate in mid- to late S phase and that agents that accelerate replication timing of EBV reduce viral genome stability. Hydroxyurea (HU) treatment, which is known to eliminate EBV episomes, shifted EBV replication to earlier times in the cell cycle. HU treatment correlated with hyperacetylation of histone H3 and loss of telomere repeat factor 2 (TRF2) binding at the EBV origin of plasmid replication (OriP). Deletion of TRF2 binding sites within OriP or short hairpin RNA depletion of TRF2 advanced the replication timing of OriP-containing plasmids. Inhibitors of class I histone deacetylases (HDACs) increased histone acetylation at OriP, advanced the replication timing of EBV, and reduced EBV genome copy number. We also show that HDAC1 and -2 form a stable complex with TRF2 at OriP and that HU treatment inhibits HDAC activity. We propose that the TRF2-HDAC complex enhances EBV episome stability by providing a checkpoint that delays replication initiation at OriP.

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 Cell Cycle Association of the Retinoblastoma Protein Rb and the Histone Demethylase LSD1 with the Epstein-Barr Virus Latency Promoter Cp

2008 ◽  
Vol 82 (7) ◽  
pp. 3428-3437 ◽  
Author(s):  
Charles M. Chau ◽  
Zhong Deng ◽  
Hyojueng Kang ◽  
Paul M. Lieberman
Keyword(s):  
Cell Cycle ◽  
Epstein Barr Virus ◽  
Affinity Purification ◽  
Histone H3 ◽  
Histone Demethylase ◽  
Stable Complex ◽  
Dependent Manner ◽  
Barr Virus ◽  
A Cell ◽  
Epstein Barr

ABSTRACT The Epstein-Barr virus C promoter (Cp) regulates the major multicistronic transcript encoding the EBNA-LP, 1, 2, and 3 genes required for B-cell proliferation during latency. The growth-transforming potential of these viral genes suggests that they must be tightly regulated with the host cell cycle and differentiation process. To better understand Cp regulation, we used DNA affinity purification to identify cellular and viral proteins that bind to Cp in latently infected cells. Several previously unknown factors were identified, including the cell cycle regulatory proteins E2F1 and Rb. E2F1 bound to a specific site in Cp located in the core Cp region 3′ of the known EBNA2-responsive RBP-Jk (CSL, CBF1) binding site. The histone H3 K4 demethylase LSD1 (BCC110) was also identified by DNA affinity and was shown to form a stable complex with Rb. Coimmunoprecipitation assays demonstrated that E2F1, Rb, and LSD1 bind to Cp in a cell cycle-dependent manner. Rb and LSD1 binding to Cp increased after the S phase, corresponding to a decrease in histone H3 K4 methylation and Cp transcription. Coimmunoprecipitation and immunofluorescence assays reveal that LSD1 interacts with Rb. Surprisingly, LSD1 did not coimmunoprecipitate with E2F1, suggesting that it associates with Rb independently of E2F1. Depletion of LSD1 by small interfering RNAs inhibited Cp basal transcription levels, and overexpression of LSD1 altered the cell cycle profile in p53-positive (p53+), but not p53-negative (p53−), HCT cells. These findings indicate that Cp is a cell cycle-regulated promoter that is under the control of Rb and the histone demethylase LSD1 in multiple latency types.

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 Origin of Plasmid Replication (OriP) by the S-Phase Checkpoint Kinase Chk2

2010 ◽  
Vol 84 (10) ◽  
pp. 4979-4987 ◽  
Author(s):  
Jing Zhou ◽  
Zhong Deng ◽  
Julie Norseen ◽  
Paul M. Lieberman
Keyword(s):  
Dna Replication ◽  
Epstein Barr Virus ◽  
Replication Timing ◽  
S Phase ◽  
Plasmid Replication ◽  
Acceptor Site ◽  
Plasmid Maintenance ◽  
Barr Virus ◽  
Amino Terminal ◽  
Epstein Barr

ABSTRACT The Epstein-Barr virus (EBV) origin of plasmid replication (OriP) is required for episome stability during latent infection. Telomere repeat factor 2 (TRF2) binds directly to OriP and facilitates DNA replication and plasmid maintenance. Recent studies have found that TRF2 interacts with the DNA damage checkpoint protein Chk2. We show here that Chk2 plays an important role in regulating OriP plasmid stability, chromatin modifications, and replication timing. The depletion of Chk2 by small interfering RNA (siRNA) leads to a reduction in DNA replication efficiency and a loss of OriP-dependent plasmid maintenance. This corresponds to a change in OriP replication timing and an increase in constitutive histone H3 acetylation. We show that Chk2 interacts with TRF2 in the early G1/S phase of the cell cycle. We also show that Chk2 can phosphorylate TRF2 in vitro at a consensus acceptor site in the amino-terminal basic domain of TRF2. TRF2 mutants with a serine-to-aspartic acid phosphomimetic substitution mutation were reduced in their ability to recruit the origin recognition complex (ORC) and stimulate OriP replication. We suggest that the Chk2 phosphorylation of TRF2 is important for coordinating ORC binding with chromatin remodeling during the early S phase and that a failure to execute these events leads to replication defects and plasmid instability.

scholarly journals Inhibition of class I histone deacetylases by romidepsin potently induces Epstein-Barr virus lytic cycle and mediates enhanced cell death with ganciclovir

2015 ◽  
Vol 138 (1) ◽  
pp. 125-136 ◽  
Author(s):  
Kwai Fung Hui ◽  
Arthur Kwok Leung Cheung ◽  
Chung King Choi ◽  
Po Ling Yeung ◽  
Jaap M. Middeldorp ◽  
...  
Keyword(s):  
Cell Death ◽  
Histone Deacetylases ◽  
Epstein Barr Virus ◽  
Lytic Cycle ◽  
Class I ◽  
Barr Virus ◽  
Epstein Barr

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 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 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 Soluble Epstein-Barr Virus Glycoproteins gH, gL, and gp42 Form a 1:1:1 Stable Complex That Acts Like Soluble gp42 in B-Cell Fusion but Not in Epithelial Cell Fusion

2006 ◽  
Vol 80 (19) ◽  
pp. 9444-9454 ◽  
Author(s):  
Austin N. Kirschner ◽  
Jasmina Omerović ◽  
Boris Popov ◽  
Richard Longnecker ◽  
Theodore S. Jardetzky
Keyword(s):  
B Cells ◽  
Epithelial Cell ◽  
Epithelial Cells ◽  
B Cell ◽  
Membrane Fusion ◽  
Cell Fusion ◽  
Epstein Barr Virus ◽  
Stable Complex ◽  
Barr Virus ◽  
Epstein Barr

ABSTRACT Epstein-Barr virus (EBV) is a herpesvirus that infects cells by fusing its lipid envelope with the target cell membrane. The fusion process requires the actions of viral glycoproteins gH, gL, and gB for entry into epithelial cells and additionally requires gp42 for entry into B cells. To further study the roles of these membrane-associated glycoproteins, purified soluble forms of gp42, gH, and gL were expressed that lack the membrane-spanning regions. The soluble gH/gL protein complex binds to soluble gp42 with high affinity, forming a stable heterotrimer with 1:1:1 stoichiometry, and this complex is not formed by an N-terminally truncated variant of gp42. The effects of adding soluble gp42, gH/gL, and gH/gL/gp42 were examined with a virus-free cell-cell fusion assay. The results demonstrate that, in contrast to gp42, membrane fusion does not proceed with secreted gH/gL. The addition of soluble gH/gL does not inhibit or enhance B-cell or epithelial cell fusion when membrane-bound gH/gL, gB, and gp42 are present. However, the soluble gH/gL/gp42 complex does activate membrane fusion with B cells, similarly to soluble gp42, but it does not inhibit fusion with epithelial cells, as observed for gp42 alone. A gp42 peptide, derived from an N-terminal segment involved in gH/gL interactions, binds to soluble gH/gL and inhibits EBV-mediated epithelial cell fusion, mimicking gp42. These observations reveal distinct functional requirements for gH/gL and gp42 complexes in EBV-mediated membrane fusion.

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