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 Nucleosome Assembly Proteins Bind to Epstein-Barr Virus Nuclear Antigen 1 and Affect Its Functions in DNA Replication and Transcriptional Activation

2009 ◽  
Vol 83 (22) ◽  
pp. 11704-11714 ◽  
Author(s):  
Shan Wang ◽  
Lori Frappier
Keyword(s):  
Dna Replication ◽  
Transcriptional Activation ◽  
Epstein Barr Virus ◽  
Nuclear Antigen ◽  
Nucleosome Assembly ◽  
Barr Virus ◽  
Ds Element ◽  
Epstein Barr ◽  
Assembly Proteins

ABSTRACT The EBNA1 protein of Epstein-Barr virus (EBV) plays several important roles in EBV latent infection, including activating DNA replication from the latent origin of replication (oriP) and activating the transcription of other latency genes within the EBV chromatin. These functions require EBNA1 binding to the DS and FR elements within oriP, respectively, although how these interactions activate these processes is not clear. We previously identified interactions of EBNA1 with the related nucleosome assembly proteins NAP1 and TAF-I, known to affect the replication and transcription of other chromatinized templates. We have further investigated these interactions, showing that EBNA1 binds directly to NAP1 and to the β isoform of TAF-I (also called SET) and that these interactions greatly increase the solubility of EBNA1 in vitro. These interactions were confirmed in EBV-infected cells, and chromatin immunoprecipitation with these cells showed that NAP1 and TAF-I both localized with EBNA1 to the FR element, while only TAF-I was detected with EBNA1 at the DS element. In keeping with these observations, alteration of the NAP1 or TAF-Iβ level by RNA interference and overexpression inhibited transcriptional activation by EBNA1 in FR reporter assays. In addition, EBNA1-mediated DNA replication was stimulated when TAF-I (but not NAP1) was downregulated and was inhibited by TAF-Iβ overexpression. The results indicate that the interaction of EBNA1 with NAP1 and TAF-I is important for transcriptional activation and that EBNA1 recruits TAF-I to the DS element, where it negatively regulates DNA replication.

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 Characterization of the Uracil-DNA Glycosylase Activity of Epstein-Barr Virus BKRF3 and Its Role in Lytic Viral DNA Replication

2006 ◽  
Vol 81 (3) ◽  
pp. 1195-1208 ◽  
Author(s):  
Chih-Chung Lu ◽  
Ho-Ting Huang ◽  
Jiin-Tarng Wang ◽  
Geir Slupphaug ◽  
Tsai-Kun Li ◽  
...  
Keyword(s):  
Dna Replication ◽  
Epstein Barr Virus ◽  
Efficient Production ◽  
Transcription Activator ◽  
Viral Dna ◽  
Barr Virus ◽  
Epstein Barr ◽  
Lytic Dna Replication

ABSTRACT Uracil-DNA glycosylases (UDGs) of the uracil-N-glycosylase (UNG) family are the primary DNA repair enzymes responsible for removal of inappropriate uracil from DNA. Recent studies further suggest that the nuclear human UNG2 and the UDGs of large DNA viruses may coordinate with their DNA polymerase accessory factors to enhance DNA replication. Based on its amino acid sequence, the putative UDG of Epstein-Barr virus (EBV), BKRF3, belongs to the UNG family of proteins, and it was demonstrated previously to enhance oriLyt-dependent DNA replication in a cotransfection replication assay. However, the expression and enzyme activity of EBV BKRF3 have not yet been characterized. In this study, His-BKRF3 was expressed in bacteria and purified for biochemical analysis. Similar to the case for the Escherichia coli and human UNG enzymes, His-BKRF3 excised uracil from single-stranded DNA more efficiently than from double-stranded DNA and was inhibited by the purified bacteriophage PBS1 inhibitor Ugi. In addition, BKRF3 was able to complement an E. coli ung mutant in rifampin and nalidixic acid resistance mutator assays. The expression kinetics and subcellular localization of BKRF3 products were detected in EBV-positive lymphoid and epithelial cells by using BKRF3-specific mouse antibodies. Expression of BKRF3 is regulated mainly by the immediate-early transcription activator Rta. The efficiency of EBV lytic DNA replication was slightly affected by BKRF3 small interfering RNA (siRNA), whereas cellular UNG2 siRNA or inhibition of cellular and viral UNG activities by expressing Ugi repressed EBV lytic DNA replication. Taking these results together, we demonstrate the UNG activity of BKRF3 in vitro and in vivo and suggest that UNGs may participate in DNA replication or repair and thereby promote efficient production of viral DNA.

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 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 Role for G-Quadruplex RNA Binding by Epstein-Barr Virus Nuclear Antigen 1 in DNA Replication and Metaphase Chromosome Attachment

2009 ◽  
Vol 83 (20) ◽  
pp. 10336-10346 ◽  
Author(s):  
Julie Norseen ◽  
F. Brad Johnson ◽  
Paul M. Lieberman
Keyword(s):  
Dna Replication ◽  
Epstein Barr Virus ◽  
Origin Recognition Complex ◽  
Rna Binding ◽  
Binding Capacity ◽  
Nuclear Antigen ◽  
Metaphase Chromosomes ◽  
Barr Virus ◽  
G Quadruplex ◽  
Epstein Barr

ABSTRACT Latent infection by Epstein-Barr virus (EBV) requires both replication and maintenance of the viral genome. EBV nuclear antigen 1 (EBNA1) is a virus-encoded protein that is critical for the replication and maintenance of the genome during latency in proliferating cells. We have previously demonstrated that EBNA1 recruits the cellular origin recognition complex (ORC) through an RNA-dependent interaction with EBNA1 linking region 1 (LR1) and LR2. We now show that LR1 and LR2 bind to G-rich RNA that is predicted to form G-quadruplex structures. Several chemically distinct G-quadruplex-interacting drugs disrupted the interaction between EBNA1 and ORC. The G-quadruplex-interacting compound BRACO-19 inhibited EBNA1-dependent stimulation of viral DNA replication and preferentially blocked proliferation of EBV-positive cells relative to EBV-negative cell lines. BRACO-19 treatment also disrupted the ability of EBNA1 to tether to metaphase chromosomes, suggesting that maintenance function is also mediated through G-quadruplex recognition. These findings suggest that the EBNA1 replication and maintenance function uses a common G-quadruplex binding capacity of LR1 and LR2, which may be targetable by small-molecule inhibitors.

scholarly journals A Redox-Sensitive Cysteine in Zta Is Required for Epstein-Barr Virus Lytic Cycle DNA Replication

2005 ◽  
Vol 79 (21) ◽  
pp. 13298-13309 ◽  
Author(s):  
Pu Wang ◽  
Latasha Day ◽  
Jayaraju Dheekollu ◽  
Paul M. Lieberman
Keyword(s):  
Dna Replication ◽  
Dna Binding ◽  
Redox Regulation ◽  
Binding Activity ◽  
Lytic Replication ◽  
Lytic Cycle ◽  
Barr Virus ◽  
Dna Binding Activity ◽  
Epstein Barr

ABSTRACT Epstein-Barr virus (EBV) reactivation from latency is known to be sensitive to redox regulation. The immediate-early protein Zta is a member of the basic-leucine zipper (bZIP) family of DNA binding proteins that stimulates viral and cellular transcription and nucleates a replication complex at the viral lytic origin. Zta shares with several members of the bZIP family a conserved cysteine residue (C189) that confers redox regulation of DNA binding. In this work, we show that replacement of C189 with serine (C189S) eliminated lytic cycle DNA replication function of Zta. The mechanistic basis for this replication defect was investigated. We show that C189S was not significantly altered for DNA binding activity in vitro or in vivo. We also show that C189S was not defective for transcription activation of EBV early gene promoters. C189S was deficient for transcription activation of several viral late genes that depend on lytic replication and therefore was consistent with a primary defect of C189S in activating lytic replication. C189S was not defective in binding methylated DNA binding sites and was capable of activating Rta from endogenous latent viral genomes, in contrast to the previously characterized S186A mutation. C189S was slightly impaired for its ability to form a stable complex with Rta, although this did not prevent Rta recruitment to OriLyt. C189S did provide some resistance to oxidation and nitrosylation, which potently inhibit Zta DNA binding activity in vitro. Interestingly, this redox sensitivity was not strictly dependent on C189S but involved additional cysteine residues in Zta. These results provide evidence that the conserved cysteine in the bZIP domain of Zta plays a primary role in EBV lytic cycle DNA replication.

Sign in / Sign up

Export Citation Format

Share Document