scholarly journals The Papillomavirus E8∧E2C Protein Represses DNA Replication from Extrachromosomal Origins

2003 ◽  
Vol 23 (22) ◽  
pp. 8352-8362 ◽  
Author(s):  
Thomas Zobel ◽  
Thomas Iftner ◽  
Frank Stubenrauch
Keyword(s):  
Dna Replication ◽  
Dna Binding ◽  
Copy Number ◽  
Human Papillomaviruses ◽  
Virus Infections ◽  
Dimerization Domain ◽  
Barr Virus ◽  
Amino Terminal ◽  
Copy Number Control ◽  
Epstein Barr

ABSTRACT Carcinogenic DNA viruses such as high-risk human papillomaviruses (HPV) and Epstein-Barr-Virus (EBV) replicate during persistent infections as low-copy-number plasmids. EBV DNA replication is restricted by host cell replication licensing mechanisms. In contrast, copy number control of HPV genomes is not under cellular control but involves the viral sequence-specific DNA-binding E2 activator and E8∧E2C repressor proteins. Analysis of HPV31 mutant genomes revealed that residues outside of the DNA-binding/dimerization domain of E8∧E2C limit viral DNA replication, indicating that binding site competition or heterodimerization among E2 and E8∧E2C proteins does not contribute to copy number control. Domain swap experiments demonstrated that the amino-terminal 21 amino acids of E8∧E2C represent a novel, transferable DNA replication repressor domain, whose activity requires conserved lysine and tryptophan residues. Furthermore, E8∧E2C(1-21)-GAL4 fusion proteins inhibited the replication of the plasmid origin of replication of EBV, suggesting that E8∧E2C functions as a general replication repressor of extrachromosomal origins. This finding could be important for the development of novel therapies against persistent DNA tumor virus infections.

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 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 Telomere Repeat Binding Factors TRF1, TRF2, and hRAP1 Modulate Replication of Epstein-Barr Virus OriP

2003 ◽  
Vol 77 (22) ◽  
pp. 11992-12001 ◽  
Author(s):  
Zhong Deng ◽  
Constandache Atanasiu ◽  
John S. Burg ◽  
Dominique Broccoli ◽  
Paul M. Lieberman
Keyword(s):  
Cell Cycle ◽  
Dna Replication ◽  
Epstein Barr Virus ◽  
Affinity Purification ◽  
Dominant Negative ◽  
Full Length ◽  
Barr Virus ◽  
Amino Terminal ◽  
Epstein Barr ◽  
Dyad Symmetry

ABSTRACT Epstein-Barr virus OriP confers cell cycle-dependent DNA replication and stable maintenance on plasmids in EBNA1-positive cells. The dyad symmetry region of OriP contains four EBNA1 binding sites that are punctuated by 9-bp repeats referred to as nonamers. Previous work has shown that the nonamers bind to cellular factors associated with human telomeres and contribute to episomal maintenance of OriP. In this work, we show that substitution mutation of all three nonamer sites reduces both DNA replication and plasmid maintenance of OriP-containing plasmids by 2.5- to 5-fold. The nonamers were required for high-affinity binding of TRF1, TRF2, and hRap1 to the dyad symmetry element but were not essential for the binding of EBNA1 as determined by DNA affinity purification from nuclear extracts. Chromatin immunoprecipitation assays indicated that TRF1, TRF2, and hRap1 bound OriP in vivo. Cell cycle studies indicate that TRF2 binding to OriP peaks in G1/S while TRF1 binding peaks in G2/M. OriP replication was inhibited by transfection of full-length TRF1 but not by deletion mutants lacking the myb DNA binding domain. In contrast, OriP replication was not affected by transfection of full-length TRF2 or hRap1 but was potently inhibited by dominant-negative TRF2 or hRap1 amino-terminal truncation mutants. Knockdown experiments with short interfering RNAs (siRNAs) directed against TRF2 and hRap1 severely reduced OriP replication, while TRF1 siRNA had a modest stimulatory effect on OriP replication. These results indicate that TRF2 and hRap1 promote, while TRF1 antagonizes, OriP-dependent DNA replication and suggest that these telomeric factors contribute to the establishment of replication competence at OriP.

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.

scholarly journals Amino Acids in the Basic Domain of Epstein-Barr Virus ZEBRA Protein Play Distinct Roles in DNA Binding, Activation of Early Lytic Gene Expression, and Promotion of Viral DNA Replication

2006 ◽  
Vol 80 (18) ◽  
pp. 9115-9133 ◽  
Author(s):  
Lee Heston ◽  
Ayman El-Guindy ◽  
Jill Countryman ◽  
Charles Dela Cruz ◽  
Henri-Jacques Delecluse ◽  
...  
Keyword(s):  
Gene Expression ◽  
Amino Acids ◽  
Dna Replication ◽  
Dna Binding ◽  
Lytic Cycle ◽  
Late Gene Expression ◽  
Lytic Gene ◽  
Basic Domain ◽  
Barr Virus ◽  
Epstein Barr

ABSTRACT The ZEBRA protein of Epstein-Barr virus (EBV) drives the viral lytic cycle cascade. The capacity of ZEBRA to recognize specific DNA sequences resides in amino acids 178 to 194, a region in which 9 of 17 residues are either lysine or arginine. To define the basic domain residues essential for activity, a series of 46 single-amino-acid-substitution mutants were examined for their ability to bind ZIIIB DNA, a high-affinity ZEBRA binding site, and for their capacity to activate early and late EBV lytic cycle gene expression. DNA binding was obligatory for the protein to activate the lytic cascade. Nineteen mutants that failed to bind DNA were unable to disrupt latency. A single acidic replacement of a basic amino acid destroyed DNA binding and the biologic activity of the protein. Four mutants that bound weakly to DNA were defective at stimulating the expression of Rta, the essential first target of ZEBRA in lytic cycle activation. Four amino acids, R183, A185, C189, and R190, are likely to contact ZIIIB DNA specifically, since alanine or valine substitutions at these positions drastically weakened or eliminated DNA binding. Twenty-three mutants were proficient in binding to ZIIIB DNA. Some DNA binding-proficient mutants were refractory to supershift by BZ-1 monoclonal antibody (epitope amino acids 214 to 230), likely as the result of the increased solubility of the mutants. Mutants competent to bind DNA could be separated into four functional groups: the wild-type group (eight mutants), a group defective at activating Rta (five mutants, all with mutations at the S186 site), a group defective at activating EA-D (three mutants with the R179A, S186T, and K192A mutations), and a group specifically defective at activating late gene expression (seven mutants). Three late mutants, with a Y180A, Y180E, or K188A mutation, were defective at stimulating EBV DNA replication. This catalogue of point mutants reveals that basic domain amino acids play distinct functions in binding to DNA, in activating Rta, in stimulating early lytic gene expression, and in promoting viral DNA replication and viral late gene expression. These results are discussed in relationship to the recently solved crystal structure of ZEBRA bound to an AP-1 site.

scholarly journals Inhibition of Epstein-Barr Virus OriP Function by Tankyrase, a Telomere-Associated Poly-ADP Ribose Polymerase That Binds and Modifies EBNA1

2005 ◽  
Vol 79 (8) ◽  
pp. 4640-4650 ◽  
Author(s):  
Zhong Deng ◽  
Constandache Atanasiu ◽  
Kehao Zhao ◽  
Ronen Marmorstein ◽  
Juan I. Sbodio ◽  
...  
Keyword(s):  
Dna Replication ◽  
Epstein Barr Virus ◽  
Affinity Purification ◽  
Dependent Manner ◽  
Telomere Repeat ◽  
Barr Virus ◽  
Amino Terminal ◽  
Epstein Barr

ABSTRACT Tankyrase (TNKS) is a telomere-associated poly-ADP ribose polymerase (PARP) that has been implicated along with several telomere repeat binding factors in the regulation of Epstein-Barr virus origin of plasmid replication (OriP). We now show that TNKS1 can bind to the family of repeats (FR) and dyad symmetry regions of OriP by using a chromatin immunoprecipitation assay and DNA affinity purification. TNKS1 and TNKS2 bound to EBNA1 in coimmunoprecipitation experiments with transfected cell lysates and with purified recombinant proteins in vitro. Two RXXPDG-like TNKS-interacting motifs in the EBNA1 amino-terminal domain mediated binding with the ankyrin repeat domain of TNKS. Mutations of both motifs at EBNA1 G81 and G425 abrogated TNKS binding and enhanced EBNA1-dependent replication of OriP. Small hairpin RNA targeted knock-down of TNKS1 enhanced OriP-dependent DNA replication. Overexpression of TNKS1 or TNKS2 inhibited OriP-dependent DNA replication, while a PARP-inactive form of TNKS2 (M1045V) was compromised for this inhibition. We show that EBNA1 is subject to PAR modification in vivo and to TNKS1-mediated PAR modification in vitro. These results indicate that TNKS proteins can interact directly with the EBNA1 protein, associate with the FR region of OriP in vivo, and inhibit OriP replication in a PARP-dependent manner.

Comparison of Co-Presence of Epstein-Barr Virus and High-Risk Human Papillomaviruses in Colorectal Cancers in the Middle East Region

2020 ◽  
Author(s):  
Karim Nagi ◽  
Ishita Gupta ◽  
Hamda A Al-Thawadi ◽  
Ayesha Jabeen ◽  
Mohammed I. Malk ◽  
...  
Keyword(s):  
Colorectal Cancer ◽  
High Risk ◽  
Epstein Barr Virus ◽  
Human Papillomaviruses ◽  
Colorectal Cancers ◽  
Barr Virus ◽  
Human Carcinomas ◽  
Epstein Barr ◽  
Invasive Carcinomas ◽  
High Risk Hpv

Background: Several studies have shown the presence of onco viral DNA in colorectal tumor tissues. Viral infection by onco-viruses such as Human papillomaviruses (HPVs) and Epstein–Barr virus (EBV) are well-known to be involved in the onset and/or progression of numerous human carcinomas. Methods: We explored the co-presence of high-risk HPVs and EBV in a cohort of colorectal cancer samples from Lebanon (94) and Syria (102) by PCR, immunohistochemistry and tissue microarray. Results: The results of the study point out that 54% of colorectal cancer cases in Syria are positive for high-risk HPVs, while 30% of the cases in Lebanon are positive for these viruses; the most frequent high-risk HPV types in these populations are 16, 18, 31, 33 and 35. Analysis of LMP1 showed similar results in both populations; 36% of Syrian and 31% of Lebanese samples. Additionally, we report that EBV and high-risk HPVs are co-present in these samples. In Syrian samples, EBV and HPVs are co-present in 16% of the population, however, in the Lebanese samples, 20% of the cases are positive for both EBV and HPVs; their co-presence is associated with high/intermediate grade invasive carcinomas. Conclusion: These data suggest that EBV and high-risk HPVs are co-present in human colorectal cancers where they can cooperate in the progression of these cancers. Nevertheless, further studies are needed to elucidate the role of those oncoviruses in the development of human colorectal carcinomas.

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