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 Reactivation of Lytic Replication from B Cells Latently Infected with Epstein-Barr Virus Occurs with High S-Phase Cyclin-Dependent Kinase Activity while Inhibiting Cellular DNA Replication

2003 ◽  
Vol 77 (2) ◽  
pp. 851-861 ◽  
Author(s):  
Ayumi Kudoh ◽  
Masatoshi Fujita ◽  
Tohru Kiyono ◽  
Kiyotaka Kuzushima ◽  
Yutaka Sugaya ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Dna Replication ◽  
Epstein Barr Virus ◽  
S Phase ◽  
Lytic Replication ◽  
Cdk Inhibitors ◽  
Cyclin Dependent Kinase ◽  
Barr Virus ◽  
Epstein Barr ◽  
Cellular Dna

ABSTRACT Productive infection and replication of herpesviruses usually occurs in growth-arrested cells, but there has been no direct evidence in the case of Epstein-Barr virus (EBV), since an efficient lytic replication system without external stimuli does not exist for the virus. Expression of the EBV lytic-switch transactivator BZLF1 protein in EBV-negative epithelial tumor cell lines, however, is known to arrest the cell cycle in G0/G1 by induction of the tumor suppressor protein p53 and the cyclin-dependent kinase (CDK) inhibitors p21WAF-1/CIP-1 and p27KIP-1, followed by the accumulation of a hypophosphorylated form of the Rb protein. In order to determine the effect of the onset of lytic viral replication on cellular events in latently EBV-infected B LCLs, a tightly controlled induction system of the EBV lytic-replication program by inducible BZLF1 protein expression was established in B95-8 cells. The induction of lytic replication completely arrested cell cycle progression and cellular DNA replication. Surprisingly, the levels of p53, p21WAF-1/CIP-1, and p27KIP-1 were constant before and after induction of the lytic program, indicating that the cell cycle arrest induced by the lytic program is not mediated through p53 and the CDK inhibitors. Furthermore, although cellular DNA replication was blocked, elevation of cyclin E/A expression and accumulation of hyperphosphorylated forms of Rb protein were observed, a post-G1/S phase characteristic of cells. Thus, while the EBV lytic program promoted specific cell cycle-associated activities involved in the progression from G1 to S phase, it inhibited cellular DNA synthesis. Such cellular conditions appear to especially favor viral lytic replication.

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 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.

scholarly journals Mre11 Complex and DNA Replication: Linkage to E2F and Sites of DNA Synthesis

2001 ◽  
Vol 21 (17) ◽  
pp. 6006-6016 ◽  
Author(s):  
Richard S. Maser ◽  
Olga K. Mirzoeva ◽  
Julie Wells ◽  
Heidi Olivares ◽  
Bret R. Williams ◽  
...  
Keyword(s):  
Dna Replication ◽  
Epstein Barr Virus ◽  
S Phase ◽  
Replication Forks ◽  
Chromosomal Replication ◽  
Checkpoint Activation ◽  
Barr Virus ◽  
Virus Origin ◽  
Mre11 Complex ◽  
Epstein Barr

ABSTRACT We show that the Mre11 complex associates with E2F family members via the Nbs1 N terminus. This association and Nbs1 phosphorylation are correlated with S-phase checkpoint proficiency, whereas neither is sufficient individually for checkpoint activation. The Nbs1 E2F interaction occurred near the Epstein-Barr virus origin of replication as well as near a chromosomal replication origin in the c-myc promoter region and was restricted to S-phase cells. The Mre11 complex colocalized with PCNA at replication forks throughout S phase, both prior to and coincident with the appearance of nascent DNA. These data suggest that the Mre11 complex suppresses genomic instability through its influence on both the regulation and progression of DNA replication.

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 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.

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