Cellular transcription factors recruit viral replication proteins to activate the Epstein–Barr virus origin of lytic DNA replication, oriLyt

2000 ◽  
Vol 19 (2) ◽  
pp. 315-315
Author(s):  
M. Baumann ◽  
R. Feederle ◽  
E. Kremmer ◽  
W. Hammerschmidt
Keyword(s):  
Transcription Factors ◽  
Dna Replication ◽  
Viral Replication ◽  
Epstein Barr Virus ◽  
Replication Proteins ◽  
Barr Virus ◽  
Virus Origin ◽  
Epstein Barr ◽  
Lytic Dna Replication ◽  
Cellular Transcription

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 Functional interaction of Oct transcription factors with the family of repeats in Epstein–Barr virus oriP

2005 ◽  
Vol 86 (5) ◽  
pp. 1261-1267 ◽  
Author(s):  
J. Almqvist ◽  
J. Zou ◽  
Y. Linderson ◽  
C. Borestrom ◽  
E. Altiok ◽  
...  
Keyword(s):  
Transcription Factors ◽  
Epstein Barr Virus ◽  
Mobility Shift ◽  
Barr Virus ◽  
Binding Factor ◽  
The Family ◽  
Epstein Barr ◽  
Mobility Shift Assay ◽  
Specific Regulation ◽  
Cellular Transcription

The family of repeats (FR) is a major upstream enhancer of the Epstein–Barr virus (EBV) latent C promoter (Cp) that controls transcription of six different latent nuclear proteins following interaction with the EBV nuclear protein EBNA1. Here, it was shown that Cp could also be activated by octamer-binding factor (Oct) proteins. Physical binding to the FR by the cellular transcription factors Oct-1 and Oct-2 was demonstrated by using an electrophoretic mobility-shift assay. Furthermore, Oct-1 in combination with co-regulator Bob.1, or Oct-2 alone, could drive transcription of a heterologous thymidine kinase promoter linked to the FR in both B cells and epithelial cells. Cp controlled by the FR was also activated by binding of Oct-2 to the FR. This may have direct implications for B cell-specific regulation of Cp.

scholarly journals Architecture of Replication Compartments Formed during Epstein-Barr Virus Lytic Replication

2005 ◽  
Vol 79 (6) ◽  
pp. 3409-3418 ◽  
Author(s):  
Tohru Daikoku ◽  
Ayumi Kudoh ◽  
Masatoshi Fujita ◽  
Yutaka Sugaya ◽  
Hiroki Isomura ◽  
...  
Keyword(s):  
Dna Replication ◽  
Viral Replication ◽  
Epstein Barr Virus ◽  
Protein A ◽  
Lytic Replication ◽  
Viral Dna ◽  
Barr Virus ◽  
Epstein Barr ◽  
Cellular Replication ◽  
Cellular Dna

ABSTRACT Epstein-Barr virus (EBV) productive DNA replication occurs at discrete sites, called replication compartments, in nuclei. In this study we performed comprehensive analyses of the architecture of the replication compartments. The BZLF1 oriLyt binding proteins showed a fine, diffuse pattern of distribution throughout the nuclei at immediate-early stages of induction and then became associated with the replicating EBV genome in the replication compartments during lytic infection. The BMRF1 polymerase (Pol) processivity factor showed a homogenous, not dot-like, distribution in the replication compartments, which completely coincided with the newly synthesized viral DNA. Inhibition of viral DNA replication with phosphonoacetic acid, a viral DNA Pol inhibitor, eliminated the DNA-bound form of the BMRF1 protein, although the protein was sufficiently expressed in the cells. These observations together with the findings that almost all abundantly expressed BMRF1 proteins existed in the DNA-bound form suggest that the BMRF1 proteins not only act at viral replication forks as Pol processive factors but also widely distribute on newly replicated EBV genomic DNA. In contrast, the BALF5 Pol catalytic protein, the BALF2 single-stranded-DNA binding protein, and the BBLF2/3 protein, a component of the helicase-primase complex, were colocalized as distinct dots distributed within replication compartments, representing viral replication factories. Whereas cellular replication factories are constructed based on nonchromatin nuclear structures and nuclear matrix, viral replication factories were easily solubilized by DNase I treatment. Thus, compared with cellular DNA replication, EBV lytic DNA replication factories would be simpler so that construction of the replication domain would be more relaxed.

scholarly journals Protein Array Identification of Substrates of the Epstein-Barr Virus Protein Kinase BGLF4

2009 ◽  
Vol 83 (10) ◽  
pp. 5219-5231 ◽  
Author(s):  
Jian Zhu ◽  
Gangling Liao ◽  
Liang Shan ◽  
Jun Zhang ◽  
Mei-Ru Chen ◽  
...  
Keyword(s):  
Dna Replication ◽  
Protein Kinase ◽  
Epstein Barr Virus ◽  
Protein Array ◽  
Lytic Cycle ◽  
Virus Protein ◽  
Barr Virus ◽  
Epstein Barr ◽  
Lytic Dna Replication ◽  
In Cells

ABSTRACT A conserved family of herpesvirus protein kinases plays a crucial role in herpesvirus DNA replication and virion production. However, despite the fact that these kinases are potential therapeutic targets, no systematic studies have been performed to identify their substrates. We generated an Epstein-Barr virus (EBV) protein array to evaluate the targets of the EBV protein kinase BGLF4. Multiple proteins involved in EBV lytic DNA replication and virion assembly were identified as previously unrecognized substrates for BGLF4, illustrating the broad role played by this protein kinase. Approximately half of the BGLF4 targets were also in vitro substrates for the cellular kinase CDK1/cyclin B. Unexpectedly, EBNA1 was identified as a substrate and binding partner of BGLF4. EBNA1 is essential for replication and maintenance of the episomal EBV genome during latency. BGLF4 did not prevent EBNA1 binding to sites in the EBV latency origin of replication, oriP. Rather, we found that BGLF4 was recruited by EBNA1 to oriP in cells transfected with an oriP vector and BGLF4 and in lytically induced EBV-positive Akata cells. In cells transfected with an oriP vector, the presence of BGLF4 led to more rapid loss of the episomal DNA, and this was dependent on BGLF4 kinase activity. Similarly, expression of doxycycline-inducible BGLF4 in Akata cells led to a reduction in episomal EBV genomes. We propose that BGLF4 contributes to effective EBV lytic cycle progression, not only through phosphorylation of EBV lytic DNA replication and virion proteins, but also by interfering with the EBNA1 replication function.

scholarly journals Essential Role of Rta in Lytic DNA Replication of Epstein-Barr Virus

2012 ◽  
Vol 87 (1) ◽  
pp. 208-223 ◽  
Author(s):  
A. El-Guindy ◽  
M. Ghiassi-Nejad ◽  
S. Golden ◽  
H.-J. Delecluse ◽  
G. Miller
Keyword(s):  
Dna Replication ◽  
Epstein Barr Virus ◽  
Essential Role ◽  
Barr Virus ◽  
Epstein Barr ◽  
Lytic Dna Replication

scholarly journals Tenofovir prodrugs potently inhibit Epstein–Barr virus lytic DNA replication by targeting the viral DNA polymerase

2020 ◽  
Vol 117 (22) ◽  
pp. 12368-12374 ◽  
Author(s):  
Natalia C. Drosu ◽  
Elazer R. Edelman ◽  
David E. Housman
Keyword(s):  
Dna Replication ◽  
Disease Prevention ◽  
Dna Polymerase ◽  
Epstein Barr Virus ◽  
Suppressive Therapy ◽  
Barr Virus ◽  
Acyclovir Treatment ◽  
Lytic Reactivation ◽  
Epstein Barr ◽  
Lytic Dna Replication

Epstein–Barr virus (EBV) is a ubiquitous human γ-herpesvirus that establishes life-long infection and increases the risk for the development of several cancers and autoimmune diseases. The mechanisms by which chronic EBV infection leads to subsequent disease remain incompletely understood. Lytic reactivation plays a central role in the development of EBV-driven cancers and may contribute to other EBV-associated diseases. Thus, the clinical use of antivirals as suppressive therapy for EBV lytic reactivation may aid efforts aimed at disease prevention. Current antivirals for EBV have shown limited clinical utility due to low potency or high toxicity, leaving open the need for potent antivirals suitable for long-term prophylaxis. In the present study, we show that tenofovir disoproxil fumarate (TDF) and tenofovir alafenamide (TAF), drugs with excellent safety profiles used clinically for HIV prevention, inhibit EBV lytic DNA replication, with respective IC50values of 0.30 μM and 84 nM. In a cell-based assay, TAF was 35- and 24-fold and TDF was 10- and 7-fold more potent than acyclovir and penciclovir, respectively, and TAF was also twice as potent as ganciclovir. The active metabolite of tenofovir prodrugs, tenofovir-diphosphate, inhibited the incorporation of dATP into a primed DNA template by the EBV DNA polymerase in vitro. In contrast to acyclovir, treatment of cells during latency for 24 h with TAF still inhibited EBV lytic DNA replication at 72 h after drug was removed. Our results suggest that tenofovir prodrugs may be particularly effective as inhibitors of EBV lytic reactivation, and that clinical studies to address critical questions about disease prevention are warranted.

scholarly journals Four-dimensional analyses show that replication compartments are clonal factories in which Epstein–Barr viral DNA amplification is coordinated

2019 ◽  
Vol 116 (49) ◽  
pp. 24630-24638 ◽  
Author(s):  
Thejaswi Nagaraju ◽  
Arthur U. Sugden ◽  
Bill Sugden
Keyword(s):  
Viral Replication ◽  
Epstein Barr Virus ◽  
Dna Amplification ◽  
Replication Proteins ◽  
Barr Virus ◽  
Viral Particles ◽  
Cell Assays ◽  
Ebv Dna ◽  
Epstein Barr ◽  
Do So

Herpesviruses must amplify their DNA to load viral particles and they do so in replication compartments. The development and functions of replication compartments during DNA amplification are poorly understood, though. Here we examine 2 functionally distinct replicons in the same cells to dissect DNA amplification within replication compartments. Using a combination of single-cell assays, computational modeling, and population approaches, we show that compartments initially were seeded by single genomes of Epstein–Barr virus (EBV). Their amplification subsequently took 13 to 14 h in individual cells during which their compartments occupied up to 30% of the nucleus and the nuclear volume grew by 50%. The compartmental volumes increased in proportion to the amount of DNA and viral replication proteins they contained. Each compartment synthesized similar levels of DNA, indicating that the total number of compartments determined the total levels of DNA amplification. Further, the amplification, which depended on the number of origins, was regulated differently early and late during the lytic phase; early during the lytic phase, the templates limited DNA synthesis, while later the templates were in excess, coinciding with a decline in levels of the viral replication protein, BMRF1, in the replication compartments. These findings show that replication compartments are factories in which EBV DNA amplification is both clonal and coordinated.

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