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 Inhibition of S-Phase Cyclin-Dependent Kinase Activity Blocks Expression of Epstein-Barr Virus Immediate-Early and Early Genes, Preventing Viral Lytic Replication

2004 ◽  
Vol 78 (1) ◽  
pp. 104-115 ◽  
Author(s):  
Ayumi Kudoh ◽  
Tohru Daikoku ◽  
Yutaka Sugaya ◽  
Hiroki Isomura ◽  
Masatoshi Fujita ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Epstein Barr Virus ◽  
S Phase ◽  
Lytic Replication ◽  
Immediate Early ◽  
Specific Cell ◽  
Cyclin Dependent Kinase ◽  
Barr Virus ◽  
Cellular Environment ◽  
Epstein Barr

ABSTRACT The induction of lytic replication of the Epstein-Barr virus (EBV) completely arrests cell cycle progression, in spite of elevation of S-phase cyclin-dependent kinase (CDK) activity, thereby causing accumulation of hyperphosphorylated forms of retinoblastoma (Rb) protein (A. Kudoh, M. Fujita, T. Kiyono, K. Kuzushima, Y. Sugaya, S. Izuta, Y. Nishiyama, and T. Tsurumi, J. Virol. 77:851-861, 2003). Thus, the EBV lytic program appears to promote specific cell cycle-associated activity involved in the progression from G1 to S phase. We have proposed that this provides a cellular environment that is advantageous for EBV productive infection. Purvalanol A and roscovitine, inhibitors of S-phase CDKs, blocked the viral lytic replication when cells were treated at the early stage of lytic infection, while well-characterized inhibitors of enzymes, such as mitogen-activated protein kinase, phosphatidylinositol 3-kinase, and protein kinase C, known to be involved in BZLF1 gene expression did not. Inhibition of CDK activity resulted in the accumulation of the hypophosphorylated form of Rb protein and inhibition of expression of EBV immediate-early and early proteins. Cycloheximide block-and-release experiments clearly demonstrated that even in the presence of enough amounts of the BZLF1 protein, purvalanol A blocked expression of lytic viral proteins at transcription level. Furthermore, reporter gene experiments confirmed that BZLF1-induced activation of early EBV promoters was impaired in the presence of the CDK inhibitor. We conclude here that the EBV lytic program promotes specific cell cycle-associated activity involved in the progression from G1 to S phase because the S-phase-like cellular environment is essential for the expression of immediate-early and early genes supplying the viral replication proteins and hence for lytic viral replication.

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 The Epstein-Barr Virus Protein BRLF1 Activates S Phase Entry through E2F1 Induction

1999 ◽  
Vol 73 (8) ◽  
pp. 6540-6550 ◽  
Author(s):  
Jennifer J. Swenson ◽  
Amy E. Mauser ◽  
William K. Kaufmann ◽  
Shannon C. Kenney
Keyword(s):  
Cell Cycle ◽  
Epstein Barr Virus ◽  
Human Fibroblasts ◽  
S Phase ◽  
Lytic Replication ◽  
Osteosarcoma Cell ◽  
Barr Virus ◽  
Infected Cells ◽  
Epstein Barr ◽  
Phase Entry

ABSTRACT The Epstein-Barr Virus (EBV) immediate-early protein BRLF1 is one of two transactivators which mediate the switch from latent to lytic replication in EBV-infected cells. DNA viruses often modulate the function of critical cell cycle proteins to maximize the efficiency of virus replication. Here we have examined the effect of BRLF1 on cell cycle progression. A replication-deficient adenovirus expressing BRLF1 (AdBRLF1) was used to infect normal human fibroblasts and various epithelial cell lines. BRLF1 expression induced S phase entry in contact-inhibited fibroblasts and in the human osteosarcoma cell line U-2 OS. AdBRLF1 infection produced a dramatic increase in the level of E2F1 but not E2F4. In contrast, the levels of Rb, p107, and p130 were decreased in AdBRLF1-infected cells. Electrophoretic mobility shift assays confirmed an increased level of free E2F1 in the AdBRLF1-infected human fibroblasts. Consistent with the previously described effect of E2F1, AdBRLF1-infected fibroblasts had increased levels of p53 and p21 and died by apoptosis. BRLF1-induced activation of E2F1 may be required for efficient EBV lytic replication, since at least one critical viral replication gene (the viral DNA polymerase) is activated by E2F (C. Liu, N. D. Sista, and J. S. Pagano, J. Virol. 70:2545–2555, 1996).

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 Effect of phosphorylation on the transactivation activity of Epstein–Barr virus BMRF1, a major target of the viral BGLF4 kinase

2008 ◽  
Vol 89 (4) ◽  
pp. 884-895 ◽  
Author(s):  
Pei-Wen Yang ◽  
Shih-Shin Chang ◽  
Ching-Hwa Tsai ◽  
Yi-Hsin Chao ◽  
Mei-Ru Chen
Keyword(s):  
Dna Replication ◽  
Nuclear Localization ◽  
Epstein Barr Virus ◽  
Human Herpesvirus ◽  
Lytic Replication ◽  
Barr Virus ◽  
Transactivation Activity ◽  
Synergistic Activation ◽  
Functional Regulation ◽  
Epstein Barr

Modification of human herpesvirus DNA polymerase processivity factors (PFs) by phosphorylation occurs frequently during viral lytic replication. However, functional regulation of the herpesvirus PFs through phosphorylation is not well understood. In addition to processivity, the PF BMRF1 of Epstein–Barr virus can function as a transactivator to activate the BHLF1 promoter within the lytic origin of replication (oriLyt), which is assumed to facilitate DNA replication through remodelling viral chromatin structure. BMRF1 is known to be phosphorylated by the viral BGLF4 kinase, but its impact on BMRF1 function is unclear. Seven candidate BGLF4 target sites were predicted within a proline-rich region between the DNA-processivity and nuclear-localization domains of BMRF1. We show that four of these residues, Ser-337, Thr-344, Ser-349 and Thr-355, are responsible for the BGLF4-induced hyperphosphorylation of BMRF1. In functional analyses, a phosphorylation-mimicking mutant of BMRF1 shows similar nuclear localization, as well as DNA-binding ability, to the wild type; however, it displays stronger synergistic activation of the BHLF1 promoter with Zta. Notably, BGLF4 downregulates BMRF1 transactivation and enhances the transactivation activity of Zta and the synergistic activation of BMRF1 and Zta on the BHLF1 promoter. Our findings suggest that BGLF4 may modulate the activation of the oriLyt BHLF1 promoter coordinately through multiple mechanisms to facilitate optimal oriLyt-dependent viral DNA replication.

scholarly journals Multiple Roles of Epstein-Barr Virus SM Protein in Lytic Replication

2007 ◽  
Vol 81 (8) ◽  
pp. 4058-4069 ◽  
Author(s):  
Zhao Han ◽  
Elessa Marendy ◽  
Yong-Dong Wang ◽  
Jing Yuan ◽  
Jeffery T. Sample ◽  
...  
Keyword(s):  
Gene Expression ◽  
Dna Replication ◽  
Epstein Barr Virus ◽  
Lytic Replication ◽  
Late Gene ◽  
Late Gene Expression ◽  
Barr Virus ◽  
Ebv Dna ◽  
Epstein Barr ◽  
Sm Protein

ABSTRACT The effect of Epstein-Barr virus (EBV) SM protein on EBV gene expression was examined using a recombinant EBV strain with the SM gene deleted and DNA microarrays representing all known EBV coding regions. Induction of lytic EBV replication in the absence of SM led to expression of approximately 40% of EBV genes, but a block in expression of over 50% of EBV genes. Contrary to previous findings, several early genes were SM dependent, and lytic EBV DNA replication did not occur in the absence of SM. Notably, two genes essential for lytic EBV DNA replication, BSLF1 and BALF5, encoding EBV DNA primase and polymerase, respectively, were SM dependent. Lytic DNA replication was partially rescued by ectopic expression of EBV primase and polymerase, but virion production was not. Rescue of DNA replication only enhanced expression of a subset of late genes, consistent with a direct requirement for SM for late gene expression in addition to its contribution to DNA replication. Therefore, while SM is essential for most late gene expression, the proximate block to virion production by the EBV SM deletion strain is an inability to replicate linear DNA. The block to DNA replication combined with the direct effect of SM on late gene expression leads to a global deficiency of late gene expression. SM also inhibited BHRF1 expression during productive replication in comparison to that of cells induced into lytic replication in the absence of SM. Thus, SM plays a role in multiple steps of lytic cycle EBV gene expression and that it is transcript-specific in both activation and repression functions.

scholarly journals Homologous Recombinational Repair Factors Are Recruited and Loaded onto the Viral DNA Genome in Epstein-Barr Virus Replication Compartments

2009 ◽  
Vol 83 (13) ◽  
pp. 6641-6651 ◽  
Author(s):  
Ayumi Kudoh ◽  
Satoko Iwahori ◽  
Yoshitaka Sato ◽  
Sanae Nakayama ◽  
Hiroki Isomura ◽  
...  
Keyword(s):  
Dna Replication ◽  
Homologous Recombination ◽  
Viral Genome ◽  
Epstein Barr Virus ◽  
Lytic Replication ◽  
Recombinational Repair ◽  
Viral Dna ◽  
Barr Virus ◽  
Homologous Recombinational Repair ◽  
Epstein Barr

ABSTRACT Homologous recombination is an important biological process that facilitates genome rearrangement and repair of DNA double-strand breaks (DSBs). The induction of Epstein-Barr virus (EBV) lytic replication induces ataxia telangiectasia-mutated (ATM)-dependent DNA damage checkpoint signaling, leading to the clustering of phosphorylated ATM and Mre11/Rad50/Nbs1 (MRN) complexes to sites of viral genome synthesis in nuclei. Here we report that homologous recombinational repair (HRR) factors such as replication protein A (RPA), Rad51, and Rad52 as well as MRN complexes are recruited and loaded onto the newly synthesized viral genome in replication compartments. The 32-kDa subunit of RPA is extensively phosphorylated at sites in accordance with those with ATM. The hyperphosphorylation of RPA32 causes a change in RPA conformation, resulting in a switch from the catalysis of DNA replication to the participation in DNA repair. The levels of Rad51 and phosphorylated RPA were found to increase with the progression of viral productive replication, while that of Rad52 proved constant. Furthermore, biochemical fractionation revealed increases in levels of DNA-bound forms of these HRRs. Bromodeoxyuridine-labeled chromatin immunoprecipitation and PCR analyses confirmed the loading of RPA, Rad 51, Rad52, and Mre11 onto newly synthesized viral DNA, and terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling analysis demonstrated DSBs in the EBV replication compartments. HRR factors might be recruited to repair DSBs on the viral genome in viral replication compartments. RNA interference knockdown of RPA32 and Rad51 prevented viral DNA synthesis remarkably, suggesting that homologous recombination and/or repair of viral DNA genome might occur, coupled with DNA replication to facilitate viral genome synthesis.

scholarly journals Evidence for DNA Hairpin Recognition by Zta at the Epstein-Barr Virus Origin of Lytic Replication

2010 ◽  
Vol 84 (14) ◽  
pp. 7073-7082 ◽  
Author(s):  
Andrew J. Rennekamp ◽  
Pu Wang ◽  
Paul M. Lieberman
Keyword(s):  
Dna Replication ◽  
Inverted Repeat ◽  
Epstein Barr Virus ◽  
Lytic Replication ◽  
Lytic Cycle ◽  
Productive Infection ◽  
Dna Hairpin ◽  
Barr Virus ◽  
Early Protein ◽  
Epstein Barr

ABSTRACT The Epstein-Barr virus immediate-early protein (Zta) plays an essential role in viral lytic activation and pathogenesis. Zta is a basic zipper (b-Zip) domain-containing protein that binds multiple sites in the viral origin of lytic replication (OriLyt) and is required for lytic-cycle DNA replication. We present evidence that Zta binds to a sequence-specific, imperfect DNA hairpin formed by an inverted repeat within the upstream essential element (UEE) of OriLyt. Mutations in the OriLyt sequence that are predicted to disrupt hairpin formation also disrupt Zta binding in vitro. Restoration of the hairpin rescues the defect. We also show that OriLyt DNA isolated from replicating cells contains a nuclease-sensitive region that overlaps with the inverted-repeat region of the UEE. Furthermore, point mutations in Zta that disrupt specific recognition of the UEE hairpin are defective for activation of lytic replication. These data suggest that Zta acts by inducing and/or stabilizing a DNA hairpin structure during productive infection. The DNA hairpin at OriLyt with which Zta interacts resembles DNA structures formed at other herpesvirus origins and may therefore represent a common secondary structure used by all herpesvirus family members during the initiation of DNA replication.

scholarly journals Epstein–Barr virus Rta-mediated transactivation of p21 and 14-3-3σ arrests cells at the G1/S transition by reducing cyclin E/CDK2 activity

2012 ◽  
Vol 93 (1) ◽  
pp. 139-149 ◽  
Author(s):  
Sheng-Yen Huang ◽  
Min-Jie Hsieh ◽  
Chu-Ying Chen ◽  
Yen-Ju Chen ◽  
Jen-Yang Chen ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cell Cycle Arrest ◽  
Epstein Barr Virus ◽  
Cyclin E ◽  
Lytic Replication ◽  
Immediate Early ◽  
Luciferase Reporter ◽  
Barr Virus ◽  
Cycle Arrest ◽  
Epstein Barr

Many herpesviral immediate-early proteins promote their robust lytic phase replications by hijacking the cell cycle machinery. Previously, lytic replication of Epstein–Barr virus (EBV) was found to be concurrent with host cell cycle arrest. In this study, we showed that ectopic expression of EBV immediate-early protein Rta in HEp-2 cells resulted in increased G1/S population, hypophosphorylation of pRb and decreased incorporation of 5-bromo-2′-deoxyuridine. In addition, EBV Rta transcriptionally upregulates the expressions of p21 and 14-3-3σ in HEp-2 cells, 293 cells and nasopharyngeal carcinoma TW01 cells. Although p21 and 14-3-3σ are known targets for p53, Rta-mediated p21 and 14-3-3σ transactivation can be detected in the absence of p53. In addition, results from luciferase reporter assays indicated that direct binding of Rta to either promoter sequences is not required for activation. On the other hand, a special class of Sp1-responsive elements was involved in Rta-mediated transcriptional activation on both promoters. Finally, Rta-induced p21 expression diminished the activity of CDK2/cyclin E complex, and, Rta-induced 14-3-3σ expression sequestered CDK1 and CDK2 in the cytoplasm. Based on these results, we hypothesize that through the disruption of CDK1 and CDK2 activities, EBV Rta might contribute to cell cycle arrest in EBV-infected epithelial cells during viral reactivation.

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