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 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 CD8+ immunodominance among Epstein-Barr virus lytic cycle antigens directly reflects the efficiency of antigen presentation in lytically infected cells

2005 ◽  
Vol 201 (3) ◽  
pp. 349-360 ◽  
Author(s):  
Victoria A. Pudney ◽  
Alison M. Leese ◽  
Alan B. Rickinson ◽  
Andrew D. Hislop
Keyword(s):  
T Cell ◽  
Epstein Barr Virus ◽  
Cd8 T Cell ◽  
Lytic Replication ◽  
Lytic Cycle ◽  
E Proteins ◽  
Barr Virus ◽  
Infected Cells ◽  
Epstein Barr ◽  
L Proteins

Antigen immunodominance is an unexplained feature of CD8+ T cell responses to herpesviruses, which are agents whose lytic replication involves the sequential expression of immediate early (IE), early (E), and late (L) proteins. Here, we analyze the primary CD8 response to Epstein-Barr virus (EBV) infection for reactivity to 2 IE proteins, 11 representative E proteins, and 10 representative L proteins, across a range of HLA backgrounds. Responses were consistently skewed toward epitopes in IE and a subset of E proteins, with only occasional responses to novel epitopes in L proteins. CD8+ T cell clones to representative IE, E, and L epitopes were assayed against EBV-transformed lymphoblastoid cell lines (LCLs) containing lytically infected cells. This showed direct recognition of lytically infected cells by all three sets of effectors but at markedly different levels, in the order IE > E ≫ L, indicating that the efficiency of epitope presentation falls dramatically with progress of the lytic cycle. Thus, EBV lytic cycle antigens display a hierarchy of immunodominance that directly reflects the efficiency of their presentation in lytically infected cells; the CD8+ T cell response thereby focuses on targets whose recognition leads to maximal biologic effect.

scholarly journals Cell Cycle Regulation by Kaposi's Sarcoma-Associated Herpesvirus K-bZIP: Direct Interaction with Cyclin-CDK2 and Induction of G1 Growth Arrest

2003 ◽  
Vol 77 (17) ◽  
pp. 9652-9661 ◽  
Author(s):  
Yoshihiro Izumiya ◽  
Su-Fang Lin ◽  
Thomas J. Ellison ◽  
Alon M. Levy ◽  
Greg L. Mayeur ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cell Cycle Regulation ◽  
Kaposi's Sarcoma ◽  
Epstein Barr Virus ◽  
Kaposi’S Sarcoma ◽  
Barr Virus ◽  
Infected Cells ◽  
Epstein Barr ◽  
Kaposi’S Sarcoma Associated Herpesvirus ◽  
Cycle Regulation

ABSTRACT In order to cope with hostile host environments, many viruses have developed strategies to perturb the cellular machinery to suit their replication needs. Some herpesvirus genes protect cells from undergoing apoptosis to prolong the lives of infected cells, while others, such as Epstein-Barr virus Zta, slow down the G1/S transition phase to allow ample opportunity for transcription and translation of viral genes before the onset of cellular genomic replication. In this study, we investigated whether Kaposi's sarcoma-associated herpesvirus (KSHV) K-bZIP, a homologue of the Epstein-Barr virus transcription factor BZLF1 (Zta), plays a role in cell cycle regulation. Here we show that K-bZIP physically associates with cyclin-CDK2 and downmodulates its kinase activity. The association can be detected in the natural environment of KSHV-infected cells without artificial overexpression of either component. With purified protein, it can be shown that the interaction between K-bZIP and cyclin-CDK2 is direct and that K-bZIP alone is sufficient to inhibit CDK2 activity. The interacting domain of K-bZIP has been mapped to the basic region. The result of these associations is a prolonged G1 phase, accompanied by the induction of p21 and p27 in a naturally infected B-cell line. Thus, in addition to the previously described transcription and genome replication functions, a new role of K-bZIP in KSHV replication is identified in this report.

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.

scholarly journals Epstein-Barr Virus Lytic Replication Elicits ATM Checkpoint Signal Transduction While Providing an S-phase-like Cellular Environment

2004 ◽  
Vol 280 (9) ◽  
pp. 8156-8163 ◽  
Author(s):  
Ayumi Kudoh ◽  
Masatoshi Fujita ◽  
Lumin Zhang ◽  
Noriko Shirata ◽  
Tohru Daikoku ◽  
...  
Keyword(s):  
Signal Transduction ◽  
Epstein Barr Virus ◽  
S Phase ◽  
Lytic Replication ◽  
Barr Virus ◽  
Cellular Environment ◽  
Epstein Barr ◽  
Checkpoint Signal

scholarly journals Expression of EBNA-1 mRNA Is Regulated by Cell Cycle during Epstein-Barr Virus Type I Latency

1999 ◽  
Vol 73 (4) ◽  
pp. 3154-3161 ◽  
Author(s):  
Matthew G. Davenport ◽  
Joseph S. Pagano
Keyword(s):  
Cell Cycle ◽  
Epstein Barr Virus ◽  
S Phase ◽  
Luciferase Reporter ◽  
Infected Cell Line ◽  
Type I ◽  
Mrna Levels ◽  
Dependent Manner ◽  
Barr Virus ◽  
Epstein Barr

ABSTRACT Expression of EBNA-1 protein is required for the establishment and maintenance of the Epstein-Barr virus (EBV) genome during latent infection. During type I latency, the BamHI Q promoter (Qp) gives rise to EBNA-1 expression. The dominant regulatory mechanism for Qp appears to be mediated through the Q locus, located immediately downstream of the transcription start site. Binding of EBNA-1 to the Q locus represses Qp constitutive activity, and repression has been reported to be overcome by an E2F family member that binds to the Q locus and displaces EBNA-1 (N. S. Sung, J. Wilson, M. Davenport, N. D. Sista, and J. S. Pagano, Mol. Cell. Biol. 14:7144–7152, 1994). These data suggest that the final outcome of Qp activity is reciprocally controlled by EBNA-1 and E2F. Since E2F activity is cell cycle regulated, Qp activity and EBNA-1 expression are predicted to be regulated in a cell cycle-dependent manner. Proliferation of the type I latently infected cell line, Akata, was synchronized with the use of the G2/M blocking agent nocodazole. From 65 to 75% of cells could be made to peak in S phase without evidence of viral reactivation. Following release from G2/M block, EBNA-1 mRNA levels declined as the synchronized cells entered the G1 phase of the cell cycle. As cells proceeded into S phase, EBNA-1 mRNA levels increased parallel to the peak in cell numbers in S phase. However, EBNA-1 protein levels showed no detectable change during the cell cycle, most likely due to the protein’s long half-life as estimated by inhibition of protein synthesis by cycloheximide. Finally, in Qp luciferase reporter assays, the activity of Qp was shown to be regulated by cell cycle and to be dependent on the E2F sites within the Q locus. These findings demonstrate that transcriptional activity of Qp is cell cycle regulated and indicated that E2F serves as the stimulus for this regulation.

scholarly journals Negative Autoregulation of Epstein-Barr Virus (EBV) Replicative Gene Expression by EBV SM Protein

2009 ◽  
Vol 83 (16) ◽  
pp. 8041-8050 ◽  
Author(s):  
Dinesh Verma ◽  
Chen Ling ◽  
Eric Johannsen ◽  
Tirumuru Nagaraja ◽  
Sankar Swaminathan
Keyword(s):  
Epstein Barr Virus ◽  
Feedback Regulation ◽  
Lytic Replication ◽  
Lytic Cycle ◽  
Immediate Early ◽  
Mrna Levels ◽  
Barr Virus ◽  
Infected Cells ◽  
Epstein Barr ◽  
Sm Protein

ABSTRACT The Epstein-Barr virus (EBV) SM protein is essential for lytic EBV DNA replication and virion production. When EBV replication is induced in cells infected with an SM-deleted recombinant EBV, approximately 50% of EBV genes are expressed inefficiently. When EBV replication is rescued by transfection of SM, SM enhances expression of these genes by direct and indirect mechanisms. While expression of most EBV genes is either unaffected or enhanced by SM, expression of several genes is decreased in the presence of SM. Expression of BHRF1, a homolog of cellular bcl-2, is particularly decreased in the presence of SM. Investigation of the mechanism of BHRF1 downregulation revealed that SM downregulates expression of the immediate-early EBV transactivator R. In EBV-infected cells, R-responsive promoters, including the BHRF1 and SM promoters, were less active in the presence of SM, consistent with SM inhibition of R expression. SM decreased spliced R mRNA levels, supporting a posttranscriptional mechanism of R inhibition. R and BHRF1 expression were also found to decrease during later stages of EBV lytic replication in EBV-infected lymphoma cells. These data indicate that feedback regulation of immediate-early and early genes occurs during the lytic cycle of EBV regulation.

scholarly journals Induction of the Lytic Cycle Sensitizes Epstein-Barr Virus-Infected B Cells to NK Cell Killing That Is Counteracted by Virus-Mediated NK Cell Evasion Mechanisms in the Late Lytic Cycle

2015 ◽  
Vol 90 (2) ◽  
pp. 947-958 ◽  
Author(s):  
Luke R. Williams ◽  
Laura L. Quinn ◽  
Martin Rowe ◽  
Jianmin Zuo
Keyword(s):  
B Cells ◽  
Nk Cells ◽  
Epstein Barr Virus ◽  
Nk Cell ◽  
Cell Killing ◽  
Lytic Replication ◽  
Lytic Cycle ◽  
Barr Virus ◽  
Infected Cells ◽  
Epstein Barr

ABSTRACTEpstein-Barr Virus (EBV) persists for the lifetime of the infected host despite eliciting strong immune responses. This persistence requires a fine balance between the host immune system and EBV immune evasion. Accumulating evidence suggests an important role for natural killer (NK) cells in this balance. NK cells can kill EBV-infected cells undergoing lytic replicationin vitro, and studies in both humans and mice with reconstituted human immune systems have shown that NK cells can limit EBV replication and prevent infectious mononucleosis. We now show that NK cells, via NKG2D and DNAM-1 interactions, recognize and kill EBV-infected cells undergoing lytic replication and that expression of a single EBV lytic gene, BZLF1, is sufficient to trigger sensitization to NK cell killing. We also present evidence suggesting the possibility of the existence of an as-yet-unidentified DNAM-1 ligand which may be particularly important for killing lytically infected normal B cells. Furthermore, while cells entering the lytic cycle become sensitized to NK cell killing, we observed that cells in the late lytic cycle are highly resistant. We identified expression of the vBcl-2 protein, BHRF1, as one effective mechanism by which EBV mediates this protection. Thus, contrary to the view expressed in some reports, EBV has evolved the ability to evade NK cell responses.IMPORTANCEThis report extends our understanding of the interaction between EBV and host innate responses. It provides the first evidence that the susceptibility to NK cell lysis of EBV-infected B cells undergoing lytic replication is dependent upon the phase of the lytic cycle. Induction of the lytic cycle is associated with acquired sensitization to NK cell killing, while progress through the late lytic cycle is associated with acquired resistance to killing. We provide mechanistic explanations for this novel observation, indicating important roles for the BZLF1 immediate early transactivator, the BHRF1 vBcl-2 homologue, and a novel ligand for the DNAM-1 NK cell receptor.

scholarly journals The Epstein-Barr Virus LF2 Protein Inhibits Viral Replication

2008 ◽  
Vol 82 (17) ◽  
pp. 8509-8519 ◽  
Author(s):  
Michael A. Calderwood ◽  
Amy M. Holthaus ◽  
Eric Johannsen
Keyword(s):  
Transcriptional Activation ◽  
Epstein Barr Virus ◽  
Reference Strain ◽  
Latent Infection ◽  
Potent Inhibitor ◽  
Lytic Replication ◽  
Future Studies ◽  
Barr Virus ◽  
Infected Cells ◽  
Epstein Barr

ABSTRACT The switch from Epstein-Barr virus (EBV) latent infection to lytic replication is governed by two transcriptional regulators, Zta and Rta. We previously reported that the EBV protein encoded by the LF2 gene binds to Rta and can inhibit Rta activity in reporter gene assays. We now report that LF2 associates with Rta in the context of EBV-infected cells induced for lytic replication. LF2 inhibition of Rta occurs in both epithelial and B cells, and this downregulation is promoter specific: LF2 decreases Rta activation of the BALF2, BMLF1, and BMRF1 promoters by 60 to 90% but does not significantly decrease Rta activation of its own promoter (Rp). LF2 decreases Rta activation by at least two mechanisms: decreased DNA binding and interference with transcriptional activation by the Rta acidic activation domain. Coexpression of LF2 also specifically induces modification of Rta by the small ubiquitin-like modifiers SUMO2 and SUMO3. We further demonstrate that LF2 overexpression blocks lytic activation in EBV-infected cells induced with Rta or Zta. Our results demonstrate that LF2, a gene deleted from the EBV reference strain B95-8, encodes a potent inhibitor of EBV replication, and they suggest that future studies of EBV replication need to account for the potential effects of LF2 on Rta activity.

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