scholarly journals The Epstein-Barr Virus Immediate-Early Protein BZLF1 Regulates p53 Function through Multiple Mechanisms

2002 ◽  
Vol 76 (24) ◽  
pp. 12503-12512 ◽  
Author(s):  
Amy Mauser ◽  
Shin'ichi Saito ◽  
Ettore Appella ◽  
Carl W. Anderson ◽  
William T. Seaman ◽  
...  
Keyword(s):  
Dna Binding ◽  
Epstein Barr Virus ◽  
Human Fibroblasts ◽  
Binding Activity ◽  
Immediate Early ◽  
Inhibitory Effects ◽  
Barr Virus ◽  
Dna Binding Activity ◽  
Early Protein ◽  
Epstein Barr

ABSTRACT The Epstein-Barr virus (EBV) immediate-early protein BZLF1 is a transcriptional activator that mediates the switch between the latent and the lytic forms of EBV infection. It was previously reported that BZLF1 inhibits p53 transcriptional function in reporter gene assays. Here we further examined the effects of BZLF1 on p53 function by using a BZLF1-expressing adenovirus vector (AdBZLF1). Infection of cells with the AdBZLF1 vector increased the level of cellular p53 but prevented the induction of p53-dependent cellular target genes, such as p21 and MDM2. BZLF1-expressing cells had increased p53-specific DNA binding activity in electrophoretic mobility shift assays, increased p53 phosphorylation at multiple residues (including serines 6, 9, 15, 33, 46, 315, and 392), and increased acetylation at lysine 320 and lysine 382. Thus, the inhibitory effects of BZLF1 on p53 transcriptional function cannot be explained by its effects on p53 phosphorylation, acetylation, or DNA binding activity. BZLF1 substantially reduced the level of cellular TATA binding protein (TBP) in both normal human fibroblasts and A549 cells, and the inhibitory effects of BZLF1 on p53 transcriptional function could be partially rescued by the overexpression of TBP. Thus, BZLF1 has numerous effects on p53 posttranslational modification but may inhibit p53 transcriptional function in part through an indirect mechanism involving the suppression of TBP expression.

scholarly journals Constitutive activation of STAT proteins in primary lymphoid and myeloid leukemia cells and in Epstein-Barr virus (EBV)-related lymphoma cell lines

Blood ◽  
1996 ◽  
Vol 88 (3) ◽  
pp. 809-816 ◽  
Author(s):  
RM Weber-Nordt ◽  
C Egen ◽  
J Wehinger ◽  
W Ludwig ◽  
V Gouilleux-Gruart ◽  
...  
Keyword(s):  
Dna Binding ◽  
Nuclear Localization ◽  
Myeloid Leukemia ◽  
Epstein Barr Virus ◽  
Binding Activity ◽  
Leukemia Cells ◽  
Stat Proteins ◽  
Barr Virus ◽  
Dna Binding Activity ◽  
Epstein Barr

Abstract Although various molecular mechanisms of STAT protein (signal transducers and activators of transcription) activation have been identified, little is known about the functional role of STAT-dependent transcriptional activation. Herein we report the constitutive nuclear localization, phosphorylation, and DNA-binding activity of STAT proteins in leukemia cells and lymphoma cell lines. With the use of oligonucleotide probes derived from the Fc gamma RI promoter, the beta- casein promoter and a STAT-binding element in the promoter of the Bci-2 gene constitutive activation of STAT proteins was detected in untreated acute T- and C/B-leukemia cells (3 of 5 and 12 of 19 patients, respectively). Supershift analyses using Stats 1–6 specific antisera showed the constitutive DNA binding activity of Stat5 in these cells. Confocal microscopy revealed the nuclear localization of Stat5 and Western blot analyses showed tyrosine phosphorylation of Stat5 in nuclear extracts of acute leukemia cells. In contrast, peripheral blood mononuclear cells did not display constitutive STAT-DNA interaction. Further studies were performed on freshly isolated acute myeloid leukemia cells as well as on cell line derived K562, lymphoblastoid cells (LCL), and Burkitt's lymphoma cells (BL). Fluorescence microscopy, gelshift, and supershift experiments showed the nuclear localization and constitutive DNA-binding activity of Stat5 in K562 cells. Stat1 and Stat3 were constitutively activated in freshly isolated AML cells (10 of 14 patients) and in Epstein Barr virus- positive or interleukin-10 expressing permanent LCL and BL cells. Thus, these data indicate a differential pattern of STAT protein activation in lymphoid or myeloid leukemia and in lymphoma cells.

scholarly journals Constitutive activation of STAT proteins in primary lymphoid and myeloid leukemia cells and in Epstein-Barr virus (EBV)-related lymphoma cell lines

Blood ◽  
1996 ◽  
Vol 88 (3) ◽  
pp. 809-816 ◽  
Author(s):  
RM Weber-Nordt ◽  
C Egen ◽  
J Wehinger ◽  
W Ludwig ◽  
V Gouilleux-Gruart ◽  
...  
Keyword(s):  
Dna Binding ◽  
Nuclear Localization ◽  
Myeloid Leukemia ◽  
Epstein Barr Virus ◽  
Binding Activity ◽  
Leukemia Cells ◽  
Stat Proteins ◽  
Barr Virus ◽  
Dna Binding Activity ◽  
Epstein Barr

Although various molecular mechanisms of STAT protein (signal transducers and activators of transcription) activation have been identified, little is known about the functional role of STAT-dependent transcriptional activation. Herein we report the constitutive nuclear localization, phosphorylation, and DNA-binding activity of STAT proteins in leukemia cells and lymphoma cell lines. With the use of oligonucleotide probes derived from the Fc gamma RI promoter, the beta- casein promoter and a STAT-binding element in the promoter of the Bci-2 gene constitutive activation of STAT proteins was detected in untreated acute T- and C/B-leukemia cells (3 of 5 and 12 of 19 patients, respectively). Supershift analyses using Stats 1–6 specific antisera showed the constitutive DNA binding activity of Stat5 in these cells. Confocal microscopy revealed the nuclear localization of Stat5 and Western blot analyses showed tyrosine phosphorylation of Stat5 in nuclear extracts of acute leukemia cells. In contrast, peripheral blood mononuclear cells did not display constitutive STAT-DNA interaction. Further studies were performed on freshly isolated acute myeloid leukemia cells as well as on cell line derived K562, lymphoblastoid cells (LCL), and Burkitt's lymphoma cells (BL). Fluorescence microscopy, gelshift, and supershift experiments showed the nuclear localization and constitutive DNA-binding activity of Stat5 in K562 cells. Stat1 and Stat3 were constitutively activated in freshly isolated AML cells (10 of 14 patients) and in Epstein Barr virus- positive or interleukin-10 expressing permanent LCL and BL cells. Thus, these data indicate a differential pattern of STAT protein activation in lymphoid or myeloid leukemia and in lymphoma cells.

scholarly journals Epstein-Barr Virus Immediate-Early Proteins BZLF1 and BRLF1 Activate the ATF2 Transcription Factor by Increasing the Levels of Phosphorylated p38 and c-Jun N-Terminal Kinases

2000 ◽  
Vol 74 (3) ◽  
pp. 1224-1233 ◽  
Author(s):  
Amy L. Adamson ◽  
Dayle Darr ◽  
Elizabeth Holley-Guthrie ◽  
Robert A. Johnson ◽  
Amy Mauser ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Epstein Barr Virus ◽  
Viral Latency ◽  
Immediate Early ◽  
Expression Vectors ◽  
Protein Z ◽  
Barr Virus ◽  
Ebv Infection ◽  
Early Protein ◽  
Epstein Barr

ABSTRACT Expression of either Epstein-Barr virus (EBV) immediate-early protein BZLF1 (Z) or BRLF1 (R) is sufficient to convert EBV infection from the latent to lytic form. Disruption of viral latency requires transcriptional activation of the Z and R promoters. The Z and R proteins are transcriptional activators, and each immediate-early protein activates expression of the other immediate-early protein. Z activates the R promoter through a direct binding mechanism. However, R does not bind directly to the Z promoter. In this study, we demonstrate that the ZII element (a cyclic AMP response element site) in the Z promoter is required for efficient activation by R. The ZII element has been shown to be important for induction of lytic EBV infection by tetradecanoyl phorbol acetate and surface immunoglobulin cross-linking and is activated by Z through an indirect mechanism. We demonstrate that both R and Z activate the cellular stress mitogen-activated protein (MAP) kinases, p38 and JNK, resulting in phosphorylation (and activation) of the cellular transcription factor ATF2. Furthermore, we show that the ability of R to induce lytic EBV infection in latently infected cells is significantly reduced by inhibition of either the p38 kinase or JNK pathways. In contrast, inhibition of stress MAP kinase pathways does not impair the ability of Z expression vectors to disrupt viral latency, presumably because expression of Z under the control of a strong heterologous promoter bypasses the need to activate Z transcription. Thus, both R and Z can activate the Z promoter indirectly by inducing ATF2 phosphorylation, and this activity appears to be important for R-induced disruption of viral latency.

scholarly journals Epstein–Barr virus nuclear antigen 1 is a DNA-binding protein with strong RNA-binding activity

2004 ◽  
Vol 85 (10) ◽  
pp. 2755-2765 ◽  
Author(s):  
Chih-Chung Lu ◽  
Chia-Wei Wu ◽  
Shin C. Chang ◽  
Tzu-Yi Chen ◽  
Chwan-Ren Hu ◽  
...  
Keyword(s):  
Dna Binding ◽  
Epstein Barr Virus ◽  
Rna Binding ◽  
Binding Protein ◽  
Dna Binding Protein ◽  
Binding Activity ◽  
Nuclear Antigen ◽  
Barr Virus ◽  
Infected Cells ◽  
Epstein Barr

Epstein–Barr virus (EBV) nuclear antigen 1 (EBNA-1) plays key roles in both the regulation of gene expression and the replication of the EBV genome in latently infected cells. To characterize the RNA-binding activity of EBNA-1, it was demonstrated that EBNA-1 binds efficiently to RNA homopolymers that are composed of poly(G) and weakly to those composed of poly(U). All three RGG boxes of EBNA-1 contributed additively to poly(G)-binding activity and could mediate RNA binding when attached to a heterologous protein in an RNA gel mobility-shift assay. In vitro-transcribed EBV and non-EBV RNA probes revealed that EBNA-1 bound to most RNAs examined and the affinity increased as the content of G and U increased, as demonstrated in competition assays. Among these probes, the 5′ non-coding region (NCR) (nt 131–278) of hepatitis C virus RNA appeared to be the strongest competitor for EBNA-1 binding to the EBV-encoded small nuclear RNA 1 (EBER1) probe, whereas a mutant 5′ NCR RNA with partially disrupted secondary structure was a weak competitor. Furthermore, the interaction of endogenous EBNA-1 and EBER1 in EBV-infected cells was demonstrated by a ribonucleoprotein immunoprecipitation assay. These results revealed that EBNA-1 is a DNA-binding protein with strong binding activity to a relatively broad spectrum of RNA and suggested an additional biological impact of EBNA-1 through its ability to bind to RNA.

scholarly journals Epstein-Barr Virus Immediate-Early Protein BZLF1 Is SUMO-1 Modified and Disrupts Promyelocytic Leukemia Bodies

2001 ◽  
Vol 75 (5) ◽  
pp. 2388-2399 ◽  
Author(s):  
Amy L. Adamson ◽  
Shannon Kenney
Keyword(s):  
Cell Lines ◽  
Transcriptional Activation ◽  
Epstein Barr Virus ◽  
Promyelocytic Leukemia ◽  
Lytic Replication ◽  
Immediate Early ◽  
Barr Virus ◽  
Early Protein ◽  
Pml Bodies ◽  
Epstein Barr

ABSTRACT Although the immediate-early proteins of both herpes simplex virus (HSV) and cytomegalovirus (CMV) are known to modify promyelocytic leukemia (PML) (ND10) bodies in the nucleus of the host cell, it has been unclear whether lytic infection with gamma herpesviruses induces a similar effect. The PML protein is induced by interferon, involved in major histocompatibility complex class I presentation, and necessary for certain types of apoptosis. Therefore, it is likely that PML bodies function in an antiviral capacity. SUMO-1 modification of PML is known to be required for the formation of PML bodies. To examine whether Epstein-Barr virus (EBV) lytic replication interferes with PML bodies, we expressed the EBV immediate-early genes BZLF1 (Z) and BRLF1 (R) in EBV-positive cell lines and examined PML localization. Both Z and R expression resulted in PML dispersion in EBV-positive cells. Z but not R expression is sufficient to disrupt PML bodies in EBV-negative cell lines. We show that dispersion of PML bodies by Z requires a portion of the transcriptional activation domain of Z but not the DNA-binding function. As was previously reported for the HSV-1 ICP0 and CMV IE1 proteins, Z reduces the amount of SUMO-1-modified PML. We also found that Z itself is SUMO-1 modified (through amino acid 12) and that Z competes with PML for limiting amounts of SUMO-1. These results suggest that disruption of PML bodies is important for efficient lytic replication of EBV. Furthermore, Z may potentially alter the function of a variety of cellular proteins by inhibiting SUMO-1 modification.

scholarly journals Inhibition of IFN-γ Signaling by an Epstein-Barr Virus Immediate-Early Protein

Immunity ◽  
2001 ◽  
Vol 15 (5) ◽  
pp. 787-799 ◽  
Author(s):  
Thomas E Morrison ◽  
Amy Mauser ◽  
Athena Wong ◽  
Jenny P.-Y Ting ◽  
Shannon C Kenney
Keyword(s):  
Epstein Barr Virus ◽  
Immediate Early ◽  
Barr Virus ◽  
Early Protein ◽  
Ifn Γ ◽  
Epstein Barr

scholarly journals Expression of Epstein–Barr virus BZLF1 immediate-early protein induces p53 degradation independent of MDM2, leading to repression of p53-mediated transcription

Virology ◽  
2009 ◽  
Vol 388 (1) ◽  
pp. 204-211 ◽  
Author(s):  
Yoshitaka Sato ◽  
Noriko Shirata ◽  
Ayumi Kudoh ◽  
Satoko Iwahori ◽  
Sanae Nakayama ◽  
...  
Keyword(s):  
Epstein Barr Virus ◽  
Immediate Early ◽  
Barr Virus ◽  
Early Protein ◽  
Epstein Barr

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.

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