scholarly journals A Noncanonical Basic Motif of Epstein-Barr Virus ZEBRA Protein Facilitates Recognition of Methylated DNA, High-Affinity DNA Binding, and Lytic Activation

2019 ◽  
Vol 93 (14) ◽  
Author(s):  
Erin Weber ◽  
Olga Buzovetsky ◽  
Lee Heston ◽  
Kuan-Ping Yu ◽  
Kirsten M. Knecht ◽  
...  
Keyword(s):  
Dna Binding ◽  
Viral Replication ◽  
Epstein Barr Virus ◽  
Lytic Cycle ◽  
Binding Motif ◽  
Methylated Dna ◽  
Barr Virus ◽  
High Affinity ◽  
Bzip Domain ◽  
Epstein Barr

ABSTRACTThe pathogenesis of Epstein-Barr virus (EBV) infection, including development of lymphomas and carcinomas, is dependent on the ability of the virus to transit from latency to the lytic phase. This conversion, and ultimately disease development, depends on the molecular switch protein, ZEBRA, a viral bZIP transcription factor that initiates transcription from promoters of viral lytic genes. By binding to the origin of viral replication, ZEBRA is also an essential replication protein. Here, we identified a novel DNA-binding motif of ZEBRA, N terminal to the canonical bZIP domain. This RRTRK motif is important for high-affinity binding to DNA and is essential for recognizing the methylation state of viral promoters. Mutations in this motif lead to deficiencies in DNA binding, recognition of DNA methylation, lytic cycle DNA replication, and viral late gene expression. This work advances our understanding of ZEBRA-dependent activation of the viral lytic cascade.IMPORTANCEThe binding of ZEBRA to methylated and unmethylated viral DNA triggers activation of the EBV lytic cycle, leading to viral replication and, in some patients, cancer development. Our work thoroughly examines how ZEBRA uses a previously unrecognized basic motif to bind nonmethylated and methylated DNA targets, leading to viral lytic activation. Our findings show that two different positively charged motifs, including the canonical BZIP domain and a newly identified RRTRK motif, contribute to the mechanism of DNA recognition by a viral AP-1 protein. This work contributes to the assessment of ZEBRA as a potential therapeutic target for antiviral and oncolytic treatments.

scholarly journals Phosphoacceptor Site S173 in the Regulatory Domain of Epstein-Barr Virus ZEBRA Protein Is Required for Lytic DNA Replication but Not for Activation of Viral Early Genes

2007 ◽  
Vol 81 (7) ◽  
pp. 3303-3316 ◽  
Author(s):  
Ayman El-Guindy ◽  
Lee Heston ◽  
Henri-Jacques Delecluse ◽  
George Miller
Keyword(s):  
Dna Binding ◽  
Viral Replication ◽  
Epstein Barr Virus ◽  
Lytic Replication ◽  
Lytic Cycle ◽  
Regulation Of Transcription ◽  
Regulatory Domain ◽  
Barr Virus ◽  
Early Genes ◽  
Epstein Barr

ABSTRACT The Epstein-Barr virus ZEBRA protein controls the viral lytic cycle. ZEBRA activates the transcription of viral genes required for replication. ZEBRA also binds to oriLyt and interacts with components of the viral replication machinery. The mechanism that differentiates the roles of ZEBRA in regulation of transcription and initiation of lytic replication is unknown. Here we show that S173, a residue in the regulatory domain, is obligatory for ZEBRA to function as an origin binding protein but is dispensable for its role as a transcriptional activator of early genes. Serine-to-alanine substitution of this residue, which prevents phosphorylation of S173, resulted in a threefold reduction in the DNA binding affinity of ZEBRA for oriLyt, as assessed by chromatin immunoprecipitation. An independent assay based on ZEBRA solubility demonstrated a marked defect in DNA binding by the Z(S173A) mutant. The phenotype of a phosphomimetic mutant, the Z(S173D) mutant, was similar to that of wild-type ZEBRA. Our findings suggest that phosphorylation of S173 promotes viral replication by enhancing ZEBRA's affinity for DNA. The results imply that stronger DNA binding is required for ZEBRA to activate replication than that required to activate transcription.

DNA-binding-defective mutants of the Epstein-Barr virus lytic switch activator Zta transactivate with altered specificities

1994 ◽  
Vol 14 (5) ◽  
pp. 3041-3052
Author(s):  
E K Flemington ◽  
J P Lytle ◽  
C Cayrol ◽  
A M Borras ◽  
S H Speck
Keyword(s):  
Dna Binding ◽  
Epstein Barr Virus ◽  
Lytic Cycle ◽  
Promoter Elements ◽  
Barr Virus ◽  
Binding Forms ◽  
Viral Genes ◽  
Direct Binding ◽  
Epstein Barr ◽  
Necessary And Sufficient

The Epstein-Barr virus BRLF1 and BZLF1 genes are the first viral genes transcribed upon induction of the viral lytic cycle. The protein products of both genes (referred to here as Rta and Zta, respectively) activate expression of other viral genes, thereby initiating the lytic cascade. Among the viral antigens expressed upon induction of the lytic cycle, however, Zta is unique in its ability to disrupt viral latency; expression of the BZLF1 gene is both necessary and sufficient for triggering the viral lytic cascade. We have previously shown that Zta can activate its own promoter (Zp), through binding to two Zta recognition sequences (ZIIIA and ZIIIB). Here we describe mutant Zta proteins that do not bind DNA (referred to as Zta DNA-binding mutants [Zdbm]) but retain the ability to transactivate Zp. Consistent with the inability of these mutants to bind DNA, transactivation of Zp by Zdbm is not dependent on the Zta recognition sequences. Instead, transactivation by Zdbm is dependent upon promoter elements that bind cellular factors. An examination of other viral and cellular promoters identified promoters that are weakly responsive or unresponsive to Zdbm. An analysis of a panel of artificial promoters containing one copy of various promoter elements demonstrated a specificity for Zdbm activation that is distinct from that of Zta. These results suggest that non-DNA-binding forms of some transactivators retain the ability to transactivate specific target promoters without direct binding to DNA.

scholarly journals DNA-binding-defective mutants of the Epstein-Barr virus lytic switch activator Zta transactivate with altered specificities.

1994 ◽  
Vol 14 (5) ◽  
pp. 3041-3052 ◽  
Author(s):  
E K Flemington ◽  
J P Lytle ◽  
C Cayrol ◽  
A M Borras ◽  
S H Speck
Keyword(s):  
Dna Binding ◽  
Epstein Barr Virus ◽  
Lytic Cycle ◽  
Promoter Elements ◽  
Barr Virus ◽  
Binding Forms ◽  
Viral Genes ◽  
Direct Binding ◽  
Epstein Barr ◽  
Necessary And Sufficient

The Epstein-Barr virus BRLF1 and BZLF1 genes are the first viral genes transcribed upon induction of the viral lytic cycle. The protein products of both genes (referred to here as Rta and Zta, respectively) activate expression of other viral genes, thereby initiating the lytic cascade. Among the viral antigens expressed upon induction of the lytic cycle, however, Zta is unique in its ability to disrupt viral latency; expression of the BZLF1 gene is both necessary and sufficient for triggering the viral lytic cascade. We have previously shown that Zta can activate its own promoter (Zp), through binding to two Zta recognition sequences (ZIIIA and ZIIIB). Here we describe mutant Zta proteins that do not bind DNA (referred to as Zta DNA-binding mutants [Zdbm]) but retain the ability to transactivate Zp. Consistent with the inability of these mutants to bind DNA, transactivation of Zp by Zdbm is not dependent on the Zta recognition sequences. Instead, transactivation by Zdbm is dependent upon promoter elements that bind cellular factors. An examination of other viral and cellular promoters identified promoters that are weakly responsive or unresponsive to Zdbm. An analysis of a panel of artificial promoters containing one copy of various promoter elements demonstrated a specificity for Zdbm activation that is distinct from that of Zta. These results suggest that non-DNA-binding forms of some transactivators retain the ability to transactivate specific target promoters without direct binding to DNA.

scholarly journals Functional Interaction between Epstein-Barr Virus Replication Protein Zta and Host DNA Damage Response Protein 53BP1

2009 ◽  
Vol 83 (21) ◽  
pp. 11116-11122 ◽  
Author(s):  
Sarah G. Bailey ◽  
Elizabeth Verrall ◽  
Celine Schelcher ◽  
Alex Rhie ◽  
Aidan J. Doherty ◽  
...  
Keyword(s):  
Dna Damage ◽  
Viral Replication ◽  
Epstein Barr Virus ◽  
Signal Transduction Pathway ◽  
Human Herpesvirus ◽  
Lytic Cycle ◽  
Barr Virus ◽  
Proteomic Approach ◽  
Viral Genes ◽  
Epstein Barr

ABSTRACT Epstein-Barr virus (EBV; human herpesvirus 4) poses major clinical problems worldwide. Following primary infection, EBV enters a form of long-lived latency in B lymphocytes, expressing few viral genes, and it persists for the lifetime of the host with sporadic bursts of viral replication. The switch between latency and replication is governed by the action of a multifunctional viral protein Zta (also called BZLF1, ZEBRA, and Z). Using a global proteomic approach, we identified a host DNA damage repair protein that specifically interacts with Zta: 53BP1. 53BP1 is intimately connected with the ATM signal transduction pathway, which is activated during EBV replication. The interaction of 53BP1 with Zta requires the C-terminal ends of both proteins. A series of Zta mutants that show a wild-type ability to perform basic functions of Zta, such as dimer formation, interaction with DNA, and the transactivation of viral genes, were shown to have lost the ability to induce the viral lytic cycle. Each of these mutants also is compromised in the C-terminal region for interaction with 53BP1. In addition, the knockdown of 53BP1 expression reduced viral replication, suggesting that the association between Zta and 53BP1 is involved in the viral replication cycle.

scholarly journals Epstein-Barr Virus BZLF1-Mediated Downregulation of Proinflammatory Factors Is Essential for Optimal Lytic Viral Replication

2015 ◽  
Vol 90 (2) ◽  
pp. 887-903 ◽  
Author(s):  
Yuqing Li ◽  
Xubing Long ◽  
Lu Huang ◽  
Mengtian Yang ◽  
Yan Yuan ◽  
...  
Keyword(s):  
Viral Replication ◽  
Epstein Barr Virus ◽  
Lytic Cycle ◽  
Inflammatory Factors ◽  
Barr Virus ◽  
Ebv Infection ◽  
Tnf Α ◽  
Ifn Γ ◽  
Epstein Barr ◽  
Proinflammatory Factors

ABSTRACTElevated secretion of inflammatory factors is associated with latent Epstein-Barr virus (EBV) infection and the pathology of EBV-associated diseases; however, knowledge of the inflammatory response and its biological significance during the lytic EBV cycle remains elusive. Here, we demonstrate that the immediate early transcriptional activator BZLF1 suppresses the proinflammatory factor tumor necrosis factor alpha (TNF-α) by binding to the promoter of TNF-α and preventing NF-κB activation. A BZLF1Δ207-210 mutant with a deletion of 4 amino acids (aa) in the protein-protein binding domain was not able to inhibit the proinflammatory factors TNF-α and gamma interferon (IFN-γ) and reduced viral DNA replication with complete transcriptional activity during EBV lytic gene expression. TNF-α depletion restored the viral replication mediated by BZLF1Δ207-210. Furthermore, a combination of TNF-α- and IFN-γ-neutralizing antibodies recovered BZLF1Δ207-210-mediated viral replication, indicating that BZLF1 attenuates the antiviral response to aid optimal lytic replication primarily through the inhibition of TNF-α and IFN-γ secretion during the lytic cycle. These results suggest that EBV BZLF1 attenuates the proinflammatory responses to facilitate viral replication.IMPORTANCEThe proinflammatory response is an antiviral and anticancer strategy following the complex inflammatory phenotype. Latent Epstein-Barr virus (EBV) infection strongly correlates with an elevated secretion of inflammatory factors in a variety of severe diseases, while the inflammatory responses during the lytic EBV cycle have not been established. Here, we demonstrate that BZLF1 acts as a transcriptional suppressor of the inflammatory factors TNF-α and IFN-γ and confirm that BZLF1-facilitated escape from the TNF-α and IFN-γ response during the EBV lytic life cycle is required for optimal viral replication. This finding implies that the EBV lytic cycle employs a distinct strategy to evade the antiviral inflammatory response.

scholarly journals Terminal Differentiation into Plasma Cells Initiates the Replicative Cycle of Epstein-Barr Virus In Vivo

2005 ◽  
Vol 79 (2) ◽  
pp. 1296-1307 ◽  
Author(s):  
Lauri L. Laichalk ◽  
David A. Thorley-Lawson
Keyword(s):  
Viral Replication ◽  
Epstein Barr Virus ◽  
Plasma Cells ◽  
Acute Stress ◽  
Cytotoxic T Lymphocyte ◽  
Lytic Cycle ◽  
Barr Virus ◽  
Epstein Barr

ABSTRACT In this paper we demonstrate that the cells which initiate replication of Epstein-Barr virus (EBV) in the tonsils of healthy carriers are plasma cells (CD38hi, CD10−, CD19+, CD20lo, surface immunoglobulin negative, and cytoplasmic immunoglobulin positive). We further conclude that differentiation into plasma cells, and not the signals that induce differentiation, initiates viral replication. This was confirmed by in vitro studies showing that the promoter for BZLF1, the gene that begins viral replication, becomes active only after memory cells differentiate into plasma cells and is also active in plasma cell lines. This differs from the reactivation of BZLF1 in vitro, which occurs acutely and is associated with apoptosis and not with differentiation. We suggest that differentiation and acute stress represent two distinct pathways of EBV reactivation in vivo. The fraction of cells replicating the virus decreases as the cells progress through the lytic cycle such that only a tiny fraction actually release infectious virus. This may reflect abortive replication or elimination of cells by the cellular immune response. Consistent with the later conclusion, the cells did not down regulate major histocompatibility complex class I molecules, suggesting that this is not an immune evasion tactic used by EBV and that the cells remain vulnerable to cytotoxic-T-lymphocyte attack.

HAUSP/USP7 as an Epstein–Barr virus target

2004 ◽  
Vol 32 (5) ◽  
pp. 731-732 ◽  
Author(s):  
M.N. Holowaty ◽  
L. Frappier
Keyword(s):  
Epstein Barr Virus ◽  
Latent Infection ◽  
Nuclear Antigen ◽  
Barr Virus ◽  
High Affinity ◽  
Cellular Immortalization ◽  
Epstein Barr ◽  
Epstein Barr Nuclear Antigen

USP7 (also called HAUSP) is a de-ubiquitinating enzyme recently identified as a key regulator of the p53–mdm2 pathway, which stabilizes both p53 and mdm2. We have discovered that the Epstein–Barr nuclear antigen 1 protein of Epstein–Barr virus binds with high affinity to USP7 and disrupts the USP7–p53 interaction. The results have important implications for the role of Epstein–Barr nuclear antigen 1 in the cellular immortalization that is typical of an Epstein–Barr virus latent infection.

scholarly journals The Epstein-Barr virus (EBV) nuclear antigen 1 BamHI F promoter is activated on entry of EBV-transformed B cells into the lytic cycle.

1992 ◽  
Vol 66 (12) ◽  
pp. 7461-7468 ◽  
Author(s):  
A L Lear ◽  
M Rowe ◽  
M G Kurilla ◽  
S Lee ◽  
S Henderson ◽  
...  
Keyword(s):  
B Cells ◽  
Epstein Barr Virus ◽  
Lytic Cycle ◽  
Nuclear Antigen ◽  
Barr Virus ◽  
Epstein Barr

scholarly journals Epstein–Barr virus induces a distinct form of DNA-bound STAT1 compared with that found in interferon-stimulated B lymphocytes

2007 ◽  
Vol 88 (7) ◽  
pp. 1876-1886 ◽  
Author(s):  
James McLaren ◽  
Martin Rowe ◽  
Paul Brennan
Keyword(s):  
Dna Binding ◽  
Epstein Barr Virus ◽  
Lymphoblastoid Cell Lines ◽  
Distinct Form ◽  
Post Translational Modifications ◽  
Constitutive Activation ◽  
Barr Virus ◽  
Molecular Identity ◽  
Infected Cells ◽  
Epstein Barr

Since ‘constitutive activation’ of STAT1 was first described in Epstein–Barr virus (EBV)-immortalized lymphoblastoid cell lines (LCLs), there has been controversy regarding the molecular identity of the STAT1 DNA-binding complex found in these cells. The post-translational modifications of STAT1 in LCLs have been analysed and an LMP1-induced STAT1 DNA-binding complex, different from that generated by alpha interferon (IFN) stimulation and not involving tyrosine phosphorylation, is demonstrated. STAT1 is serine-phosphorylated downstream of PI3K and MEK in LCLs and this modification restricts IFN-stimulated STAT1–DNA binding. These data suggest that EBV induces a distinct form of DNA-bound STAT1 in virus-infected cells.

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