Epstein-Barr Virus ZEBRA Protein Research Articles

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

The 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.IMPORTANCE The 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.

Role of Epstein-Barr Virus Zebra protein in induction of t(8;14) translocation.

Role of Epstein-Barr Virus Zebra protein in induction of t(8;14) translocation.

ZEBRA cell‐penetrating peptide as an efficient delivery system in Candida albicans

There is increasing interest in drug delivery systems, such as nanoparticles, liposomes, and cell-penetrating peptides, for the development of new antimicrobial treatments. In this study, we investigated the transduction capacity of a carrier peptide derived from the Epstein-Barr virus ZEBRA protein in the pathogenic fungus Candida albicans. ZEBRA-minimal domain (MD) was able to cross the cell wall and cell membrane, delivering eGFP to the cytoplasm. Uptake into up to 70% of the cells was observed within two hours, without toxicity. This new delivery system could be used in C. albicans as a carrier for different biological molecules including peptides, proteins, and nucleic acids. Thereby, in antifungal therapy, MD may carry promising bioactive fungal inhibitors that otherwise penetrate poorly into the cells. Furthermore, MD will be of interest for deciphering molecular pathways involving cell-cycle control in yeast or signaling pathways. Short interfering peptides could be internalized using MD, providing new tools for the inhibition of metabolic or signaling cascades essential for the growth and virulence of C. albicans, such as yeast-to-hyphae transition, cell wall remodeling, stress signaling and antifungal resistance. These findings create new possibilities for the internalization of cargo molecules, with applications for both treatment and functional analyses.

Analysis of an ankyrin-like region in Epstein Barr Virus encoded (EBV) BZLF-1 (ZEBRA) protein: implications for interactions with NF-κB and p53

BackgroundThe carboxyl terminal of Epstein-Barr virus (EBV) ZEBRA protein (also termed BZLF-1 encoded replication protein Zta or ZEBRA) binds to both NF-κB and p53. The authors have previously suggested that this interaction results from an ankyrin-like region of the ZEBRA protein since ankyrin proteins such as IκB interact with NF-κB and p53 proteins. These interactions may play a role in immunopathology and viral carcinogenesis in B lymphocytes as well as other cell types transiently infected by EBV such as T lymphocytes, macrophages and epithelial cells.MethodsRandomization of the ZEBRA terminal amino acid sequence followed by statistical analysis suggest that the ZEBRA carboxyl terminus is most closely related to ankyrins of the invertebrate cactus IκB-like protein. This observation is consistent with an ancient origin of ZEBRA resulting from a recombination event between an ankyrin regulatory protein and a fos/jun DNA binding factor. In silico modeling of the partially solved ZEBRA carboxyl terminus structure using PyMOL software demonstrate that the carboxyl terminus region of ZEBRA can form a polymorphic structure termed ZANK (ZEBRA ANKyrin-like region) similar to two adjacent IκB ankyrin domains.ConclusionsViral capture of an ankyrin-like domain provides a mechanism for ZEBRA binding to proteins in the NF-κB and p53 transcription factor families, and also provides support for a process termed "Ping-Pong Evolution" in which DNA viruses such as EBV are formed by exchange of information with the host genome. An amino acid polymorphism in the ZANK region is identified in ZEBRA from tumor cell lines including Akata that could alter binding of Akata ZEBRA to the p53 tumor suppressor and other ankyrin binding protein, and a novel model of antagonistic binding interactions between ZANK and the DNA binding regions of ZEBRA is suggested that may be explored in further biochemical and molecular biological models of viral replication.

A Subset of Replication Proteins Enhances Origin Recognition and Lytic Replication by the Epstein-Barr Virus ZEBRA Protein

ZEBRA is a site-specific DNA binding protein that functions as a transcriptional activator and as an origin binding protein. Both activities require that ZEBRA recognizes DNA motifs that are scattered along the viral genome. The mechanism by which ZEBRA discriminates between the origin of lytic replication and promoters of EBV early genes is not well understood. We explored the hypothesis that activation of replication requires stronger association between ZEBRA and DNA than does transcription. A ZEBRA mutant, Z(S173A), at a phosphorylation site and three point mutants in the DNA recognition domain of ZEBRA, namely Z(Y180E), Z(R187K) and Z(K188A), were similarly deficient at activating lytic DNA replication and expression of late gene expression but were competent to activate transcription of viral early lytic genes. These mutants all exhibited reduced capacity to interact with DNA as assessed by EMSA, ChIP and an in vivo biotinylated DNA pull-down assay. Over-expression of three virally encoded replication proteins, namely the primase (BSLF1), the single-stranded DNA-binding protein (BALF2) and the DNA polymerase processivity factor (BMRF1), partially rescued the replication defect in these mutants and enhanced ZEBRA's interaction with oriLyt. The findings demonstrate a functional role of replication proteins in stabilizing the association of ZEBRA with viral DNA. Enhanced binding of ZEBRA to oriLyt is crucial for lytic viral DNA replication.

Expression and purification of ZEBRA fusion proteins and applications for the delivery of macromolecules into mammalian cells.

The recent development of peptide carriers for efficient and specific delivery of biologically active molecules into mammalian cells represents a major advance in the study of both normal and uncontrolled cell growth. In the past few years, this technology has been successfully applied to the delivery of therapeutic molecules in animal models, and now some of these carriers are available in the clinic for the treatment of some human diseases. This unit describes the production, in a bacterial expression system, of reporter proteins (EGFP and beta-galactosidase) fused to a transduction domain of the Epstein-Barr virus ZEBRA protein, as well as purification of the fusion proteins. The purified fusion proteins can be added to any of a large spectrum of mammalian cells and the internalization process measured by flow cytometry and fluorescence microscopy on live cells. Fluorescence microscopy on fixed cells is used to study their intracellular distribution.

Mutations of amino acids in the DNA-recognition domain of Epstein–Barr virus ZEBRA protein alter its sub-nuclear localization and affect formation of replication compartments

ZEBRA, a transcription factor and DNA replication protein encoded by the Epstein–Barr virus (EBV) BZLF1 gene, plays indispensable roles in the EBV lytic cycle. We recently described the phenotypes of 46 single amino acid substitutions introduced into the DNA-recognition region of ZEBRA [Heston, L., El-Guindy, A., Countryman, J., Dela Cruz, C., Delecluse, H.J., and Miller, G. 2006]. The 27 DNA-binding-proficient mutants exhibited distinct defects in their ability to activate expression of the kinetic classes of viral genes. Four phenotypic variants could be discerned: wild-type, defective at activating Rta, defective at activating early genes, and defective at activating late genes. Here we analyze the distribution of ZEBRA within the nucleus and the localization of EA-D (the viral DNA polymerase processivity factor), an indicator of the development of replication compartments, in representatives of each phenotypic group. Plasmids encoding wild-type (WT) and mutant ZEBRA were transfected into 293 cells containing EBV-bacmids. WT ZEBRA protein was diffusely and smoothly distributed throughout the nucleus, sparing nucleoli, and partially recruited to globular replication compartments. EA-D induced by WT ZEBRA was present diffusely in some cells and concentrated in globular replication compartments in other cells. The distribution of ZEBRA and EA-D proteins was identical to WT following transfection of K188R, a mutant with a conservative change. The distribution of S186A mutant ZEBRA protein, defective for activation of Rta and EA-D, was identical to WT, except that the mutant ZEBRA was never found in globular compartments. Co-expression of Rta with S186A mutant rescued diffuse EA-D but not globular replication compartments. The most striking observation was that several mutant ZEBRA proteins defective in activating EA-D (R179A, K181A and A185V) and defective in activating lytic viral DNA replication and late genes (Y180E and K188A) were localized to numerous punctate foci. The speckled appearance of R179A and Y180E was more regular and clearly defined in EBV-positive than in EBV-negative 293 cells. The Y180E late-mutant induced EA-D, but prevented EA-D from localizing to globular replication compartments. These results show that individual amino acids within the basic domain influence localization of the ZEBRA protein and its capacity to induce EA-D to become located in mature viral replication compartments. Furthermore, these mutant ZEBRA proteins delineate several stages in the processes of nuclear re-organization which accompany lytic EBV replication.

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

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.

Amino Acids in the Basic Domain of Epstein-Barr Virus ZEBRA Protein Play Distinct Roles in DNA Binding, Activation of Early Lytic Gene Expression, and Promotion of Viral DNA Replication

The ZEBRA protein of Epstein-Barr virus (EBV) drives the viral lytic cycle cascade. The capacity of ZEBRA to recognize specific DNA sequences resides in amino acids 178 to 194, a region in which 9 of 17 residues are either lysine or arginine. To define the basic domain residues essential for activity, a series of 46 single-amino-acid-substitution mutants were examined for their ability to bind ZIIIB DNA, a high-affinity ZEBRA binding site, and for their capacity to activate early and late EBV lytic cycle gene expression. DNA binding was obligatory for the protein to activate the lytic cascade. Nineteen mutants that failed to bind DNA were unable to disrupt latency. A single acidic replacement of a basic amino acid destroyed DNA binding and the biologic activity of the protein. Four mutants that bound weakly to DNA were defective at stimulating the expression of Rta, the essential first target of ZEBRA in lytic cycle activation. Four amino acids, R183, A185, C189, and R190, are likely to contact ZIIIB DNA specifically, since alanine or valine substitutions at these positions drastically weakened or eliminated DNA binding. Twenty-three mutants were proficient in binding to ZIIIB DNA. Some DNA binding-proficient mutants were refractory to supershift by BZ-1 monoclonal antibody (epitope amino acids 214 to 230), likely as the result of the increased solubility of the mutants. Mutants competent to bind DNA could be separated into four functional groups: the wild-type group (eight mutants), a group defective at activating Rta (five mutants, all with mutations at the S186 site), a group defective at activating EA-D (three mutants with the R179A, S186T, and K192A mutations), and a group specifically defective at activating late gene expression (seven mutants). Three late mutants, with a Y180A, Y180E, or K188A mutation, were defective at stimulating EBV DNA replication. This catalogue of point mutants reveals that basic domain amino acids play distinct functions in binding to DNA, in activating Rta, in stimulating early lytic gene expression, and in promoting viral DNA replication and viral late gene expression. These results are discussed in relationship to the recently solved crystal structure of ZEBRA bound to an AP-1 site.

Expression, purification, crystallization and preliminary X-ray analysis of a C-terminal fragment of the Epstein–Barr virus ZEBRA protein

A C-terminal fragment of the Epstein-Barr virus immediate-early transcription factor ZEBRA has been expressed as a recombinant protein in Escherichia coli and purified to homogeneity. The fragment behaves as a dimer in solution, consistent with the presence of a basic region leucine-zipper (bZIP) domain. Crystals of the fragment in complex with a DNA duplex were grown by the hanging-drop vapour-diffusion technique using polyethylene glycol 4000 and magnesium acetate as crystallization agents. Crystals diffract to better than 2.5 A resolution using synchrotron radiation (lambda = 0.976 A). Crystals belong to space group C2, with unit-cell parameters a = 94.2, b = 26.5, c = 98.1 A, beta = 103.9 degrees.

Identification of Constitutive Phosphorylation Sites on the Epstein-Barr Virus ZEBRA Protein

ZEBRA, the product of the Epstein-Barr virus gene bzlf1, and a member of the AP-1 subfamily of basic zipper (bZIP) transcription factors, is necessary and sufficient to disrupt viral latency and to initiate the viral lytic cycle. Two serine residues of ZEBRA, Ser167 and Ser173, are substrates for casein kinase 2 (CK2) and are constitutively phosphorylated in vivo. Phosphorylation of ZEBRA at its CK2 sites is required for proper temporal regulation of viral gene expression. Phosphopeptide analysis indicated that ZEBRA contains additional constitutive phosphorylation sites. Here we employed a co-migration strategy to map these sites in vivo. The cornerstone of this strategy was to correlate the migration of 32P- and 35S-labeled tryptic peptides of ZEBRA. The identity of the peptides was revealed by mutagenesis of methionine and cysteine residues present in each peptide. Phosphorylation sites within the peptide were identified by mutagenesis of serines and threonines. ZEBRA was shown to be phosphorylated at serine and threonine residues, but not tyrosine. Two previously unrecognized phosphorylation sites of ZEBRA were identified in the NH2-terminal region of the transactivation domain: a cluster of weak phosphorylation sites at Ser6, Thr7, and Ser8 and a strong phosphorylation site at Thr14. Thr14 was embedded in a MAP kinase consensus sequence and could be phosphorylated in vitro by JNK, despite the absence of a canonical JNK docking site. Thus ZEBRA is now known to be constitutively phosphorylated at three distinct sites.

Structural Basis of Lytic Cycle Activation by the Epstein-Barr Virus ZEBRA Protein

Epstein-Barr virus (EBV) causes infectious mononucleosis and is linked to several human malignancies. EBV has a biphasic infection cycle consisting of a latent and a lytic, replicative phase. The switch from latent to lytic infection is triggered by the EBV immediate-early transcription factor ZEBRA (BZLF1, Zta, Z, EB1). We present the crystal structure of ZEBRA's DNA binding domain bound to an EBV lytic gene promoter element. ZEBRA exhibits a variant of the basic-region leucine zipper (bZIP) fold in which a C-terminal moiety stabilizes the coiled coil involved in dimer formation. The structure provides insights into ZEBRA's broad target site specificity, preferential activation of specific EBV promoters in their methylated state, ability to dimerize despite lacking a leucine zipper motif, and failure to heterodimerize with cellular bZIP proteins. The structure will allow for the design of new therapeutic agents that block activation of the EBV lytic cycle.

Phosphorylation of Epstein-Barr virus ZEBRA protein at its casein kinase 2 sites mediates its ability to repress activation of a viral lytic cycle late gene by Rta.

ZEBRA, a member of the bZIP family, serves as a master switch between latent and lytic cycle Epstein-Barr virus (EBV) gene expression. ZEBRA influences the activity of another viral transactivator, Rta, in a gene-specific manner. Some early lytic cycle genes, such as BMRF1, are activated in synergy by ZEBRA and Rta. However, ZEBRA suppresses Rta's ability to activate a late gene, BLRF2. Here we show that this repressive activity is dependent on the phosphorylation state of ZEBRA. We find that two residues of ZEBRA, S167 and S173, that are phosphorylated by casein kinase 2 (CK2) in vitro are also phosphorylated in vivo. Inhibition of ZEBRA phosphorylation at the CK2 substrate motif, either by serine-to-alanine substitutions or by use of a specific inhibitor of CK2, abolished ZEBRA's capacity to repress Rta activation of the BLRF2 gene, but did not alter its ability to initiate the lytic cycle or to synergize with Rta in activation of the BMRF1 early-lytic-cycle gene. These studies illustrate how the phosphorylation state of a transcriptional activator can modulate its behavior as an activator or repressor of gene expression. Phosphorylation of ZEBRA at its CK2 sites is likely to play an essential role in proper temporal control of the EBV lytic life cycle.

The Epstein-Barr virus ZEBRA protein activates transcription from the early lytic F promoter by binding to a promoter-proximal AP-1-like site.

The ZEBRA protein encoded by the Epstein-Barr virus (EBV) genome activates a switch from the latent to the lytic gene expression programme of the virus. ZEBRA, a member of the basic leucine zipper family of DNA-binding proteins, is a transcriptional activator capable of inducing expression from several virus lytic cycle promoters by binding to activator protein 1 (AP-1)-like sites. The Epstein-Barr virus BamHI F promoter, Fp, was for some time believed to initiate EBNA1-specific transcription in EBV-transformed latent cells. More recent data, however, show that Fp is an early lytic promoter and that the dominant EBNA1 gene promoter in latent cells is Qp, located about 200 bp downstream of Fp. In the present investigation we confirm that Fp displays the characteristics of a lytic promoter. Fp is downregulated in latently EBV-infected cells, both in the endogenous virus genome and in reporter plasmids that carry Fp regulatory sequences upstream of position -136 and down to +10 relative to the Fp transcription start site (+1), and is activated on induction of the virus lytic cycle. We show that the repression of Fp in latent stages of infection can be abolished by ZEBRA, and demonstrate that ZEBRA activates Fp through a direct interaction with an AP-1-like site at position -52/-46 in the promoter-proximal Fp region.

Dimerization of the Epstein-Barr virus ZEBRA protein in the yeast two-hybrid system. Comparison of a ZEBRA variant with the B95-8 form

Epstein-Barr virus (EBV) is a herpes virus associated with several human tumors. The EBV protein, ZEBRA, is a transactivator of the basic leucine zipper family (bZip). It binds to specific sequences on DNA and is able to interact with cellular proteins such as p53. The interaction of the ZEBRA protein with its cognate DNA sequences is stable as long as the dimerization domain is functional. Recent work from this laboratory identified a ZEBRA variant (Z206) with a single amino acid change at residue 206. An alanine is substituted for a serine, and this replacement is present in 72% of nasopharyngeal carcinoma from Europe and North Africa. As amino acid 206 lies within the dimerization domain it could be instrumental in interactions with other proteins. The yeast two-hybrid system was used to study ZEBRA-protein interactions. As ZEBRA by itself is a transactivator in yeast, it cannot be used directly in this assay. This paper describes modifications in ZEBRA amino acid sequences, rendering it usable in the yeast two-hybrid assay. We compared the dimerization capacity of the Z206 variant to that of ZEBRA from B95-8 (Z95) and observed that reporter gene activity with Z206 was consistently lower than that of Z95 ( P < 0.05). Furthermore, no interaction was found to occur between either form of ZEBRA (Z206 or Z95) and the tumor suppressor, p53 in the yeast two-hybrid system.

Compensatory Energetic Relationships between Upstream Activators and the RNA Polymerase II General Transcription Machinery

Activation of RNA polymerase II transcription in vivo and in vitro is synergistic with respect to increasing numbers of activator binding sites or increasing concentrations of activator. The Epstein-Barr virus ZEBRA protein manifests both forms of synergy during activation of genes involved in the viral lytic cycle. The synergy has an underlying mechanistic basis that we and others have proposed is founded largely on the energetic contributions of (i) upstream ZEBRA binding to its sites, (ii) the general pol II machinery binding to the core promoter, and (iii) interactions between ZEBRA and the general machinery. We hypothesize that these interactions form a network for which a minimum stability must be attained to activate transcription. One prediction of this model is that the energetic contributions should be reciprocal, such that a strong core promoter linked to a weak upstream promoter would be functionally analogous to a weak core linked to a strong upstream promoter. We tested this view by measuring the transcriptional response after systematically altering the upstream and core promoters. Our data provide strong qualitative support for this hypothesis and provide a theoretical basis for analyzing Epstein-Barr virus gene regulation.

Alteration of a single serine in the basic domain of the Epstein-Barr virus ZEBRA protein separates its functions of transcriptional activation and disruption of latency.

The ZEBRA protein from Epstein-Barr virus (EBV) activates a switch from the latent to the lytic expression program of the virus. ZEBRA, a member of the bZIP family of DNA-binding proteins, is a transcriptional activator capable of inducing expression from viral lytic cycle promoters. It had previously been thought that ZEBRA's capacity to disrupt EBV latency resided primarily in its ability to activate transcription of genes that encode products required for lytic replication. We generated a point mutant of ZEBRA, Z(S186A), that was not impaired in its ability to activate transcription; however, this mutation abolished its ability to initiate the viral lytic cascade. The mutant, containing a serine-to-alanine substitution in the DNA-binding domain of the protein, bound to several known ZEBRA-binding sites and activated transcription from reporters bearing known ZEBRA-responsive promoters but did not disrupt latency in EBV-infected cell lines. Therefore, initiation of the EBV lytic cycle by the ZEBRA protein requires a function in addition to transcriptional activation; a change of serine 186 to alanine in the DNA-binding domain of ZEBRA abolished this additional function and uncovered a new role for the ZEBRA protein in disruption of EBV latency. The additional function that is required for initiation of the lytic viral life cycle is likely to require phosphorylation of serine 186 of the ZEBRA protein, which may influence either DNA recognition or transcriptional activation of lytic viral promoters in a chromatinized viral episome.

Assembly of the isomerized TFIIA--TFIID--TATA ternary complex is necessary and sufficient for gene activation.

The prevailing view of eukaryotic gene activation poses that activators stimulate transcription by recruiting limiting components of the general transcription machinery to a core promoter. In one such model case, activation by the Epstein-Barr virus ZEBRA protein correlated closely with recruitment of the general transcription factors TFIIA and TFIID (the DA complex) as measured by DNase I footprinting and gel mobility shift assays. We now report that simple recruitment is not sufficient for full-level activation. An additional concentration-independent, rate-limiting step is activator-mediated isomerization of the DA complex characterized by an extended TFIID footprint. The isomerized complex supports both binding of TFIIB in gel mobility shift assays and activated transcription in heat-treated nuclear extracts, even after removal of ZEBRA. Surprisingly, the regulatory phenomenon of synergy was manifested only when the concentration of TFIID was limiting. When the DA complex was saturating, transcription was not synergistic, as indicated by the ability of a single activator to induce isomerization effectively and turn on a gene. On the basis of these observations, we propose a new biochemical model for eukaryotic gene activation and synergy.

Restoration of the Epstein-Barr Virus ZEBRA Protein's Capacity to Disrupt Latency by the Addition of Heterologous Activation Regions

The ZEBRA protein has a unique biological function among herpesviral proteins. It is responsible for the disruption of Epstein-Barr virus (EBV) latency and the induction of the lytic cycle. ZEBRA is a bZIP transcriptional activator which binds as a dimer to 7-bp response elements within EBV promoters and is directly involved in the stimulation of virus replication at the EBV lytic origin. We have employed the ZEBRA/EBV biological system to test whether a heterologous activation domain can substitute for another activation domain (the ZEBRA domain). The ZEBRA activation region was replaced with the potent acid activation region from the herpes simplex virus VP16 protein or with the activation region of the EBV R protein. Both chimeras were found to transactivate model and native promoters at equivalent or better levels than ZEBRA itself. Activation was not target- or cell-type dependent, nor was it dependent on the presence of virus. These activation domains restored ZEBRA's ability to induce early antigen and to stimulate origin replication to levels that were equal to or greater than those of wild type. These studies suggest that the specificities of some of the known biological functions of ZEBRA are not dependent upon the nature of the activation domain present within ZEBRA.

Serine-173 of the Epstein-Barr virus ZEBRA protein is required for DNA binding and is a target for casein kinase II phosphorylation.

An Epstein-Barr virus-encoded protein, ZEBRA, mediates the switch from latency to the viral lytic life cycle. ZEBRA's domain structure and DNA binding specificity resemble that of cellular transcriptional activators such as c-Fos/c-Jun. We show that ZEBRA, like c-Jun, is phosphorylated by casein kinase II (CKII). The principal site of phosphorylation is serine-173 (S173), five amino acids upstream of the basic DNA recognition domain. CKII phosphorylation abrogated ZEBRA's capacity to bind its target DNA sequences. S173 is a functional component of ZEBRA's DNA binding domain, since mutation of S173 to alanine (S173A) reduced DNA binding in vitro to 10% of wild-type levels. Transcriptional activation of a native viral promoter in vivo by mutant S173A was also reduced markedly. Reversible phosphorylation of S173 is likely to be an important means of regulating ZEBRA's activity in vivo.