We previously found that a form of EpsteinBarr virus with rearranged DNA induces replication of latent Epstein-Barr virus. We now have found that one of three fragments of this rearranged DNA, when cloned in recombinant plasmids and used to transfect cells, can activate expression of several polypeptides from a latent viral genome. The 33-kDa protein that is the product of the active fragment is likely to be responsible for disruption of latency.Latency is a biological property common to the herpesviruses, which persist in their host after initial infection. Viral genomes, which are quiescent during latency, can be activated to replicate mature virus by a variety of stimuli. In the familiar case of herpes simplex virus, such stimuli include sunlight, elevated temperature, and section of the trigeminal nerve (1).The biochemical mechanisms that maintain latency and that are modulated upon reactivation are not understood. Excellent experimental models exist for establishment and maintenance of herpes simplex latency in ganglionic neurons of the mouse, but it is difficult to carry out biochemical analysis in vivo (2). Progress has been made in development of in vitro models of herpes simplex latency in human fibroblasts and, recently, in neuronal cultures (3). However, viral replication must be suppressed by addition of interferon and inhibitors of viral DNA synthesis; furthermore, only some of the cultured cells contain viral genomes (4).Latency of the Epstein-Barr herpesvirus (EBV) in human B lymphocytes can be established, manipulated, and analyzed in cell culture. A few viral functions are expressed during latency, but mature transcripts of genes representing products made when virus is synthesized are not present. EBV latency can be activated by a number of diverse stimuli, including phorbol ester tumor promoters, butyrate, antiimmunoglobulins, and a factor in serum (5-8). Induction of viral replication is accompanied by synthesis of many new mRNAs and polypeptides (9).Findings in a series of recent experiments indicated that a form of rearranged EBV DNA, which we call heterogeneous or het DNA, might provide clues about viral genes and gene products that play a role in latency of EBV. het DNA was found in a cell line designated P3J-HR-1 (HR-1) which spontaneously synthesizes considerable amounts of EBV. It was initially found that het DNA was not associated with all HR-1 cells; cellular subclones of HR-1 cells lacking het DNA could readily be isolated. Those cell clones which lacked het DNA spontaneously synthesized small amounts of virus, although much more virus could be recovered after induction with phorbol ester (10). Virus released from HR-1 cells did not immortalize lymphocytes, a defect which correlated with a sizeable genomic deletion (11). Instead, HR-1 virus is measured by its capacity to induce early antigens (EA) in Raji cells, which already contain an EBV genome. Subcloned HR-1 virus without het DNA did not cause EA expression in Raji cells. A rare HR-1 subclone with large amounts of het D...

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Activation of the Epstein-Barr virus (EBV) lytic cycle is mediated through the combined actions of ZEBRA and Rta, the products of the viral BZLF1 and BRLF1 genes. During latency, these two genes are tightly repressed. Histone deacetylase inhibitors (HDACi) can activate viral lytic gene expression. Therefore, a widely held hypothesis is that Zp and Rp, the promoters for BZLF1 and BRLF1, are repressed by chromatin and that hyperacetylation of histone tails, by allowing the access of positively acting factors, leads to transcription of BZLF1 and BRLF1. To investigate this hypothesis, we used chromatin immunoprecipitation (ChIP) to examine the acetylation and phosphorylation states of histones H3 and H4 on Zp and Rp in three cell lines, Raji, B95-8, and HH514-16, which differ in their response to EBV lytic induction by HDACi. We studied the effects of three HDACi, sodium butyrate (NaB), trichostatin A (TSA), and valproic acid (VPA). We also examined the effects of tetradecanoyl phorbol acetate (TPA) and 5-aza-2-deoxycytidine, a DNA methyltransferase inhibitor, on histone modification. In Raji cells, TPA and NaB act synergistically to activate the EBV lytic cycle and promote an increase in histone H3 and H4 acetylation and phosphorylation at Zp and Rp. Surprisingly, however, when Raji cells were treated with NaB or TSA, neither of which is sufficient to activate the lytic cycle, an increase of comparable magnitude of hyperacetylated and phosphorylated histone H3 at Zp and Rp was observed. In B95-8 cells, NaB inhibited lytic induction by TPA, yet NaB promoted hyperacetylation of H3 and H4. In HH514-16 cells, NaB and TSA strongly activated the EBV lytic cycle and caused hyperacetylation of histone H3 on Zp and Rp. However, when HH514-16 cells were treated with VPA, lytic cycle mRNAs or proteins were not induced, although histone H3 was hyperacetylated as measured by immunoblotting or by ChIP on Zp and Rp. Taken together, our data suggest that open chromatin at EBV BZLF1 and BRLF1 promoters is not sufficient to activate EBV lytic cycle gene expression.

An Epstein-Barr viral gene (ZEBRA) is identified that, in human lymphoblastoid cells, activates a switch causing the virus to shift from the latent to the replicative phase of its life cycle. We have shown that a 2.7-kilobase-pair rearranged Epstein-Barr virus DNA fragment of this gene (BamHI fragment WZhet) induced transient expression of viral replicative antigens and polypeptides when it was transfected into a somatic cell hybrid, which was derived from the fusion of an epithelial line cell with a Burkitt lymphoma cell. We now show that this rearranged WZhet fragment, when introduced stably into lymphoblastoid cells, will activate expression of the complete viral replicative cycle in 1-10% of the lymphoblastoid cells, leading to production of biologically active virions that can immortalize primary lymphocytes. The transfected plasmid appears to be regulated in a manner analogous to the complete Epstein-Barr virus genome.

These experiments identify an Epstein-Barr virus-encoded gene product, called ZEBRA (BamHI fragment Z Epstein-Barr replication activator) protein, which activates a switch between the latent and replicative life cycle of the virus. Our previous work had shown that the 2.7-kilobase-pair WZhet piece of rearranged Epstein-Barr virus DNA from a defective virus activated replication when introduced into cells with a latent genome, but it was not clear whether a protein product was required for the phenomenon. We now use deletional, site-directed, and chimeric mutagenesis, together with gene transfer, to show that a 43-kilodalton protein, encoded in the BZLF1 open reading frame of het DNA, is responsible for this process. The rearrangement in defective DNA does not contribute to the structural gene for the protein. Similar proteins with variable electrophoretic mobility (37 to 39 kilodaltons) were encoded by BamHI Z fragments from standard, nondefective Epstein-Barr virus genomes. Plasmids expressing the ZEBRA proteins from B95-8 and HR-1 viruses were less efficient at activating replication in D98/HR-1 cells than those which contained the ZEBRA gene from the defective virus. It is not yet known whether these functional differences are due to variations in expression of the plasmids or to intrinsic differences in the activity of these polymorphic polypeptides.

A defective Epstein-Barr virus (EBV) containing a deleted and rearranged genome (het DNA) causes latent EBV to replicate. This activity maps to the 2. Previous work identified an EBV gene product that switches the virus from latency into replication (12)(13)(14)(15) Fig. 2 legend).Transfection. Stationary-phase cells were exposed for 30 min at 37°C to 1-5 ug of DNA in RPMI medium containing 100 ,ug of DEAE-dextran per ml. Thereafter, cells were washed and resuspended in conditioned growth medium with or without phorbol 12-myristate 13-acetate (PMA) at 4 ng/ml for 48 hr.Polypeptide Detection. ZEBRA was detected by Western blotting with monospecific antibodies raised in rabbits immunized with a TrpE-BZLF1 fusion protein (N.T., J.C., C.R., D. Katz, and G.M., unpublished data). Replicative polypeptides were detected with human WC antiserum (26). RESULTSRearranged Sequences from BamHI W Enhance ZEBRA Expression After Transfection of BL Cells. Countryman et al. had shown that rearranged sequences in WZhet did not contribute to the structural gene for the ZEBRA protein nor were they obligatory for activating EBV replication in a somatic cell hybrid (D98/HR-1) (13). These conclusions were based on the use ofdeletion mutants in which sequences were progressively removed from the BamHI W region of pSV2neo WZhet (Fig. 1 A and B).To determine whether rearranged sequences from BamHI W affected expression of ZEBRA in B cells the same mutants were transfected into BL41/CL16 cells. Expression of ZEBRA was monitored 48 hr after transfection by using monospecific anti-BZLF1 antibody (Fig. 1C) 9801The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

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-aminoacid-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.

The lytic cycle of Epstein-Barr virus (EBV) can be activated by transfection of the gene for ZEBRA, a viral basic-zipper (bZip) transcriptional activator. ZEBRA and cellular AP-1 bZip activators, such as c-Fos, have homologous DNA-binding domains, and their DNA-binding specificities overlap. Moreover, EBV latency can also be disrupted by phorbol esters, which act, in part, through AP-1 activators. It is not known whether ZEBRA and AP-1 factors play equivalent roles in the initial stages of reactivation. Here, the contribution of ZEBRA's basic DNA recognition domain to disruption of latency was analyzed by comparing ZEBRA with chimeric mutants in which the DNA recognition domain of ZEBRA was replaced with the analogous domain of c-Fos. Chimeric ZEBRA/c-Fos proteins overexpressed in Escherichia coli bound DNA with the specificity of c-Fos; they bound a heptamer AP-1 site and an octamer TPA response element (TRE). ZEBRA bound the AP-1 site and an array of ZEBRA response elements (ZREs). In assays with reporter genes, both ZEBRA and ZEBRA/c-Fos chimeric mutants activated transcription from Zp, a promoter of the ZEBRA gene (BZLF1) that contains the TRE and multiple ZREs. However, despite their capacity to activate reporters bearing Zp, neither ZEBRA nor the c-Fos chimeras activated transcription from Zp in the context of the intact latent viral genome. In contrast, ZEBRA but not ZEBRA/c-Fos chimeras activated Rp, a second viral promoter that controls ZEBRA expression. Hence, transcriptional autostimulation by transfected ZEBRA occurred preferentially at Rp. Both ZEBRA and the ZEBRA/c-Fos chimeras activated transcription from reporters with multimerized AP-1 sites. However, in the context of the virus, only ZEBRA activated the promoters of two early lytic cycle genes, BMRF1 and BMLF1, that contain an AP-1 site. Thus, overexpression of an activator that recognized AP-1 and TRE sites was not sufficient to activate EBV early lytic cycle genes.