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.