Epstein-Barr virus (EBV) causes infectious mononucleosis and is associated with various malignancies, including Burkitt's lymphoma and nasopharyngeal carcinoma. Like all herpesviruses, the EBV life cycle alternates between latency and lytic replication. During latency, the viral genome is largely silenced by host-driven methylation of CpG motifs and, in the switch to the lytic cycle, this epigenetic silencing is overturned. A key event is the activation of the viral BRLF1 gene by the immediate-early protein Zta. Zta is a bZIP transcription factor that preferentially binds to specific response elements (ZREs) in the BRLF1 promoter (Rp) when these elements are methylated. Zta's ability to trigger lytic cycle activation is severely compromised when a cysteine residue in its bZIP domain is mutated to serine (C189S), but the molecular basis for this effect is unknown. Here we show that the C189S mutant is defective for activating Rp in a Burkitt's lymphoma cell line. The mutant is compromised both in vitro and in vivo for binding two methylated ZREs in Rp (ZRE2 and ZRE3), although the effect is striking only for ZRE3. Molecular modeling of Zta bound to methylated ZRE3, together with biochemical data, indicate that C189 directly contacts one of the two methyl cytosines within a specific CpG motif. The motif's second methyl cytosine (on the complementary DNA strand) is predicted to contact S186, a residue known to regulate methyl-ZRE recognition. Our results suggest that C189 regulates the enhanced interaction of Zta with methylated DNA in overturning the epigenetic control of viral latency. As C189 is conserved in many bZIP proteins, the selectivity of Zta for methylated DNA may be a paradigm for a more general phenomenon.

BackgroundDespite an intense interest in the biological functions of the phosphoinositide 3-kinase (PI3K) signalling enzymes, little is known about the regulation of PI3K gene expression. This also applies to the leukocyte-enriched p110δ catalytic subunit of PI3K, an enzyme that has attracted widespread interest because of its role in immunity and allergy.Principal FindingsWe show that p110δ expression is mainly regulated at the transcriptional level. In fibroblasts, lymphocytes and myeloid cells, p110δ gene transcription appears to be constitutive and not subject to acute stimulation. 5′RACE experiments revealed that p110δ mRNA transcripts contain distinct upstream untranslated exons (named exon -1, -2a, -2b, -2c and -2d), which are located up to 81 kb upstream of the translational start codon in exon 1. The levels of all the different p110δ transcripts are higher in leukocytes compared to non-leukocytes, with the p110δ transcript containing exon -2a most abundantly expressed. We have identified a highly conserved transcription factor (TF) binding cluster in the p110δ gene which has enhanced promoter activity in leukocytes compared to non-leukocytes. In human, this TF cluster is located immediately upstream of exon -2a whilst in mouse, it is located within exon -2a.ConclusionThis study identifies a conserved PIK3CD promoter region that may account for the predominant leukocyte expression of p110δ.

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 itpersists 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.The Epstein-Barr virus (EBV) life cycle is divided temporally into two phases, latency and the lytic cycle. Following the infection of epithelial cells of the oropharynx, EBV enters the lytic cycle, where the expression of approximately 80 genes and numerous rounds of genome replication occur, culminating in the production of infectious virions. The infection of B lymphocytes results in the establishment of viral latency with a restricted gene expression pattern; these cells sporadically enter the lytic cycle and reproduce infectious virus (27,53).The EBV gene BZLF1 has been associated specifically with the disruption of latency (reviewed in references 34 and 50). This gene encodes the protein Zta (ZEBRA, BZLF1, Z), which has an undisputed role in activating the viral lytic cycle. Not only is the enforced expression of Zta in cells harboring the latent virus able to induce the viral lytic cycle, but a mutant virus where BZLF1 has been inactivated also is unable to replicate the viral genome (10). Zta has homology to the bZIP family of transcription factors whose general structure includes a transactivation domain and a bZIP domain consisting of a basic DNA contact region and a coiled-coil dimerization motif, termed a leucine zipper (24,49,50). Zta has a more complex dimerization domain than other bZIP family members, consisting of a dimeric leucine zipper entwined with an adjacent carboxyl-terminal region (35,38,44,50). Zta is multifunctional; through its basic region, it interacts with specific sequence DNA motifs (ZREs) that occur in the promoters of several viral and cellular genes (49) and in the viral origin of lytic replication (Ori-lyt) (46,47). Through its bZIP domain, Zta interacts with cellular transcription factors such as p53, RAR, NF-B, CBP, and C/EBP␣ (7), ...

The Epstein-Barr virus transcription factor Zta (encoded by BZLF1) is a bZIP protein containing an ␣-helical coiled-coil homodimerization motif (zipper). The Zta zipper forms less-stable dimers than other bZIP proteins, and an adjacent region (CT) interacts with the zipper to form a novel structure that is proposed to strengthen the dimer. Here we question the role of the CT region for Zta function. Cross-linking experiments demonstrate that the entire CT region lies adjacent to the zipper. Detailed analyses of Zta truncation mutations identify an involvement of the proximal CT region (221 to 230) in dimer formation with a further contribution from the distal region (236 to 243). Biophysical analyses reveal that residues 221 to 230 enhance the stability of the coiled coil. The ability of the Zta truncation mutants to interact with three Zta-binding sites also requires the proximal CT region. Fine mapping of DNA-binding requirements highlighted the contribution of these amino acids for Zta function. Thus, the proximal part of the CT region is required to aid the dimerization of Zta and thereby its DNA-binding ability. In contrast, although the distal part of the CT region aids dimerization, it promotes only a modest increase in DNA binding. To probe this further, we defined the contribution from the CT region for Zta to transactivate a promoter embedded within the viral genome. From this we conclude that the proximal part of the CT region is absolutely required, whereas the distal part is dispensable.

EBV (Epstein-Barr virus) alternates between latency and lytic replication. During latency, the viral genome is largely silenced by host-driven methylation of CpG motifs and in the switch to the lytic cycle this epigenetic silencing is overturned. A key event is the activation of the viral protein Zta with three ZREs (Zta-response elements) from the BRLF1 promoter (referred to as Rp). Two of these ZREs contain CpG motifs and are methylated in the latent genome. Biochemical analyses and molecular modelling of Zta bound to methylated RpZRE3 indicate the precise contacts made between a serine and a cysteine residue of Zta with methyl cytosines. A single point mutant of Zta, C189S, is defective in binding to the methylated ZREs both in vitro and in vivo. This was used to probe the functional relevance of the interaction. ZtaC189S was not able to activate Rp in a B-cell line, demonstrating the relevance of the interaction with methylated ZREs. This demonstrates that Zta plays a role in overturning the epigenetic control of viral latency.