Abstract
Epstein-Barr virus (EBV) encoded transcription factor Zta (BZLF1, ZEBRA, EB1) is the prototype of a class of transcription factor (including C/EBPalpha) that interact with CpG-containing DNA response elements in a methylation-dependent manner. The EBV genome undergoes a biphasic methylation cycle; it is extensively methylated during viral latency but is reset to an unmethylated state following viral lytic replication. Zta is expressed transiently following infection and again during the switch between latency and lytic replication. The requirement for CpG-methylation at critical Zta response elements (ZREs) has been proposed to regulate EBV replication, specifically it could aid the activation of viral lytic gene expression from silenced promoters on the methylated genome during latency in addition to preventing full lytic reactivation from the non-methylated EBV genome immediately following infection. We developed a computational approach to predict the location of ZREs which we experimentally assessed using in vitro and in vivo DNA association assays. A remarkably different binding motif is apparent for the CpG and non-CpG ZREs. Computational prediction of the location of these binding motifs in EBV revealed that the majority of lytic cycle genes have at least one and many have multiple copies of methylation-dependent CpG ZREs within their promoters. This suggests that the abundance of Zta protein coupled with the methylation status of the EBV genome act together to co-ordinate the expression of lytic cycle genes at the majority of EBV promoters.
Highlights
Infection of human B-lymphocytes by Epstein-Barr virus results in the establishment of a latent state in which a highly restricted set of viral genes are expressed [1]
6 novel sites where predicted (Table S2).The ability of Zta to interact with each predicted site was assessed using electrophoretic mobility shift assays (EMSA) (Figure 1, Table S2), three novel sites were identified, eight known sites were missed and three false positives were predicted indicating that the PFM used had a low sensitivity
Within the Epstein-Barr virus (EBV) genome a total of 16 novel sequence variants of CpG Zta response elements (ZREs) were predicted (A–P) (Figure 3)
Summary
Infection of human B-lymphocytes by Epstein-Barr virus results in the establishment of a latent state in which a highly restricted set of viral genes are expressed [1]. This is accompanied by extensive methylation of CpG motifs in non-expressed viral genes [2,3,4]. The ability of Zta to bind to methylated ZREs suggests that Zta may have a direct role in overriding the epigenetic silencing of the viral genome to activate expression of viral genes required for lytic replication
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