Analysis of global methylation using a Zta-expressing nasopharyngeal carcinoma cell line

EBV infects more than 90% of the human population and persists in most individuals as a latent infection where the viral genome is silenced by host-driven methylation. The lytic cycle is initiated when the viral protein Zta binds to methylated BRLF1 and BRRF1 promoters. Although studies reveal the role of Zta and methylation changes in the viral genome upon EBV infection to reactivation, whether Zta plays any role in alteration of methylation in the host genome remains unknown. Using an inducible model, we demonstrate that global DNA methylation, based on whole-genome 5-methylcytosine content, and regional DNA methylation in repetitive elements, imprinting genes and the X chromosome, remains unchanged in response to Zta expression. Expression of DNA methyltransferases was also unaffected by ectopically expressed Zta. Our data imply that alteration of host gene expression following EBV reactivation may reflect methylation-independent Zta-mediated gene activation and not epigenetic modification of the host genome.     

Interplay between PKCδ and Sp1 on histone deacetylase inhibitor-mediated Epstein-Barr virus reactivation

Epstein-Barr virus (EBV) undergoes latent and lytic replication cycles, and its reactivation from latency to lytic replication is initiated by expression of the two viral immediate-early transactivators, Zta and Rta. In vitro, reactivation of EBV can be induced by anti-immunoglobulin, tetradecanoyl phorbol acetate, and histone deacetylase inhibitor (HDACi). We have discovered that protein kinase C delta (PKCδ) is required specifically for EBV reactivation by HDACi. Overexpression of PKCδ is sufficient to induce the activity of the Zta promoter (Zp) but not of the Rta promoter (Rp). Deletion analysis revealed that the ZID element of Zp is important for PKCδ activation. Moreover, the Sp1 putative sequence on ZID is essential for PKCδ-induced Zp activity, and the physiological binding of Sp1 on ZID has been confirmed. After HDACi treatment, activated PKCδ can phosphorylate Sp1 at serine residues and might result in dissociation of the HDAC2 repressor from ZID. HDACi-mediated HDAC2-Sp1 dissociation can be inhibited by the PKCδ inhibitor, Rotterlin. Furthermore, overexpression of HDAC2 can suppress the HDACi-induced Zp activity. Consequently, we hypothesize that HDACi induces PKCδ activation, causing phosphorylation of Sp1, and that the interplay between PKCδ and Sp1 results in the release of HDAC2 repressor from Zp and initiation of Zta expression.