Cyclic AMP-responsive element-dependent activation of Epstein-Barr virus zebra promoter by human herpesvirus 6

We have recently shown that infection of Epstein-Barr virus (EBV) genome-positive B cells by human herpesvirus 6 (HHV-6) results in the expression of the immediate-early EBV Zebra gene, followed by virus replication (L. Flamand, I. Stefanescu, D. V. Ablashi, and J, Menezes, J, Virol, 67:6768-6777, 1993), Here we show that HHV-6 upregulates Zebra gene transcription through a cyclic AMP-responsive element (CRE) located within the Zebra promoter (Zp), Using human B- or T-cell lines transfected with ZpCat reporter gene constructs, we demonstrate that a region designated the ZII domain of Zp is the target of HHV-6 transactivation, Mutation of the consensus AP-1/CRE site within ZII abolished the inducibility of Zp by HHV-6, whereas positioning of the ZII domain upstream of the beta-globin minimal promoter conferred responsiveness following HHV-6 infection, Binding of transcription factors to ZII were not induced by HHV-6 or tetradecanoyl phorbol acetate treatment, Binding of these factors to ZII was prevented by oligonucleotides containing CRE but not by AP-1 consensus sequences, Antibodies against CRE-binding (CREB) protein but not against c-Fos or c-Jun were able to supershift the DNA-protein complex, identifying the nature of the transcription factor which hinds to ZII as a member of the CREB family of proteins, Finally, transfection of CREB protein and protein kinase A expression vectors were found to activate Zp in Jurkat cells, suggesting that phosphorylated form of CREB protein can play a determining role in the EBV reactivation process.