Abstract
Induction of Epstein-Barr Virus (EBV) to enter viral lytic cycle is initiated by expression of a latent-to-lytic switching gene, BZLF1. BZLF1 gene product, Zta, is a transactivator which transactivates its own promoter Zp and a number of viral lytic gene promoters, thereby initiating a cascade of viral gene expression. BZLF1 transcription is normally suppressed in EBV-transformed B cells but can be induced by inducers such as transfoming growth factor-beta(TGF-beta) and 12-o-tetradecanoylphorbol-13-acetate (TPA). TPA responsive element at Zp is the AP-1-like binding motif. TGF-beta-induced transcription activation can be mediated via Smad4 binding element (SBE) or via Smad3 and Smad4 cooperation with c-Jun/c-Fos of AP-1 complex (Zhang et al., 1998). In this study, I generated a 269-bp Zp reporter construct Zp-236 covering –236 to +13 region of transcription initiation site) and the activity which is responsive to TPA is also activated in response to TGF-beta treatment. Sequence analysis reveals several potential SBE site or SBE-like sequence within Zp. In order to determine the role of SBE site or SBE-like sequence and AP-1 binding motif within the in TGF-beta and TPA induced activation, deletion and site-specific mutants of Zp-236 were generated. Transient transfection of deletion mutants, SBE site-specific mutants and AP-1 site-specific mutants of Zp reporter constructs into P3HR1 cells showed that Zp is synergistically activated by TGF-beta and TPA via the SBE and AP-1 binding sites. Electrophoretic mobility shift assay indicated that Smad4 associate with SBE in response to TGF-beta treatment and co-transfection experiments indicated that Zp is activated by TGF-beta-induced Smad3/Smad4 and c-Jun/c-Fos. The results suggest that the mechanism of transcriptional activation of Zp by TGF-beta is through cooperation between Smad4 binding to SBE and interaction of Smad3 and c-Jun/c-Fos at AP-1 binding motif.