12-O-Tetradecanoylphorbol-13-acetate Induces Epstein–Barr Virus Reactivation via NF-κB and AP-1 as Regulated by Protein Kinase C and Mitogen-Activated Protein Kinase

Abstract Signaling pathway components mediating Epstein–Barr virus (EBV) reactivation by 12- O -tetradecanoylphorbol-13-acetate (TPA) were characterized in terms of induction and modification of specific transacting factors. The consequences of protein kinase C (PKC) activation by TPA in inhibiting inducible nitric oxide synthase (iNOS) mRNA expression were analyzed in the EBV-infected gastric epithelial cell line GT38. Spontaneous expression of the EBV BZLF1 gene product ZEBRA became undetectable upon long-term culturing of GT38 cells, while iNOS mRNA expression increased. In such cells the PKC inhibitors 1-(5-isoquinolinesulphonyl)-2,5-dimethylpiperazine (H7) and staurosporine inhibited TPA-induced expression of BZLF1 and BRLF1 and reversed TPA-mediated inhibition of iNOS gene expression. The mitogen-activated protein kinase inhibitor PD98059 inhibited TPA-induced BZLF1 expression. Electrophoretic mobility shift assays demonstrated that transcription factors NF-κB and AP-1 were also activated by TPA in a time-dependent manner. The TPA-induced NF-κB activation was inhibited by prior treatment of the cells with the NF-κB inhibitor pyrrolidine dithiocarbamate (PDTC). TPA-induced BZLF1 expression was also inhibited by the treatment with PDTC. Northern blot analyses characterized changes in levels of the c- jun and jun B expressions of the AP-1 family. These results show that TPA induces EBV reactivation via NF-κB and AP-1 and that PKC is an important mediator in regulating gene expression leading to EBV reactivation after TPA treatment of GT38 cells.