Transforming growth factor beta 1 stimulates expression of the Epstein-Barr virus BZLF1 immediate-early gene product ZEBRA by an indirect mechanism which requires the MAPK kinase pathway

Disruption of Epstein-Barr virus (EBV) latency is mediated by ZEBRA the protein product of the immediate-early EBV gene, BZLF1. In vitro, phorbol It-myristate 13-acetate (PIMA), a potent activator of protein kinase C (PKC), induces reactivation of EBV. However, the physiological stimuli responsible for the disruption of viral latency are not well characterized. Transforming growth factor beta 1 (TGF-beta 1) has also been shown to trigger the reactivation of EBV in Burkitt Lymphoma cell lines; however, the effect of TGF-beta 1 on ZEBRA expression has not been reported. To further understand this phenomenon, we have investigated the effect of TGF-beta 1 on ZEBRA expression. Our results indicate that the treatment of different EBV-positive Burkitt's lymphoma cell lines with TGF-beta 1 induces a time-dependent activation of BZLF1 transcription with a corresponding increase in the production of the protein ZEBRA. TGF-beta 1 has been shown to exert its effects through a wide range of intracellular routes; in the present study, we have explored these pathways. Transient expression of Smad proteins on their own had no effect on ZEBRA expression, A specific inhibitor of p38 mitogen-activated protein kinase (MAPK), SB203580, did not affect TGF-beta 1-induced ZEBRA expression, whereas treatment with the MAPK/ERK kinase inhibitors, PD98059 and U0126, dramatically decreased this induction, This suggests that TGF-beta 1 effect on BZLF1 expression requires the MAPK pathway. However, in Raji and B95-8 cells additional routes can be used, as (i) the inhibition of ZEBRA induction by PD98059 or U0126 was incomplete, whereas these inhibitors completely abolished PMA-induced ZEBRA expression, (ii) TGF-beta 1 induction of ZEBRA expression occurs in PKC-depleted cells, (iii) in Raji and in B95-8 cells, the effect of TGF-beta 1 and PMA are additive. Transient transfection of the EBV-negative B-cell line DG75 with a BZLF1 promoter-fusion construct (Zp-CAT) showed that under conditions where the BZLF1 promoter is activated by PMA treatment, TGF-beta 1 had no significant effect on the expression of the chloramphenicol acetyltransferase gene. Furthermore, TGF-beta 1 induction of BZLF1 transcripts is dependent on de novo protein synthesis, which suggests that TGF-beta 1 induces BZLF1 expression by an indirect mechanism.