CHARACTERIZATION OF THE ZI DOMAINS IN THE EPSTEIN-BARR-VIRUS BZLF1 GENE PROMOTER - ROLE IN PHORBOL ESTER INDUCTION

Induction of the Epstein-Barr virus lytic cycle is mediated through th e immediate-early BZLF1 gene and the coordinately regulated BRLF1 gene , The BZLF1 gene product, Zta, transactivates its own promoter, as wel l as the promoters of a number of lytic genes, thereby initiating a ca scade of viral gene expression. Previous work identified four related elements (ZIA, ZIB, ZIG, and ZID) and a cyclic AMP response element bi nding-AP-1 element (ZII) that are involved in the induction of the BZL F1 promoter (Zp) by the phorbol ester 12-0-tetradecanoylphorbol-13-ace tate (TPA) (E. FLemington and S. H. Speck, J. Virol. 64:1217-1226, 199 0), Here we report a detailed characterization of TPA induction mediat ed by the ZI domains. Mutation of individual ZI domains within the con text of the intact promoter significantly diminished TPA induction. Cl oning of individual ZI domains upstream of a minimal promoter demonstr ated that the ZIA, ZIC, and ZID domains, but not the ZIB domain, are T PA responsive. Furthermore, cloning of the ZII domain downstream of th e ZI domains significantly augmented TPA induction. The critical regio ns within the ZIA and ZIC elements involved in binding of cellular fac tors were identified by using methylation interference and electrophor etic mobility shift analyses of ZI domain mutants. Four specific compl exes were observed with the ZIA and ZID domains, all of which could be specifically competed for by either the ZIA or ZID domain. Methylatio n interference analyses of bound complexes revealed the presence of tw o overlapping binding sites for cellular factors in the ZIA domain, an d functional studies provided evidence that both of these sites are in volved in TPA induction. Functional analyses of the ZIC domain reveale d that the 5' region of this domain is largely responsible for mediati ng TPA induction. Binding data correlated well with functional activit y and revealed that the ZIC domain binds only a subset of the cellular factors that bind to the ZIA and ZID domains. Analysis of factor bind ing to the ZIB domain revealed only a single shifted complex, which co rrelated with the most slowly migrating complex observed with the ZIA and ZID domains, These data provide a direct demonstration of TPA indu ction mediated by the ZIA, ZIC, and ZID domains and also provide the f irst evidence that the ZI domains exhibit distinct functional characte ristics.