THE EPSTEIN-BARR-VIRUS LYTIC TRANSACTIVATOR ZTA INTERACTS WITH THE HELICASE-PRIMASE REPLICATION PROTEINS

The Epstein-Barr virus transactivator Zta triggers lytic gene expressi on and is essential for replication of the lytic origin, oriLyt. Previ ous analysis indicated that the Zta activation domain contributed a re plication-specific function. We now show that the Zta activation domai n interacts with components of the EBV helicase-primase complex. The t hree helicase primase proteins BBLF4 (helicase), BSLF1 (primase), and BBLF2/3 (primase-associated factor) were expressed fused to the Myc ep itope. When expression plasmids for BBLF4 or BBLF2/3 plus BSLF1 (prima se subcomplex) were separately transfected, the proteins localized to the cytoplasm, Interaction between Zta and the components of the helic ase-primase complex was tested by examining the ability of Zta to alte r the intracellular localization of these proteins. Cotransfection of Zta with Myc-BBLF4 resulted in nuclear translocation of Myc-BBLF4; sim ilarly, cotransfection of Zta,vith the primase subcomplex led to nucle ar translocation of the Myc-BSLF1 and Myc-BBLF2/3 proteins. This reloc alization provides evidence for an interaction between Zta and the hel icase and Zta and the primase subcomplex, An affinity assay using glut athione S-transferase-Zta fusion proteins demonstrated that Myc-BBLF4 and Myc-BBLF2/3 plus BSLF1 bound to the Zta activation domain (amino a cids 1 to 133), In the nuclear relocalization assay, the amino termina l 25 amino acids of Zta were required for efficient interaction with t he primase subcomplex but not for interaction with BBLF4. Evidence for interaction between orilyt bound Zta and the helicase-primase complex was obtained in a superactivation assay using an oriLyt-chloramphenic ol acetyltransferase (CAT) reporter. Zta activated expression from a C AT reporter containing the complete orilyt region and regulated by the orilyt BHLF1 promoter. Cotransfection of the helicase primase protein s, one of which was fused to a heterologous activation domain, led to Zta-dependent superactivation of CAT expression, This assay also provi ded evidence for an interaction between the single-stranded DNA bindin g protein, BALF2, and the Zta-tethered helicase-primase complex. The h elicase-primase interaction is consistent with a role for Zta in stabi lizing the formation of an origin-bound replication complex.