The Epstein-Barr Virus Lytic Transactivator Zta Interacts with the Helicase-Primase Replication Proteins

Abstract

The Epstein-Barr virus transactivator Zta triggers lytic gene expression and is essential for replication of the lytic origin, oriLyt. Previous analysis indicated that the Zta activation domain contributed a replication-specific function. We now show that the Zta activation domain interacts with components of the EBV helicase-primase complex. The three helicase-primase proteins BBLF4 (helicase), BSLF1 (primase), and BBLF2/3 (primase-associated factor) were expressed fused to the Myc epitope. When expression plasmids for BBLF4 or BBLF2/3 plus BSLF1 (primase subcomplex) were separately transfected, the proteins localized to the cytoplasm. Interaction between Zta and the components of the helicase-primase complex was tested by examining the ability of Zta to alter the intracellular localization of these proteins. Cotransfection of Zta with Myc-BBLF4 resulted in nuclear translocation of Myc-BBLF4; similarly, cotransfection of Zta with the primase subcomplex led to nuclear translocation of the Myc-BSLF1 and Myc-BBLF2/3 proteins. This relocalization provides evidence for an interaction between Zta and the helicase and Zta and the primase subcomplex. An affinity assay using glutathioneS-transferase–Zta fusion proteins demonstrated that Myc-BBLF4 and Myc-BBLF2/3 plus BSLF1 bound to the Zta activation domain (amino acids 1 to 133). In the nuclear relocalization assay, the amino-terminal 25 amino acids of Zta were required for efficient interaction with the 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-chloramphenicol acetyltransferase (CAT) reporter. Zta activated expression from a CAT reporter containing the complete oriLyt region and regulated by the oriLyt BHLF1 promoter. Cotransfection of the helicase-primase proteins, one of which was fused to a heterologous activation domain, led to Zta-dependent superactivation of CAT expression. This assay also provided evidence for an interaction between the single-stranded DNA binding protein, BALF2, and the Zta-tethered helicase-primase complex. The helicase-primase interaction is consistent with a role for Zta in stabilizing the formation of an origin-bound replication complex.

Cellular transcription factors recruit viral replication proteins to activate the Epstein–Barr virus origin of lytic DNA replication, oriLyt

“…Numerous interactions between these proteins have been documented, consistent with the existence of a replication complex that is essentially viral in nature (Tsurumi et al, 1993;Zeng et al, 1997;Gao et al, 1998).…”

“…BZLF1 is also capable of activating oriLyt directly by binding to DNA motifs, socalled BZLF1-responsive DNA elements (ZRE), located in the upstream component (Lieberman et al, 1990;Schepers et al, 1993aSchepers et al, , 1996Sarisky et al, 1996;Askovic and Baumann, 1997). Proteinprotein interactions have been demonstrated to exist between the DNA-binding domain of BZLF1 and the DNA polymerase accessory factor (BMRF1) (Zhang et al, 1996), and between the transactivation domain of BZLF1 and both the viral helicase (BBLF4) and the primase subcomplex (composed of BSLF1 and BBLF2/3) (Gao et al, 1998). The single-stranded DNA-binding protein BALF2 may in turn contact the helicase-primase subcomplex (Gao et al, 1998).…”

“…Proteinprotein interactions have been demonstrated to exist between the DNA-binding domain of BZLF1 and the DNA polymerase accessory factor (BMRF1) (Zhang et al, 1996), and between the transactivation domain of BZLF1 and both the viral helicase (BBLF4) and the primase subcomplex (composed of BSLF1 and BBLF2/3) (Gao et al, 1998). The single-stranded DNA-binding protein BALF2 may in turn contact the helicase-primase subcomplex (Gao et al, 1998). Our data provide evidence for additional interactions of the viral DNA polymerase complex composed of the catalytic subunit BALF5 and BMRF1 (Kiehl and Dorsky, 1991) with the cellular transcription factors Sp1 and ZBP-89.…”

Herpes Virus Replication

“…The initiation process in other herpesviruses is less well understood and possibly more complicated. In EBV for example, it is known that Rta and Zta proteins are required for lytic cycle induction (70)(71)(72). Zta has also been shown to possess oriLyt-binding activity and to recruit other replication factors (helicase-primase and SSB) presumably to initiate assembly of a replisome (71).…”

“…KSHV transactivator ORF50 (EBV Rta homologue (72)) induces the viral lytic cycle (99). Additional factors such as ORF-K8 (EBV Zta homologue (70,71)) may also be required for oriLyt-dependent DNA replication (49). 5 It is thought that the mature protein is formed from a spliced transcript involving these ORFs.…”