Restoration of the Epstein-Barr Virus ZEBRA Protein's Capacity to Disrupt Latency by the Addition of Heterologous Activation Regions

Abstract

The ZEBRA protein has a unique biological function among herpesviral proteins. It is responsible for the disruption of Epstein-Barr virus (EBV) latency and the induction of the lytic cycle. ZEBRA is a bZIP transcriptional activator which binds as a dimer to 7-bp response elements within EBV promoters and is directly involved in the stimulation of virus replication at the EBV lytic origin. We have employed the ZEBRA/EBV biological system to test whether a heterologous activation domain can substitute for another activation domain (the ZEBRA domain). The ZEBRA activation region was replaced with the potent acid activation region from the herpes simplex virus VP16 protein or with the activation region of the EBV R protein. Both chimeras were found to transactivate model and native promoters at equivalent or better levels than ZEBRA itself. Activation was not target- or cell-type dependent, nor was it dependent on the presence of virus. These activation domains restored ZEBRA's ability to induce early antigen and to stimulate origin replication to levels that were equal to or greater than those of wild type. These studies suggest that the specificities of some of the known biological functions of ZEBRA are not dependent upon the nature of the activation domain present within ZEBRA.

Activation domain requirements for disruption of Epstein-Barr virus latency by ZEBRA

“…In these studies we address the domain requirements of ZEBRA in human B lymphocytes latently infected with EBV. Previous work from this laboratory has shown that ZEBRA's activation domain can be replaced by potent acidic activation regions (5). Here those studies are extended to nonacidic activation domains.…”

“…B-cell lines were transfected by using a Bio-Rad Gene Pulser apparatus with a capacitance extender, at 960 F and 0.25 kV in 0.4 ml of complete medium. After electroporation, cells were maintained in RPMI medium with 8% fetal calf serum for 2 to 4 days at 37°C under a 5% CO 2 atmosphere (5).…”

“…It contains amino acids 1 to 25 and 142 to 227 of the wild-type ZEBRA protein ( Fig. 1) (3,5). The ZdVP and ZdR clones have been described before (5); briefly, ZdVP contains amino acids 400 to 479 of herpes simplex virus's VP16 protein fused to the carboxyl terminus of Zd, and ZdR contains amino acids 480 to 605 of EBV's R protein fused to the carboxyl terminus of Zd.…”

“…1) (3,5). The ZdVP and ZdR clones have been described before (5); briefly, ZdVP contains amino acids 400 to 479 of herpes simplex virus's VP16 protein fused to the carboxyl terminus of Zd, and ZdR contains amino acids 480 to 605 of EBV's R protein fused to the carboxyl terminus of Zd. The Zd chimeras, ZdMeqII339 and Zd3C, contain the proline-rich region of the Marek's disease virus Meq protein (amino acids 142 to 339) (35) and the glutamine-rich region of EBV's EBNA3C protein (amino acids 724 to 826) (32), respectively.…”

“…CAT assays. Chloramphenicol acetyltransferase (CAT) assays were performed by using a liquid phase assay as described previously (3,5). Briefly, separate plasmids containing the effector (30 g) and the target (5 g) were electroporated into 5 ϫ 10 6 to 10 ϫ 10 6 cells.…”