Regulation of Epstein-Barr Virus BZLF1

Abstract

Epstein-Barr virus (EBV) establishes a latent infection in the human host. In order to produce infectious virus particles EBV must reactivate from latency and enter its lytic cycle. BZLF1 is the immediate early gene in EBV that mediates the switch between latency and the lytic cycle. The BZLF1 gene is under the control of the Zp promoter. Reactivation from latency is studied in EBV positive Akata cell lines where EBV can be reactivated by crosslinking the B-cell receptor (BCR) using antibodies to mimic antigen binding. This system uses stably transfected reporter plasmids to study Zp regulation. Mutagenesis identified additional regions of the promoter that contribute to regulation and the ZID MEF2 binding site was demonstrated to be functionally important during the initial stages of Zp activation. XBP-1 splicing, previously implicated in Zp reactivation, was found to occur rapidly in this system in response to BCR crosslinking and parallels the transient induction of Zp. Chromatin remodelling also plays an important role in Zp regulation. An inducible BZLF1 expression system, independent of BCR signalling, was developed in Akata cells that accurately mimics BZLF1 activity and provides a novel approach to study repression at Zp. The BZLF1 protein is related to the bZIP family of transcription factors. BZLF1 contains a bZIP motif in which C-terminal residues fold back against a zipper region that forms an a-helical coiled-coil. The 208SSENDRLR215 sequence in the zipper region is conserved between BZLF1 and C/EBP. Point mutagenesis in this sequence revealed the importance of individual residues for transactivation and progression to DNA replication. The restoration of BZLF1 DNA replication activity by complementation of two deleterious mutations (S208E and D236K) indicated that the interaction of the C-terminal tail and the core zipper region is required for DNA replication, identifying a functional role for this structural feature unique to BZLF1.