The ZIIR Element of the Epstein-Barr Virus BZLF1 Promoter Plays a Central Role in Establishment and Maintenance of Viral Latency

Abstract

The Epstein-Barr virus (EBV)BZLF1Epstein-Barr virus (EBV), a human gammaherpesvirus, infects approximately 90% of the world's population. It is a causative agent of infectious mononucleosis and is associated with several epithelial and B-cell cancers, including Burkitt's lymphoma (BL), Hodgkin's disease, nasopharygeal carcinoma, and some gastric carcinomas (reviewed in reference 41). EBV can infect primary B lymphocytes, establishing a latent form of infection in which its genome is maintained as an episome, replicating in synchrony with its host's cellular DNA. EBV can also infect epithelial cells, leading to a latent or lytic form of infection, depending upon the specific cell type and state of cellular differentiation (reviewed in reference 28).The switch from EBV latency to lytic replication in B cells is usually initiated via activating expression of the immediateearly (IE) BZLF1 gene (11). The BZLF1 gene encodes a sequence-specific DNA-binding protein, Zta (also called Z, Zebra, and EB1), a member of the bZIP family of leucine-zipper transactivators. The activities of Zta include direct participation in EBV replication via binding to the viral DNA origin of lytic replication, ori-lyt, downregulating the latency-associated promoters Cp and Wp, and serving as a transcriptional transactivator of its own promoter, other IE and early (E) viral promoters, including the BRLF1 promoter, Rp, and several cellular promoters (reviewed in references 26 and 31). The BRLF1 gene encodes a second viral transactivator, Rta (also called R). Acting together, Zta and Rta play multiple roles in lytic replication of EBV (17). While highly quiescent during latency, transcription from the BZLF1 promoter Zp can be activated in some cells by incubation with various inducers, including phorbol esters such as 12-O-tetradecanoyl-phorbol-13-acetate (TPA), calcium ionophores such as ionomycin, histone deacatylase (HDAC) inhibitors such as valproic acid (VPA) and sodium butyrate, transforming growth factor ␤ (TGF-␤), and anti-immunoglobulins (anti-Igs) (reviewed in references 26 and 31).The cis-acting elements of Zp sufficient for both basal and induced activity lie within the nucleotide (nt) Ϫ221 to ϩ12 region (mini-Zp) relative to Zp's transcription initiation site (reviewed in references 26 and 45) (Fig. 1). Three positive regulatory cis-acting elements, named ZI, ZII, and ZIII, have been identified within mini-Zp. The ubiquitous transcription factors Sp1/Sp3 can bind to the AT-rich ZIA, ZIC, and ZID elements, while the myocyte enhancer factor 2D (MEF2D) can bind to the ZIA, ZIB, and ZID elements (6,33,34). ZII is a cyclic AMP response element (CRE)-like motif that binds CREB, ATF family members, C/EBPs, and the AP-1 family of transcription factors (5,6,19,24,46,50,53). Although the ZIA-ZID and ZII elements have distinct DNA sequences and are bound by different regulatory factors, they both function as sites through which induction can be mediated by chemical inducers such as TPA and ionomycin in reporter assays (20). These inducers ac...

Shutoff of BZLF1 Gene Expression Is Necessary for Immortalization of Primary B Cells by Epstein-Barr Virus

“…Human primary blood B cells were purchased from AllCells (Emeryville, CA), infected with the indicated amount of virus in green Raji units (GRU)/cell, aliquoted at 1 ϫ 10 4 cells per well in 96-well plates, incubated at 37°C for 6 weeks, and examined for the percentage of wells containing proliferating, GFP-positive B cells as previously described (53).…”

“…Immunofluorescence staining (IFS) was performed as previously described (53,54). Titers of infectious virus were quantified by a Raji assay using 3-fold serial dilutions of each sample on two or more occasions as previously described (1,54).…”

“…We reported previously that both the ZV and ZIIR elements function as potent silencers of Zp in the context of an intact EBV genome (53,54). To begin to understand how the ZV, ZV=, and ZIIR elements collectively function to regulate transcription from Zp, we first assessed the effects of mutations in these elements individually versus concurrently using a transient-transfection assay.…”

“…This end is achieved by the presence within Zp of multiple negative regulatory elements. Three silencing elements have been identified to date within the mini-Zp region: ZIIR, HIε, and ZV (30,31,35,46,53). In addition, a phosphorylated form of MEF-2D bound to ZIA, ZIB, and ZID can also repress Zp by recruiting histone deacetylases (HDACs) to maintain chromatin in a repressed state (7).…”

“…1). We recently reported that variants of EBV strain B95.8 harboring base pair substitution mutations in the ZIIR element are somewhat defective in establishing proliferating B-cell colonies; the EBV-infected cells that do manage to grow out spontaneously reactivate into lytic replication, doing so at a very high frequency in the presence of 12-Otetradecanoyl-phorbol-13-acetate (TPA) (53). The identity of the cellular factor(s) through which ZIIR silences BZLF1 gene expression remains unknown.…”