Epstein-Barr virus BZLF1 trans activator induces the promoter of a cellular cognate gene, c-fos.

The Epstein-Barr virus BZLF1 gene product ZEBRA is a DNA-binding protein that is partially homologous to c-Fos, binds specifically to AP-1 sites, and can induce the lytic cycle in latently infected B lymphocytes. Induction of the viral lytic cycle can also be achieved by treatment with the phorbol ester 12-O-tetrade-canoylphorbol-13-acetate, a reagent which activates gene expression in part through AP-1 (Jun/Fos). In this article the interrelationship between ZEBRA and AP-1 is extended by the demonstration that ZEBRA can induce c-Fos expression through AP-1 and "AP-1-like" sites present in the c-fos promoter. Induction of c-Fos may be necessary for the expression of other viral lytic genes and perhaps cellular genes whose products are required for viral replication.     

Differentiation-associated expression of the Epstein-Barr virus BZLF1 transactivator protein in oral hairy leukoplakia.

The BZLF1 protein of Epstein-Barr virus (EBV) is a key immediate-early protein which has been shown to disrupt virus latency in EBV-infected B cells. We have generated a monoclonal antibody, BZ1, to BZLF1 which reacts in immunohistology, immunoblotting, and immunoprecipitation and which recognizes both the active, dimeric form and the inactive, monomeric form of the protein. Biopsies of oral hairy leukoplakia, an AIDS-associated lesion characterized by high-level EBV replication, were examined by immunohistochemistry using the BZ1 monoclonal antibody. A differentiation-associated pattern of BZLF1 expression was observed, BZ1 reacting with nuclei of the upper spinous layer of the lesion. This finding suggests that the BZLF1 promoter may be regulated by the degree of squamous differentiation. A comparison of in situ hybridization to EBV DNA and viral capsid antigen staining with BZ1 reactivity suggested that BZLF1 expression precedes rampant virus replication. The inability to detect EBV in the lower epithelial layers of oral hairy leukoplakia raises questions concerning the nature of EBV latency and persistence in stratified squamous epithelium.     

Analysis of the BZLF1 promoter of Epstein-Barr virus: identification of an anti-immunoglobulin response sequence.

The induction of the viral lytic cycle in latently Epstein-Barr virus (EBV)-infected B cells is initiated by activation of the BZLF1 gene, whose expression is sufficient to disrupt EBV latency, suggesting that BZLF1 acts as the switch to change from a latent to a lytic replicative cycle. In the present studies, a series of deletion plasmids encompassing positions bp -552 to +12 of the BZLF1 promoter were constructed and tested for their response to anti-immunoglobulin (anti-Ig), an inducer of the viral lytic cycle, upon transfection into EBV-negative and -positive lymphoid cells. The promoter consisted of three functionally distinct regions. Region I (bp -552 to -221) had a negative influence on promoter activity; its deletion made the promoter highly responsive to anti-Ig. Region II (bp -203 to -177) was important for conferring responsiveness to anti-Ig. The response to anti-Ig did not require the presence of the EBV genome or EBV gene products. This sequence also enhanced expression of the chloramphenicol acetyltransferase (cat) gene from the simian virus 40 promoter in response to anti-Ig, even when inserted downstream of the cat gene. Region III (-134 to -116) was a positive element that was transactivated by the BZLF1 gene product.     

Plasma cell-specific transcription factor XBP-1s binds to and transactivates the Epstein-Barr virus BZLF1 promoter

Epstein-Barr virus (EBV) in vivo is known to establish persistent infection in resting, circulating memory B cells and to productively replicate in plasma cells. Until now, the molecular mechanism of how EBV switches from latency to lytic replication in vivo was not known. Here, we report that the plasma cell differentiation factor, XBP-1s, activates the expression of the master regulator of EBV lytic activation, BZLF1. Using reporter assays, we observed that XBP-1s was able to transactivate the BZLF1 promoter, Zp, in a plasma cell line and other lymphoid cell lines but, interestingly, not in epithelial cell lines. We have identified an XBP-1s binding site on the ZID/ZII region of Zp, which when abolished by site-directed mutagenesis led to abrogation of XBP-1s binding and promoter activation. Using the chromatin immunoprecipitation assay, we observed direct binding of XBP-1s to endogenous Zp in an EBV-infected plasma cell line. Finally, in the same cell line, we observed that overexpression of XBP-1s resulted in increased expression of BZLF1, while knockdown of XBP-1s with short hairpin RNA drastically reduces BZLF1 expression. We suggest that EBV harnesses the B-cell terminal differentiation pathway via XBP-1s as a physiological signal to reactivate and begin viral replication. We are currently investigating other signals, such as the endoplasmic reticulum stress response proteins, which act upstream of XBP-1s, to identify other interacting factors that initiate and/or amplify the lytic switch.     

Characterization of the ZI domains in the Epstein-Barr virus BZLF1 gene promoter: role in phorbol ester induction.

Induction of the Epstein-Barr virus lytic cycle is mediated through the immediate-early BZLF1 gene and the coordinately regulated BRLF1 gene. The BZLF1 gene product, Zta, transactivates its own promoter, as well as the promoters of a number of lytic genes, thereby initiating a cascade of viral gene expression. Previous work identified four related elements (ZIA, ZIB, ZIC, and ZID) and a cyclic AMP response element binding-AP-1 element (ZII) that are involved in the induction of the BZLF1 promoter (Zp) by the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) (E. Flemington and S. H. Speck, J. Virol. 64:1217-1226, 1990). Here we report a detailed characterization of TPA induction mediated by the ZI domains. Mutation of individual ZI domains within the context of the intact promoter significantly diminished TPA induction. Cloning of individual ZI domains upstream of a minimal promoter demonstrated that the ZIA, ZIC, and ZID domains, but not the ZIB domain, are TPA responsive. Furthermore, cloning of the ZII domain downstream of the ZI domains significantly augmented TPA induction. The critical regions within the ZIA and ZIC elements involved in binding of cellular factors were identified by using methylation interference and electrophoretic mobility shift analyses of ZI domain mutants. Four specific complexes were observed with the ZIA and ZID domains, all of which could be specifically competed for by either the ZIA or ZID domain. Methylation interference analyses of bound complexes revealed the presence of two overlapping binding sites for cellular factors in the ZIA domain, and functional studies provided evidence that both of these sites are involved in TPA induction. Functional analyses of the ZIC domain revealed that the 5' region of this domain is largely responsible for mediating TPA induction. Binding data correlated well with functional activity and revealed that the ZIC domain binds only a subset of the cellular factors that bind to the ZIA and ZID domains. Analysis of factor binding to the ZIB domain revealed only a single shifted complex, which correlated with the most slowly migrating complex observed with the ZIA and ZID domains. These data provide a direct demonstration of TPA induction mediated by the ZIA, ZIC, and ZID domains and also provide the first evidence that the ZI domains exhibit distinct functional characteristics.     

Interaction of the lymphocyte-derived Epstein-Barr virus nuclear antigen EBNA-1 with its DNA-binding sites.

The Epstein-Barr virus (EBV) nuclear antigen EBNA-1 plays an integral role in the maintenance of latency in EBV-infected B lymphocytes. EBNA-1 binds to sequences within the plasmid origin of replication (oriP). It is essential for the replication of the latent episomal form of EBV DNA and may also regulate the expression of the EBNA group of latency gene products. We have used sequence-specific DNA-binding assays to purify EBNA-1 away from nonspecific DNA-binding proteins in a B-lymphocyte cell extract. The availability of this eucaryotic protein has allowed an examination of the interaction of EBNA-1 with its specific DNA-binding sites and an evaluation of possible roles for the different binding loci within the EBV genome. DNA filter binding assays and DNase I footprinting experiments showed that the intact Raji EBNA-1 protein recognized the two binding site loci in oriP and the BamHI-Q locus and no other sites in the EBV genome. Competition filter binding experiments with monomer and multimer region I consensus binding sites indicated that cooperative interactions between binding sites have relatively little impact on EBNA-1 binding to region I. An analysis of the binding parameters of the Raji EBNA-1 to the three naturally occurring binding loci revealed that the affinity of EBNA-1 for the three loci differed. The affinity for the sites in region I of oriP was greater than the affinity for the dyad symmetry sites (region II) of oriP, while the physically distant region III locus showed the lowest affinity. This arrangement may provide a mechanism whereby EBNA-1 can lowest affinity. This arrangement may provide a mechanism whereby EBNA-1 can mediate differing regulatory functions through differential binding to its recognition sequence.     

The Epstein-Barr virus BMLF1 promoter contains an enhancer element that is responsive to the BZLF1 and BRLF1 transactivators.

We have previously shown that the Epstein-Barr virus (EBV) immediate-early gene product, BZLF1, can activate expression of the EBV BMLF1 immediate-early promoter in EBV-positive, but not EBV-negative, B cells, suggesting that the BZLF1 effect may be mediated through another EBV gene product (S. Kenney, J. Kamine, E. Holley-Guthrie, J.-C. Lin, E.-C. Mar, and J. S. Pagano, J. Virol. 63:1729-1736, 1989). Here, we show that the EBV BRLF1 immediate-early gene product transactivates the BMLF1 promoter in either EBV-positive or EBV-negative B cells. Deletional analysis revealed that both the BZLF1-responsive region and the BRLF1-responsive region of the BMLF1 promoter are contained within the same 140-base-pair FokI-PvuII fragment located 300 base pairs upstream of the mRNA start site. This FokI-PvuII fragment functions as an enhancer element in the presence of the BRLF1 transactivator and contains the sequence CCGTGGAGA ATGTC, which is strikingly similar to the BRLF1-responsive region of the EBV DR/DL enhancer (A. Chevallier-Greco, H. Gruffat, E. Manet, A. Calender, and A. Sergeant, J. Virol. 63:615-623, 1989). The effect of BZLF1 on the BMLF1 promoter is likely to be indirect and mediated through the BRLF1 transactivator.