Urea-mediated cross-presentation of soluble Epstein-Barr virus BZLF1 protein

Soluble extracellular proteins usually do not enter the endogenous human leukocyte antigen (HLA) I-dependent presentation pathway of antigen-presenting cells, strictly impeding their applicability for the re-stimulation of protein-specific CD8(+) cytotoxic T lymphocytes (CTL). Here we present for the Epstein-Barr virus (EBV) BZLF1 a novel strategy that facilitates protein translocation into antigen-presenting cells by its solubilisation in high molar urea and subsequent pulsing of cells in presence of low molar urea. Stimulation of PBMC from HLA-matched EBV-seropositive individuals with urea-treated BZLF1 but not untreated BZLF1 induces an efficient reactivation of BZLF1-specific CTL. Urea-treated BZLF1 (uBZLF1) enters antigen-presenting cells in a temperature-dependent manner by clathrin-mediated endocytosis and is processed by the proteasome into peptides that are bound to nascent HLA I molecules. Dendritic cells and monocytes but also B cells can cross-present uBZLF1 in vitro. The strategy described here has potential for use in the development of improved technologies for the monitoring of protein-specific CTL.     

Epstein-Barr virus BZLF1 protein binds to mitotic chromosomes

Epstein-Barr virus (EBV) is a human herpesvirus that causes infectious mononucleosis and is associated with several types of cancers, including nasopharyngeal carcinoma and Burkitt's lymphoma. An EBV protein that plays an integral role during lytic replication is the immediate-early protein BZLF1. Our laboratory has found that BZLF1 (Z) localizes to host chromosomes during mitosis. Two Z-interacting proteins are also found localized to mitotic chromosomes in the presence of Z. The association between Z and mitotic chromosomes may lead to the sequestering of Z-interacting proteins within the cell and potentially cause an alteration of chromosome compaction or chromatin structure.     

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.     

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.     

Epstein-Barr virus nuclear protein 2 transactivation of the latent membrane protein 1 promoter is mediated by J kappa and PU.1.

Expression of the Epstein-Barr virus (EBV) latent membrane protein 1 (LMP-1) oncogene is regulated by the EBV nuclear protein 2 (EBNA-2) transactivator. EBNA-2 is known to interact with the cellular DNA-binding protein J kappa and is recruited to promoters containing the GTGGGAA J kappa recognition sequence. The minimal EBNA-2-responsive LMP-1 promoter includes one J kappa-binding site, and we now show that mutation of that site, such that J kappa cannot bind, reduces EBNA-2 responsiveness by 60%. To identify other factors which interact with the LMP-1 EBNA-2 response element (E2RE), a -236/-145 minimal E2RE was used as a probe in an electrophoretic mobility shift assay. The previously characterized factors J kappa, PU.1, and AML1 bind to the LMP-1 E2RE, along with six other unidentified factors (LBF2 to LBF7). Binding sites were mapped for each factor. LBF4 is B- and T-cell specific and recognizes the PU.1 GGAA core sequence as shown by methylation interference. LBF4 has a molecular mass of 105 kDa and is probably unrelated to PU.1. LBF2 was found only in epithelial cell lines, whereas LBF3, LBF5, LBF6, and LBF7 were not cell type specific. Mutations of the AML1- or LBF4-binding sites had no effect on EBNA-2 transactivation, whereas mutation of the PU.1-binding site completely eliminated EBNA-2 responses. A gst-EBNA-2 fusion protein specifically depleted PU.1 from nuclear extracts and bound in vitro translated PU.1, providing biochemical evidence for a direct EBNA-2-PU.1 interaction. Thus, EBNA-2 transactivation of the LMP-1 promoter is dependent on interaction with at least two distinct sequence-specific DNA-binding proteins, J kappa and PU.1. LBF3, LBF5, LBF6, or LBF7 may also be involved, since their binding sites also contribute to EBNA-2 responsiveness.