A new Epstein-Barr virus transactivator, R, induces expression of a cytoplasmic early antigen.

Several Epstein-Barr virus (EBV) early promoters respond to a new EBV transactivator encoded by BRLF1, designated R. Transactivation was measured in chloramphenicol acetyltransferase assays on Raji, BHK, and Vero cells that were cotransfected with the transactivator and target promoters linked to the cat gene. The divergent promoter of BamHI-H was particularly responsive to R transactivation. This large promoter region consists of a leftward TATA box for the NotI repeat gene (BHLF1) and a probable rightward TATA box for the EA-R gene (BHRF1) separated by 940 base pairs of unusual sequence complexity. Sequences within this divergent promoter region appear to confer inducibility by EBV transactivators R and Z (BZLF1). The Z transactivator stimulated expression in both the leftward and rightward directions, and R stimulated expression primarily in the rightward direction, but the MS transactivator (BMLF1) had no activity in either direction. The adenovirus E3 promoter also responded to the R transactivator, but several other herpesvirus and human promoters were nonresponsive. When the divergent promoter was linked to the EA-R gene as it is in the EBV genome, the R and Z transactivators also induced the expression of EA-R in cotransfected cells. This cytoplasmic early antigen is encoded by BHRF1 and may be anchored in intracellular membranes by a carboxy-terminal transmembrane region.     

Identification of the 12-O-tetradecanoylphorbol-13-acetate-responsive enhancer of the MS gene of the Epstein-Barr virus.

We previously located two 12-O-tetradecanoylphorbol-13-acetate (TPA)-responsive enhancers, MSTRE-I and MSTRE-II, in the upstream sequence of the MS gene of Epstein-Barr virus (Liu, Q., and Summers, W.C. (1989) J. Virol. 63, 5062-5068). The core sequence of the MSTRE-I enhancer is now determined to be between -718 and -708 of the upstream sequence of the MS gene. The activity of the enhancer is also sensitive to its immediate surrounding sequence on either side. A single copy of a 30-base pair (bp) fragment containing the MSTRE-I sequence was able to confer TPA responsiveness upon the MS promoter even in the absence of an AP-1 binding site. Multiple tandem copies of this 30-bp fragment, regardless of their relative orientations to each other, could function synergistically to enhance the MS promoter activity. At least two copies of the 30-bp fragment were required to bestow TPA induction upon the thymidine kinase gene promoter of herpes simplex virus type 1. The MSTRE-I sequence could also be bound by a Fos-GCN4 chimeric protein but with an affinity much lower than that between the chimeric protein and the AP-1 binding site. This MSTRE-I region has strong homology to one of the TPA-responsive elements (the ZII domain) in the upstream sequence of the EBV BZLF1 gene. In addition, a putative negative regulatory region or silencer was found immediately downstream of the MSTRE-I enhancer. This potential silencer region contains a 14-bp sequence that is homologous to the silencer consensus sequence of the BZLF1 gene. Therefore, the regulation of the MS gene may share the same pathway with the immediate early gene BZLF1.     

The Epstein-Barr virus BRLF1 immediate-early gene product transactivates the human immunodeficiency virus type 1 long terminal repeat by a mechanism which is enhancer independent.

The Epstein-Barr virus (EBV) immediate-early gene product, BRLF1, transactivates the human immunodeficiency virus type 1 (HIV-1) long terminal repeat. BRLF1-induced transactivation of HIV-1 promoter constructs is accompanied by an increase in plasmid mRNA and is reporter gene independent. Previously, BRLF1 transactivation of EBV promoters has been mapped to regions which function as enhancer elements. Deletional analysis demonstrates that BRLF1 transactivation of the HIV-1 promoter does not require the HIV-1 enhancer. Thus, the EBV BRLF1 gene product may transactivate by at least two different mechanisms, one mechanism involving certain enhancer elements and another mechanism which is enhancer independent.     

The Epstein-Barr virus (EBV) BZLF1 immediate-early gene product differentially affects latent versus productive EBV promoters.

The Epstein-Barr virus (EBV) BZLF1 gene product is thought to mediate the disruption of latent EBV infection. We have examined the regulatory effects of BZLF1 by studying its transactivating effects on seven different EBV promoters. We find that whereas the BZLF1 gene product increases the activity of the two early promoters, BMLF1 and BMRF1, it decreases the activity of three latent promoters (the BamHI-C and BamHI-W Epstein-Barr nuclear antigen promoters and the latent membrane protein promoter). The BZLF1-induced changes in promoter-directed chloramphenicol acetyltransferase activity occur in EBV-negative as well as EBV-positive cell lines and are accompanied by a similar change in chloramphenicol acetyltransferase mRNA. Deletion analysis of the BamHI Z fragment indicates that in a portion of the amino-terminal half of the BZLF1 gene product (amino acids 24 to 86) is not essential for positive transactivating effects but is required for down-regulating effects. Thus, different domains of the same EBV immediate-early gene product can either increase the function of EBV promoters active in productive infection or decrease the function of key promoters active in latent infection.     

The spliced BZLF1 gene of Epstein-Barr virus (EBV) transactivates an early EBV promoter and induces the virus productive cycle.

A spliced cDNA spanning the Epstein-Barr virus BZLF1 gene expresses the BZLF1 protein and is active in inducing the virus productive cycle. A deletion mutant which lacks the N-terminal half of the protein is inactive. Cotransfection experiments in EBV-negative B-lymphocyte cell lines demonstrated that the BZLF1 gene activates the promoter for the BSLF2 + BMLF1 gene in the absence of any other EBV gene product. These results confirmed that the spliced BZLF1 gene is the transactivating gene structure in BamHI-Z.     

Epstein-Barr virus (EBV)-negative B-lymphoma cell lines for clonal isolation and replication of EBV recombinants.

Previous experiments have demonstrated that positive selection markers recombined into the Epstein-Barr virus (EBV) genome enable the isolation of transforming or nontransforming mutant EBV recombinants in EBV-negative B-lymphoma (BL) cell lines (A. Marchini, J. I. Cohen, and E. Kieff, J. Virol. 66:3214-3219, 1992; F. Wang, A. Marchini, and E. Kieff, J. Virol. 65:1701-1709, 1991). However, virus has been recovered from a BL cell clone (BL41) infected with an EBV recombinant in only one instance (Wang et al., J. Virol. 65:1701-1709, 1991). We now compare the utility of four EBV-negative BL lines, BJAB, BL30, BL41, and Loukes, for isolating EBV recombinants and supporting their subsequent replication. Transforming or nontransforming EBV recombinants carrying a simian virus 40 promoter-hygromycin phosphotransferase (HYG) cassette were cloned by selecting newly infected BL cells for HYG expression. Most of the infected BL clones contained EBV episomes, and EBV gene expression was largely restricted to EBNA-1. Although the BJAB cell line was a particularly good host for isolating EBV recombinants (Marchini et al., J. Virol. 66:3214-3219, 1992), it was largely nonpermissive for virus replication, even in response to heterologous expression of the BZLF1 immediate-early transactivator. In contrast, approximately 50% of infected BL41, BL30, or Loukes cell clones responded to lytic cycle induction. Frequently, a substantial fraction of infected cells expressed the late lytic infection viral protein, gp350/220, and released infectious virus. Since BL cells do not depend on EBV for growth, transforming and nontransforming EBV recombinants were isolated and passaged.     

Use of adenovirus vectors expressing Epstein-Barr virus (EBV) immediate-early protein BZLF1 or BRLF1 to treat EBV-positive tumors

The Epstein-Barr virus (EBV) genome is present in a variety of tumor types, including virtually all undifferentiated nasopharyngeal carcinomas (NPC) and a portion of gastric carcinomas. The uniform presence of the EBV genome in certain tumors (versus only a very small number of normal B cells) suggests that novel therapies which specifically target EBV-positive cells for destruction might be effective for treating such tumors. Although the great majority of EBV-positive tumor cells are infected with one of the latent forms of EBV infection, expression of either viral immediate-early protein (BZLF1 or BRLF1) is sufficient to convert the virus to the lytic form of infection. Induction of the lytic form of EBV infection could potentially result in death of the tumor cell. Here we have examined the efficacy of adenovirus vectors expressing the BZLF1 or BRLF1 proteins for treatment of EBV-positive epithelial tumors. The BZLF1 and BRLF1 vectors induced preferential killing of EBV-positive, versus EBV-negative, gastric carcinoma cells in vitro. Infection of C18 NPC tumors (grown in nude mice) with either the BZLF1 or BRLF1 vector, but not a control adenovirus vector, induced expression of early lytic EBV genes in tumor cells. Injection of C18 tumors with the BZLF1 or BRLF1 adenovirus vector, but not the control vector, also significantly inhibited growth of the tumors in nude mice. The addition of ganciclovir did not significantly affect the antitumor effect of the BZLF1 and BRLF1 adenovirus vectors. These results suggest a potential cancer therapy against EBV-related tumors.     

EBNA-2 transactivates a lymphoid-specific enhancer in the BamHI C promoter of Epstein-Barr virus.

Among the few Epstein-Barr virus (EBV) genes expressed during latency are the Epstein-Barr nuclear antigens (EBNAs), at least one of which contributes to the ability of the virus to transform B lymphocytes. We have analyzed a promoter located in the BamHI-C fragment of EBV which is responsible for the expression of EBNA-1 in some cell lines. Deletion analysis of a 1.4-kb region 5' of the RNA start site has identified a 700-bp fragment that is required for optimal promoter activity in latently infected B lymphocytes, as shown by promoter constructs linked to the chloramphenicol acetyltransferase reporter gene. This fragment is also able to enhance activity, in an orientation-independent manner, of the simian virus 40 early promoter linked to the chloramphenicol acetyltransferase gene. The enhancer element has some constitutive activity in EBV-negative lymphoid cells, which is increased in the presence of the EBNA-2 gene product. Further deletions have shown that the EBNA-2-responsive region requires a 98-bp region that contains a degenerate octamer-binding motif. In epithelial cells there was no enhancer activity regardless of the presence of EBNA-2. These results demonstrate that BamHI-C promoter activity may be dependent not on an enhancer contained in the ori-P, as was previously assumed, but rather on EBNA-2 transactivation of this more proximal enhancer located in the upstream region of the BamHI C promoter itself.     

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.