The bZIP transactivator of Epstein-Barr virus, BZLF1, functionally and physically interacts with the p65 subunit of NF-kappa B.

The Epstein-Barr virus (EBV) BZLF1 (Z) immediate-early transactivator initiates the switch between latent and productive infection in B cells. The Z protein, which has homology to the basic leucine zipper protein c-Fos, transactivates the promoters of several replicative cycle proteins. Transactivation efficiency of the EBV BMRF1 promoter by Z is cell type dependent. In B cells, in which EBV typically exists in a latent form, Z activates the BMRF1 promoter inefficiently. We have discovered that the p65 component of the cellular factor NF-kappa B inhibits transactivation of several EBV promoters by Z. Furthermore, the inhibitor of NF-kappa B, I kappa B alpha, can augment Z-induced transactivation in the B-cell line Raji. Using glutathione S-transferase fusion proteins and coimmunoprecipitation studies, we demonstrate a direct interaction between Z and p65. This physical interaction, which requires the dimerization domain of Z and the Rel homology domain of p65, can be demonstrated both in vitro and in vivo. Inhibition of Z transactivation function by NF-kappa B p65, or possibly by other Rel family proteins, may contribute to the inefficiency of Z transactivator function in B cells and may be a mechanism of maintaining B-cell-specific viral latency.

The cellular oncogene c-myb can interact synergistically with the Epstein-Barr virus BZLF1 transactivator in lymphoid cells.

Regulation of replicative functions in the Epstein-Barr virus (EBV) genome is mediated through activation of a virally encoded transcription factor, Z (BZLF1). We have shown that the Z gene product, which binds to AP-1 sites as a homodimer and has sequence similarity to c-Fos, can efficiently activate the EBV early promoter, BMRF1, in certain cell types (i.e., HeLa cells) but not others (i.e., Jurkat cells). Here we demonstrate that the c-myb proto-oncogene product, which is itself a DNA-binding protein and transcriptional transactivator, can interact synergistically with Z in activating the BMRF1 promoter in Jurkat cells (a T-cell line) or Raji cells (an EBV-positive B-cell), whereas the c-myb gene product by itself has little effect. The simian virus 40 early promoter is also synergistically activated by the Z/c-myb combination. Synergistic transactivation of the BMRF1 promoter by the Z/c-myb combination appears to involve direct binding by the Z protein but not the c-myb protein. A 30-bp sequence in the BMRF1 promoter which contains a Z binding site (a consensus AP-1 site) is sufficient to transfer high-level lymphoid-specific responsiveness to the Z/c-myb combination to a heterologous promoter. That the c-myb oncogene product can interact synergistically with an EBV-encoded member of the leucine zipper protein family suggests c-myb is likely to engage in similar interactions with cellularly encoded transcription factors.

Killing Epstein-Barr virus-positive B lymphocytes by gene therapy: comparing the efficacy of cytosine deaminase and herpes simplex virus thymidine kinase.

Epstein-Barr virus (EBV)-positive lymphomas are frequent among immunosuppressed patients. We have examined the feasibility of killing EBV-immortalized B lymphocytes by gene transfer involving the use of "suicide" genes whose expression in target cells renders them susceptible to killing by a prodrug. We examined two gene/prodrug pairs: the Escherichia coli cytosine deaminase (CD) gene with the prodrug 5-fluorocytosine (5-FC), and the herpes simplex virus thymidine kinase (HSV-TK) gene with the prodrug ganciclovir. Retroviral vectors and drug selection were used to obtain CD or HSV-TK expression in cells. Both the CD/5-FC and the HSV-TK/ganciclovir combinations yielded substantial killing of EBV-immortalized B lymphocytes in vitro, although the CD/5-FC regimen had a significantly greater therapeutic margin than the HSV-TK/ganciclovir combination. The CD/5-FC pair, but not the HSV-TK/ganciclovir pair, was shown to have a "bystander killing effect" in vitro. When only 30% of the cells expressed the suicide gene, scid mouse tumors regressed in both the CD/5-FC regimen and the HSV-TK/ganciclovir regimen, documenting an in vivo bystander effect with both regimens. However, a greater percentage of tumors completely regressed with the CD/5-FC regimen. Overall, the sum of our data indicates that the CD/5-FC combination is the more promising regimen for treatment of EBV-associated lymphomas in vivo.

ASC-dependent RIP2 kinase regulates reduced PGE2 production in chronic periodontitis.

Levels of prostaglandin E(2) (PGE(2)) and its processing enzyme, prostaglandin-endoperoxide-synthase-2/ cyclooxygenase-2 (PTGS2/COX-2), are elevated in actively progressing periodontal lesions, but suppressed in chronic disease. COX-2 expression is regulated through inflammatory signaling that converges on the mitogen-activated protein kinase (MAPK) pathway. Emerging evidence suggests a role for the inflammatory adaptor protein, ASC/Pycard, in MAPK activation. We postulated that ASC may represent a mediator of the MAPK-mediated regulatory network of PGE(2) production. Using RNAi-mediated gene slicing, we demonstrated that ASC regulates COX-2 expression and PGE(2) production in THP1 monocytic cells following infection with Porphyromonas gingivalis (Pg). Production of PGE(2) did not require the inflammasome adaptor function of ASC, but was dependent on MAPK activation. Furthermore, the MAP kinase kinase kinase CARD domain-containing protein RIPK2 was induced by Pg in an ASC-dependent manner. Reduced ASC and RIPK2 levels were revealed by orthogonal comparison of the expression of the RIPK family in ASC-deficient THP1 cells with that in chronic periodontitis patients. We show that pharmacological inhibition of RIPK2 represses PGE(2) secretion, and RNAi-mediated silencing of RIPK2 leads to diminished MAPK activation and PGE(2) secretion. These findings identify a novel ASC-RIPK2 axis in the generation of PGE(2) that is repressed in patients diagnosed with chronic adult periodontitis.

The bZip dimerization domain of the Epstein-Barr virus BZLF1 (Z) protein mediates lymphoid-specific negative regulation.

The Epstein-Barr virus (EBV) immediate-early (IE) protein, BZLF1 (Z), initiates the switch from latent to lytic infection, Z transactivation of an early viral promoter, BMRF1, is relatively inefficient in lymphoid cells (compared with epithelial cells), unless the other EBV IE protein, BRLF1, is also present. Cellular proteins, including the p65 component of NF-kappa B, have been shown to interact directly with Z in vitro through the bZip dimerization domain and inhibit Z-induced transactivation. Here we precisely define a residue within the bZip dimerization domain of Z (amino acid 200) which is required for interaction in vitro with the p65 component of NF-kappa B, but is not essential for Z homodimerization. In lymphoid cells, a Z mutant which has been altered at amino acid 200 (tyrosine to glutamic acid) transactivates both the early BMRF1 promoter and the immediate-early BZLF1 promoter (Zp) four- to fivefold better than wild-type Z. In contrast, mutation of amino acid 200 does not affect Z transactivator function in epithelial cells. The results suggest that Z function is specifically inhibited by a lymphoid-specific protein(s) through amino acid 200 in the bZip dimerization domain. Modulation of Z's activator function may help to regulate the stringency of viral latency in lymphocytes.

The Zif268 cellular transcription factor activates expression of the Epstein-Barr virus immediate-early BRLF1 promoter.

The Epstein-Barr virus immediate-early protein BZLF1 mediates the switch from latent to lytic infection. BZLF1 transcription can be derived from either the BZLF1 promoter or the BRLF1 promoter (Rp). Productive viral infection of EBV-infected B cells can be induced by 12-O-tetradecanoylphorbol-13-acetate (TPA) treatment, as well as cross-linking of surface immunoglobulin with antiimmunoglobulin antibody. Both TPA and antiimmunoglobulin antibody are known to activate expression of the cellular transcription factor Zif268 in B cells. In this study, we have examined the regulation of BZLF1 transcription by Zif268. We show that Rp (but not the BZLF1 promoter) is activated by Zif268. Bacterially synthesized Zif268 binds strongly to an upstream sequence in the Rp promoter (located from -131 to -123 relative to the start site) and more weakly to a proximal sequence (-49 to -40). Zif268 activation of Rp requires these two Zif268 binding sites. TPA treatment of B cells induces the expression of Zif268 protein, which binds to Rp. Furthermore, TPA activation of Rp requires the upstream Zif268 site. These findings indicate that Zif268 can activate a critical Epstein-Barr virus immediate-early promoter and, therefore, may play a key role in the regulation of viral latency.

The cellular YY1 transcription factor binds a cis-acting, negatively regulating element in the Epstein-Barr virus BRLF1 promoter.

Disruption of Epstein-Barr virus latency is induced by expression of either the BZLF1 (in B cells and epithelial cells) or BRLF1 (in epithelial cells only) immediate-early protein. Regulation of BZLF1 and BRLF1 transcription may therefore modulate the stringency of viral latency. The cellular transcription factor YY1 negatively regulates BZLF1 transcription. Here we show that the BRLF1 promoter (Rp) sequences from -206 to -227 (relative to the mRNA start site) and from -7 to +6 are directly bound by YY1. Mutation of the upstream YY1 binding site increases constitutive Rp activity in epithelial cells and B cells, while mutation of the downstream YY1 binding site does not significantly affect Rp activity. Negative regulation of BZLF1 and BRLF1 transcription by YY1 may act to maintain viral latency.

Regulation of NAD metabolism in Salmonella typhimurium: genetic analysis and cloning of the nadR repressor locus.

The nadR locus (99 min) controls the transcription of several genes involved with either the biosynthesis (nadAB) or recycling (pncB) of NAD in Salmonella typhimurium. Point mutations in this locus were found to cause defects either in the transport of nicotinamide mononucleotide (PnuA-), the regulation of nadAB (NadR-) or both transport and regulation (PnuA-NadR-). Deletions or insertions into nadR always resulted in the PnuA- NadR- phenotypes. Merodiploids constructed with various combinations of PnuA-, NadR- or PnuA- NadR- strains indicate a single complementation group. The results suggest the NadR product is a bifunctional regulatory protein. Operon fusions to lacZ (nadR :: Mud1-8) were used to show that nadR is not autoregulated and is transcribed in a clockwise direction. The gene was also cloned and located within a 2 kb EcoR1-Bg/II fragment.

Identification of transactivator and nuclear localization domains in the Epstein-Barr virus DNA polymerase accessory protein, BMRF1.

The Epstein-Barr virus (EBV) BMRF1 gene product is an essential component of the viral DNA polymerase and is absolutely required for lytic virus replication. In addition to its polymerase accessory protein function, we recently demonstrated that BMRF1 is a transactivator, inducing expression of the essential oriLyt promoter, BHLF1. However, the regions of BMRF1 required for transactivation of BHLF1 are unknown. Here we demonstrate that the carboxy-terminal portion of the BMRF1 protein (amino acids 378404), although not required for DNA binding or polymerase processivity function, is required for transactivator function as well as nuclear localization. Site-directed mutagenesis of this region allowed us to separate the transactivator and nuclear localization motifs of BMRF1. The two DNA-binding domains of BMRF1 are also required for efficient transactivation of the BHLF1 promoter.

Epstein-Barr viral latency is disrupted by the immediate-early BRLF1 protein through a cell-specific mechanism.

Epstein-Barr virus (EBV), the causative agent of infectious mononucleosis, is a human herpesvirus associated with epithelial cell malignancies (nasopharyngeal carcinoma) as well as B-cell malignancies. Understanding how viral latency is disrupted is a central issue in herpesvirus biology. Epithelial cells are the major site of lytic EBV replication within the human host, and viral reactivation occurs in EBV-associated nasopharyngeal carcinomas. It is known that expression of a single viral immediate-early protein, BZLF1, is sufficient to initiate the switch from latent to lytic infection in B cells. Cellular regulation of BZLF1 transcription is therefore thought to play a key role in regulating the stringency of viral latency. Here we show that, unexpectedly, expression of another viral immediate-early protein, BRLF1, can disrupt viral latency in an epithelial cell-specific fashion. Therefore, the mechanisms leading to disruption of EBV latency appear to be cell-type specific.

Epstein-Barr virus immediate-early protein BRLF1 interacts with CBP, promoting enhanced BRLF1 transactivation.

The Epstein-Barr virus (EBV) immediate-early protein BRLF1 is a transcriptional activator that mediates the switch from latent to lytic viral replication. Many transcriptional activators function, in part, due to an interaction with histone acetylases, such as CREB-binding protein (CBP). Here we demonstrate that BRLF1 interacts with the amino and carboxy termini of CBP and that multiple domains of the BRLF1 protein are necessary for this interaction. Furthermore, we show that the interaction between BRLF1 and CBP is important for BRLF1-induced activation of the early lytic EBV gene SM in Raji cells.

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.

The Epstein-Barr virus (EBV) DNA polymerase accessory protein, BMRF1, activates the essential downstream component of the EBV oriLyt.

The EBV DNA polymerase accessory protein, BMRF1, is an essential component of the viral DNA polymerase and is required for lytic EBV replication. In addition to its polymerase accessory protein function, we have recently reported that BMRF1 is a transcriptional activator, inducing expression of the essential oriLyt promoter, BHLF1. Here we have precisely mapped the BMRF1-response element in the BHLF1 promoter. We demonstrate that a region of oriLyt (the "downstream component"), previously shown to be one of two domains absolutely essential for oriLyt replication, is required for BMRF1-induced activation of the BHLF1 promoter. Furthermore, the downstream component of oriLyt is sufficient to confer BMRF1-responsiveness to a heterologous promoter. The downstream component contains Sp1 binding sites, and confers Sp1-responsiveness to a heterologous promoter. A series of plasmids containing various protions of the oriLyt downstream component were constructed and analyzed for their ability to respond to the BMRF1 versus Sp1 transactivators. Although the BMRF1-responsive region of the downstream component overlaps the Sp1-responsive element, certain oriLyt sequences required for maximal BMRF1-responsiveness were not required for maximal Sp1-responsiveness. In particular, a site-directed mutation altering the downstream component sequence GATGG (located from -588 to -592 relative to the BHLF1 transcription initiation site) did not affect Sp1-responsiveness, but reduced BMRF-1-responsiveness by 75% and abolished oriLyt replication. Although BMRF1 possesses nonspecific DNA binding activity, were unable to demonstrate specific BMRF1 binding to the downstream component of oriLyt. Our results suggest that BMRF1-induced activation of the essential downstream component of oriLyt may play an important role in oriLyt replication.

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) BMRF1 promoter for early antigen (EA-D) is regulated by the EBV transactivators, BRLF1 and BZLF1, in a cell-specific manner.

The Epstein-Barr virus early antigen diffuse component (EA-D) is essential for Epstein-Barr virus DNA polymerase activity, and its activity is suppressed during latent infection. We investigated the regulation of the promoter (BMRF1) for this early gene by studying its responsiveness in vitro to two immediate-early viral transactivators, BZLF1 (Z) and BRLF1 (R), focusing on the differences in response in lymphoid cells and epithelial cells. In lymphoid cells, Z or R alone produced only small increases in EA-D promoter activity, whereas both transactivators together produced a large stimulatory effect. In epithelial cells, the Z transactivator alone produced maximal stimulation of the EA-D promoter; the effect of R and Z together was no greater than that of Z alone. Deletional analysis and site-directed mutagenesis of the EA-D promoter demonstrated that in epithelial cells the potential AP-1 binding site plays an essential role in Z responsiveness, although sequences further upstream are also important. In lymphoid cells, only the upstream sequences are required for transactivation by the Z/R combination, and the AP-1 site is dispensable. These data suggest that EA-D (BMRF1) promoter regulation by Z and R is cell type specific and appears to involve different mechanisms in each cell type.

The Epstein-Barr virus immediate-early promoter BRLF1 can be activated by the cellular Sp1 transcription factor.

Disruption of viral latency in Epstein-Barr virus-infected cells is mediated through the activation of the BZLF1 (Z) immediate-early gene product. The Z protein can be derived from either of two promoters: the BZLF1 promoter, which directs transcription of a 1.0-kb mRNA encoding the Z gene product alone, or the upstream BRLF1 promoter, which directs transcription of a 2.8-kb bicistronic mRNA encoding the BRLF1 and BZLF1 immediate-early proteins. In this study we have examined the regulation of the BRLF1 promoter by viral and cellular factors. We found that the BRLF1 promoter is autoregulated by the BRLF1 transactivator through a nonbinding mechanism. We show that the BRLF1 (but not the BZLF1) promoter is highly responsive to the Sp1 transcription factor. Sp1 activation of the BRLF1 promoter is mediated through a consensus Sp1-binding site located from -39 to -44 (relative to the mRNA start site). We demonstrate that the BRLF1 promoter has high constitutive activity in C-33 cells (an epithelial cell line) and that the proximal Sp1-binding site is required for this activity. Despite the ubiquitous presence of Sp1 in many cell types, we found that the BRLF1 promoter has essentially no activity in lymphoid cell lines, suggesting that factors other than Sp1 may negatively regulate the BRLF1 promoter in these cells. Our findings demonstrate that the two potential promoters directing BZLF1 transcription are differentially regulated and that Sp1 can activate the BRLF1 promoter but not the BZLF1 promoter.

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 Epstein-Barr virus immediate-early gene product, BMLF1, acts in trans by a posttranscriptional mechanism which is reporter gene dependent.

In DNA cotransfection experiments, the Epstein-Barr virus immediate-early gene product, BMLF1, stimulated the chloramphenicol acetyltransferase (CAT) activity of both latent and productive EBV promoters linked to CAT. This BMLF1-induced increase in CAT activity was out of proportion to the effect on CAT mRNA, suggesting a posttranscriptional mechanism. Furthermore, when growth hormone was used as a reporter gene instead of CAT, BMLF1 no longer functioned. Thus, the BMLF1 effect was reporter-gene dependent. The effect of the BMLF1 gene product does not then appear to be directed at promoter activation, but instead may function to increase the level of an as yet unidentified protein(s) required for Epstein-Barr virus infection.

Direct BRLF1 binding is required for cooperative BZLF1/BRLF1 activation of the Epstein-Barr virus early promoter, BMRF1.

Disruption of Epstein-Barr virus (EBV) latency is mediated through the activation of the viral immediate-early proteins, BZLF1 (Z) and BRLF1 (R).i.; (Chevallier-Greco, A., et al., (1986) EMBO J., 5, 3243-9; Countryman, and Miller, G. (1985) Proc. Natl. Acad. Sci. USA, 82, 4085-4089). We have previously demonstrated that these proteins cooperatively activate the EBV early promoter BMRF1 in lymphoid cells but not in epithelial cells. Although cooperative transactivation by these proteins has been demonstrated with a number of EBV promoters, the mechanism of this interaction is not well understood. We now show that the cooperative activation of the BMRF1 promoter by Z-plus-R requires an intact R binding site and at least one functional Z response element (ZRE). Despite the presence of an R binding site, the BMRF1 promoter is only moderately responsive to R alone in either HeLa or Jurkat cells. Efficient activation of the BMRF1 promoter by Z alone in HeLa cells requires two ZREs (located at -59 and -106), whereas two additional Z binding sites (located at -42 and -170) contribute very little to Z-induced activation. In the absence of ZREs, Z acted as a repressor of R-induced transactivation. These observations, along with observations made by other investigators (Giot, J.F. et al., (1991) Nucleic Acids Res., 19, 1251-8), suggest that Z-plus-R cooperative activation is dependent upon 1) direct binding by R and Z to responsive promoter elements and 2) contributions by cell-specific factors.

Epstein-Barr virus immediate-early proteins BZLF1 and BRLF1 activate the ATF2 transcription factor by increasing the levels of phosphorylated p38 and c-Jun N-terminal kinases.

Expression of either Epstein-Barr virus (EBV) immediate-early protein BZLF1 (Z) or BRLF1 (R) is sufficient to convert EBV infection from the latent to lytic form. Disruption of viral latency requires transcriptional activation of the Z and R promoters. The Z and R proteins are transcriptional activators, and each immediate-early protein activates expression of the other immediate-early protein. Z activates the R promoter through a direct binding mechanism. However, R does not bind directly to the Z promoter. In this study, we demonstrate that the ZII element (a cyclic AMP response element site) in the Z promoter is required for efficient activation by R. The ZII element has been shown to be important for induction of lytic EBV infection by tetradecanoyl phorbol acetate and surface immunoglobulin cross-linking and is activated by Z through an indirect mechanism. We demonstrate that both R and Z activate the cellular stress mitogen-activated protein (MAP) kinases, p38 and JNK, resulting in phosphorylation (and activation) of the cellular transcription factor ATF2. Furthermore, we show that the ability of R to induce lytic EBV infection in latently infected cells is significantly reduced by inhibition of either the p38 kinase or JNK pathways. In contrast, inhibition of stress MAP kinase pathways does not impair the ability of Z expression vectors to disrupt viral latency, presumably because expression of Z under the control of a strong heterologous promoter bypasses the need to activate Z transcription. Thus, both R and Z can activate the Z promoter indirectly by inducing ATF2 phosphorylation, and this activity appears to be important for R-induced disruption of viral latency.