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.

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.

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) 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.

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.