The RelA subunit of NF-B and the glucocorticoid receptor mutually repress each others transcriptional activity, thus providing a mechanism for immunosuppression. Deletion analysis of the glucocorticoid receptor has shown that the DNA binding domain and the ligand binding domain are essential components for repression. Here, we show by deletions and point mutations that both the Rel homology domain and the transactivation domains of RelA are required for repression of the transcriptional activity of the glucocorticoid receptor in intact cells. However, only the Rel homology domain of RelA was found to associate with the glucocorticoid receptor in vitro. RelA mutants, not able to repress glucocorticoid receptor activity, but still able to dimerize, behaved as transdominant inhibitors of the repressive activity of wild type RelA. Furthermore, we show that the 13 S E1A protein is able to interfere with the transrepressive activity of RelA. We propose that negative cross-talk between the glucocorticoid receptor and RelA is due to direct interaction via the Rel homology domain of RelA and the DNA binding domain of the glucocorticoid receptor in combination with interference by the transactivation domains of RelA with the transcriptional activity of the glucocorticoid receptor.

Repression of nuclear factor (NF)-kappaB-dependent gene expression is one of the key characteristics by which glucocorticoids exert their antiinflammatory and immunosuppressive effects. In vitro studies have shown protein-protein interactions between NF-kappaB and the glucocorticoid receptor, possibly explaining their mutual repression of transcriptional activity. Furthermore, glucocorticoid-induced transcription of IkappaBalpha was presented as a mechanism in mediation of immunosuppression by glucocorticoids. At present, the relative contribution of each mechanism has not been investigated. We show that dexamethasone induced IkappaBalpha gene transcription in human pulmonary epithelial A549 cells. However, this enhanced IkappaBalpha synthesis did not cause repression of NF-kappaB DNA-binding activity. In addition, dexamethasone was still able to inhibit the expression of NF-kappaB target genes (cyclooxygenase-2, intercellular adhesion molecule-1) in the absence of protein synthesis. Furthermore, we show that the antihormone RU486 did not induce IkappaBalpha expression. However, RU486 was still able to induce, albeit less efficiently, both glucocorticoid- and progesterone receptor-mediated repression of endogenous NF-kappaB target gene expression in A549 cells and the breast cancer cell line T47D, respectively. Taken together, these results indicate that induced IkappaBalpha expression accounts for only part of the repression of NF-kappaB activity by glucocorticoids and progestins. In addition, protein-protein interactions between NF-kappaB and the glucocorticoid or progesterone receptor, resulting in repression of NF-kappaB activity, seem also to be involved. We therefore conclude that NF-kappaB activity is repressed via a dual mechanism involving both protein-protein interactions and induction of IkappaBalpha.

The genomic localization of two immunodominant genes encoding two proteins of the Epstein-Barr virus capsid antigen (VCA) complex, VCA-p18 and VCA-p40, has been identified. For that purpose, lambda gtll-based cDNA libraries were constructed from HH514.c16 cells induced for virus production. The libraries were screened with a monoclonal antibody, EBV.OT41A, directed against VCA-p40 or with affinity-purified human antibodies against VCA-p18. Sequencing of the inserts of positive plaques showed that VCA-p18 and VCA-p40 are encoded within open reading frames (ORFs) BFRF3 and BdRF1, respectively. Peptide scanning analysis of the predicted protein of ORF BdRF1 resulted in defining the epitope of monoclonal antibody EBV.OT41A at the C-terminal region. The dominant VCA-p18 reactivity of human sera can be completely inhibited by preadsorption with Escherichia coli-expressed BFRF3-13-galactosidase. Serum of a rabbit immunized with BFRF3-13galactosidase reacts with a VCA-specific protein of 18 kDa. In addition, BFRF3-1-galactosidase affinity-purified antibodies react with VCA-p18 of virus-producing cells (HH514.c16). Complete inhibition of viral DNA polymerase activity by phosphonoacetic acid is associated with the absence of RNAs and protein products of both ORFs, indicating that VCA-p18 and VCA-p40 are true late antigens. Epstein-Barr virus (EBV) is a ubiquitous member of the human herpesvirus family and is associated with a still increasing number of disease syndromes. EBV-specific diagnosis is based on the combination of different serological parameters. These parameters include the detection of antibodies of different classes against early antigens (EA), Epstein-Barr nuclear antigens (EBNA), viral capsid antigens (VCA), and membrane antigens. Polypeptides belonging to the EBNA and EA complexes and their role in immunodiagnosis has been studied extensively (24, 29, 39). In contrast, only few studies have addressed in detail the human immune response to viral structural polypeptides. Studies of the structural polypeptides are hampered by the molecular complexity of the VCA complex, the polyspecificity of the human sera used to detect them, and the lack of an efficient virus production system. EBV virions may consist of some 30 polypeptides (9). Localization within the virion and a role in penetration, assembly, and budding have been suggested for only a few. The viral capsid may be composed of at least seven proteins (7), of which the major capsid protein encoded by open reading frame (ORF) BcLF1 (37) and a protein of 36 kDa encoded by BGLF2 (5, 33) have been described in detail. Another VCA protein, gpllO, is encoded within the BALF4 reading frame (34). This protein remains associated with cellular cytoplasmic and nuclear membranes and is not associated with the virion (15). Recently, we have identified two new immunologically dominant VCA proteins with molecular sizes of 18 and 40 kDa (36). These two proteins are structurally associated with the viral capsid and are recognized by antibodies from almost all EBV carriers.

The all-trans retinoic acid and 9-cis retinoic acid receptors (RAR and RXR, respectively) belong to a family of ligand inducible transcription factors, which exert their effect via binding to hormone response elements. Both are members of the class II sub-family of nuclear receptors, which bind DNA as dimers, on tandem repeats of a hexamer motif separated by a variable spacer. The variability in spacer length and the head-to-tail organization of the hormone response elements result in different protein-protein interactions in each of the complexes. We show that the zinc-coordinating loop regions of RXR and RAR DNA-binding domains exhibit dynamics on the millisecond to microsecond time scale. The highly dynamic second zinc finger of RXR constitutes the primary protein-protein interface in many nuclear receptor assemblies on DNA. Dynamics is also observed in the first and second zinc fingers of RAR, which are implicated in dimeric interactions with RXR on response elements with spacers of 5 base pairs and 1 base pair, respectively. The striking correspondence between the regions that exhibit conformational exchange and the dimer interfaces of the proteins complexed with DNA suggests a functional role for the dynamics. The observed flexibility may allow the proteins to adapt to various partners and with different orientations upon assembly on DNA. Furthermore, the more extensive dynamics observed for RXR may reflect the greater ability of this protein to modulate its interaction surface since it participates in a wide variety of receptor complexes.