Induction of the RelB NF-κB Subunit by the Cytomegalovirus IE1 Protein Is Mediated via Jun Kinase and c-Jun/Fra-2 AP-1 Complexes

Cytomegalovirus (CMV) is a betaherpesvirus that latently infects a majority of adults worldwide (24). Although most healthy immunocompetent individuals are asymptomatic (6), enhanced risk of atherosclerosis and restenosis after coronary angioplasty has been observed in patients with prior human CMV (HCMV) infection (23, 40). Infection by CMV has also been shown to stimulate migration and proliferation of smooth muscle cells (SMCs) in culture (38, 58, 60). HCMV has also been implicated in the transformation and pathogenesis of many malignancies (13, 19, 26, 46, 50, 53). Furthermore, increased morbidity and mortality is frequent in immunocompromised and immunosuppressed individuals, e.g., patients with AIDS or after transplantation (6). The CMV immediate-early protein 1 (IE1), a 491-amino-acid nuclear phosphoprotein, is one of two major products of the ie gene and is a predominant protein expressed during the immediate-early phase of CMV infection (22, 40). Although IE1 was first found to regulate viral gene expression by positive feedback regulation of the ie promoter (29), other functions have been reported recently, including the induction of NF-κB activity (14, 30, 47) and posttranscriptional activation of the c-Fos and c-Jun subunits of AP-1 (35). NF-κB/Rel is a family of dimeric transcription factors whose DNA-binding domains have considerable homology with an approximately 300-amino-acid region of the v-Rel oncoprotein and was thus termed the Rel homology region (28, 64). NF-κB plays a pivotal role in the control of cell proliferation, survival, neoplastic transformation, and immune and inflammation responses (25, 27, 43, 56). Members of the mammalian Rel family include p65 (RelA), RelB, c-Rel, p50, and p52 (28). In most non-B cells, NF-κB factors are inactive and sequestered in the cytoplasm by direct interaction with specific inhibitor proteins, termed IκBs, which need to be phosphorylated and degraded in order for NF-κB to transfer into the nucleus (12, 64). IE1 has been found to induce NF-κB activity, as judged by NF-κB element driven constructs, in fibroblasts and Jurkat T cells (47). Previous work from our laboratory found that IE1 selectively induces nuclear RelB and p50 in SMCs and NIH 3T3 cells (30). The increase in RelB protein mediated by IE1 could be related to an increase in steady-state relB mRNA levels. Although the relB promoter contains two proximal NF-κB elements (11), IE1 was unable to activate an NF-κB element driven construct in relB−/− mouse embryo fibroblasts. Thus, the data suggested that relB was the primary NF-κB target gene of IE1 activity (30), although the mechanism controlling relB induction by IE1 was unclear. Interestingly, RelB/p50 complexes are somewhat unique among the NF-κB family in that IκB-α binds only poorly (39), allowing them to almost freely translocate to the nucleus (20). In the mouse, high levels of RelB expression are restricted to specific regions of the thymus and spleen (65), and RelB complexes represent constitutive NF-κB activity in these tissues (66), suggesting a role for RelB in control of gene expression in these tissues. Importantly, RelB has recently been shown to display specificity of NF-κB regulated gene transcription (30). AP-1 is a transcription factor composed of members of the Jun and Fos families (2) that binds and activates transcription at TPA response elements (TRE), with the consensus sequence of 5′-TGACTCA-3′ (1). These proteins belong to the bZIP group of DNA-binding proteins, which associate to form a variety of homo- and heterodimers (2). Members of the Jun family include c-Jun, JunB, and JunD, and members of the Fos family include c-Fos, FosB, Fra-1, and Fra-2. The transcriptional activity of c-Jun is regulated by phosphorylation at Ser63 and Ser73 (9, 54). Extracellular signals, including growth factors, transforming oncoproteins, and UV irradiation, stimulate this phosphorylation of c-Jun, thereby activating c-Jun-dependent transcription (17, 35, 36). For example, the mitogen-activated protein kinase homologue JNK/SAPK binds to the N-terminal region of c-Jun and phosphorylates c-Jun at Ser63/73 (17, 36). Furthermore, Kim et al. (35) reported that HCMV IE1 protein induced AP-1 activity via a posttranscriptional, rather than transcriptional, mechanism. Here we have explored the mechanism of RelB induction by IE1. We report that IE1 activates JNK and thereby AP-1 activity, which leads to enhanced relB promoter transcription via binding to an upstream AP-1 element.