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

Abstract

We recently demonstrated that the cytomegalovirus (CMV) immediate-early 1 (IE1) protein induces transcription of the gene encoding the RelB NF-κB subunit. The mechanism of this activation has been explored here. We report that the induction of therelBpromoter by IE1 protein is mediated via activation of JNK and AP-1. The region controllingrelBpromoter induction was mapped to the upstream ∼600-bp region between −1694 and −1096 bp. IE1 stimulated AP-1 activity in NIH 3T3 cells. Competition electrophoretic mobility shift assay (EMSA) confirmed the presence of one bona fide AP-1 element centered at −1503 bp. Introduction of a G-to-C mutation in the AP-1 binding site within the distal region of therelBpromoter eliminated its activation by IE1 in both NIH 3T3 fibroblasts and vascular smooth muscle cells (SMCs). Supershift EMSA identified c-Jun, Fra-2, and c-Fos in AP-1 binding complexes in IE1 transfected NIH 3T3 cells. IE1 induced c-Jun phosphorylation, and treatment with SP600125, a selective JNK inhibitor, as well as overexpression of JNK-binding domain of JIP1, blocked IE1-mediated induction of AP-1 andrelBpromoter activity in NIH 3T3 cells and SMCs. Ectopic expression of c-Jun plus Fra-2, but not c-Fos, inducedrelBpromoter activity. TherelBpromoter has two proximal NF-κB elements, and c-Jun/Fra-2 worked in synergy with p50/p65 NF-κB complexes. Overall, these findings demonstrate for the first time the role of AP-1 in transcriptional regulation of a gene encoding an NF-κB subunit, and its involvement in induction of RelB activity by the CMV IE1 protein.