CCAAT/Enhancer-binding Proteins α and/3 β Negatively Influence the Capacity of Tumor Necrosis Factor α to Up-regulate the Human Cytomegalovirus IE1/2 Enhancer/Promoter by Nuclear Factor κB during Monocyte Differentiation

Abstract

Recently we demonstrated that the ability of tumor necrosis factor α (TNFα) to stimulate the human cyto-megalovirus (HCMV) IE1/2 enhancer/promoter activity in myeloid progenitor-like cells decreases when these cells differentiate into promonocytic cells. In addition, TNFα stimulation in the progenitor-like cell line HL-60 was shown to be mediated by nuclear factor κB (NF-κB) activation and its binding to the 18-base pair sequence motifs of the IE1/2 enhancer. We demonstrate here that the cell differentiation-dependent reduction of TNFα stimulation is not due to insufficient NF-κB activation but correlates with increased synthesis of the monocyte differentiation-associated factors CCAAT/enhancer-binding protein (C/EBP) κ and β. Overexpression of C/EBPα/β in HL-60 cells, which normally produce only very small amounts of C/EBP, stimulated the basal activity of the promoter in the absence of NF-κB but suppressed the stimulatory effect of TNFα. A novel C/EBP-binding site was identified in the IE1/2 enhancer directly downstream of a NF-κB site. In order to understand the mechanisms of interaction, we used an IE1/2 promoter mutant that failed to bind C/EBP at this position and several constructs that contained exclusively NF-κB- and/or C/EBP-binding sites upstream of the minimal IE1/2 promoter. We could demonstrate that C/EBPα/β interacts with NF-κB p65 and displays inhibitory activity even in the absence of direct DNA binding by forming p65-C/EBP-containing protein complexes bound to the NF-κB site. Moreover, C/EBP binding to the DNA adjacent to NF-κB supports the down-regulatory effect of C/EBPs possibly due to stabilization of a multimeric NF-κB-C/EBP complex. Our results show that cell differentiation factors may interfere with TNFα-induced human cytomegalovirus gene (re)activation.