Multiple cis regulatory elements control RANTES promoter activity in alveolar epithelial cells infected with respiratory syncytial virus

Respiratory syncytial virus (RSV) produces intense pulmonary inflammation, in part through its ability to induce chemokine synthesis in infected airwa y epithelial cells, RANTES (regulated upon activation, normally T-cell expr essed and presumably secreted) is a CC chemokine which recruits and activat es monocytes, lymphocytes, and eosinophils, all cell types present in the l ung inflammatory infiltrate induced by RSV infection. In this study, we ana lyzed the mechanism of RSV-induced RANTES promoter activation in human type II alveolar epithelial cells (A549 cells). Promoter deletion and mutagenes is experiments indicate that RSV requires the presence of five different ci s regulatory elements, located in the promoter fragment spanning from -220 to +55 nucleotides, corresponding to NF-kappaB, C/EBP, Jun/CREB/ATF, and in terferon regulatory factor (IRF) binding sites, Although site mutations of the NF-KB, C/EBP, and CREB/AP-1 like sites reduce RSV-induced RANTES gene t ranscription to 50% or less, only mutations affecting IRF binding completel y abolish RANTES inducibility. Supershift and microaffinity isolation assay s were used to identify the different transcription factor family members w hose DNA binding activity was RSV inducible. Expression of dominant negativ e mutants of these transcription factors further established their central role in virus induced RANTES promoter activation. Our finding that the pres ence of multiple cis regulatory elements is required for full activation of the RANTES promoter in RSV-infected alveolar epithelial cells supports the enhanceosome model for RANTES gene transcription, which is absolutely depe ndent on binding of IRF transcription factors, The identification of regula tory mechanisms of RANTES gene expression is fundamental for rational desig n of inhibitors of RSV-induced lung inflammation.