VIRAL INDUCTION OF THE HUMAN BETA-INTERFERON PROMOTER - MODULATION OFTRANSCRIPTION BY NF-KAPPA-B REL PROTEINS AND INTERFERON REGULATORY FACTORS/

Multiple regulatory domains within the -100 region of the beta interfe ron (IFN-beta) promoter control the inducible response of the IFN gene to virus infection. In this study, we demonstrate that the formation of NF-kappa B specific complexes on the positive regulatory domain II (PRDII) precedes the onset of detectable IFN-beta transcription in Sen dai virus-infected cells. By using NF-kappa B subunit-specific antibod ies, a temporal shift in the composition of NF-kappa B subunits in ass ociation with the PRDII domain is detected as a function of time after virus infection. Furthermore, a virus-induced degradation of I kappa B alpha (MAD3) protein is observed between 2 and 8 h after infection; at later times, de novo synthesis of I kappa B alpha restores I kappa B alpha to levels found in uninduced cells and correlates with the dow n regulation of IFN-beta transcription. In cotransfection experiments using various NF-kappa B subunit expression plasmids and two copies of PRDII/NF-kappa B linked to a chloramphenicol acetyltransferase report er gene, we demonstrate that expression of p65, c-Rel, or p50 or combi nations of p50-p65 and p65-c-Rel differentially stimulated PRDII-depen dent transcription. Coexpression of I kappa B alpha completely abrogat ed p65-, c-Rel-, or p65-p50-induced gene activity. When the entire IFN -beta promoter (-281 to f19) was used in coexpression studies, synergi stic stimulation of IFN-beta promoter activity was obtained when NF-ka ppa B subunits were coexpressed together with the IFN regulatory facto r 1 (IRF-1) transcription factor. Overexpression of either I kappa B o r the IRF-2 repressor was able to abrogate inducibility of the IFN-bet a promoter. Thus, multiple regulatory events-including differential ac tivation of DNA-binding NF-kappa B heterodimers, degradation of I kapp a B alpha, synergistic interaction between IRF-1 and NF-kappa B, and d ecreased repression by I kappa B and IRF-2-are all required for the tr anscriptional activation of the IFN-beta promoter.