INTERFERON-ALPHA INHIBITS THE MURINE CYTOMEGALOVIRUS IMMEDIATE-EARLY GENE-EXPRESSION BY DOWN-REGULATING NF-KAPPA-B ACTIVITY

Transcription of murine cytomegalovirus (MCMV) immediate-early (IE) ge nes is regulated by the interaction of cellular transcription factors with a strong viral enhancer controlling promoters flanking both sides of the regulatory sequence. We have previously demonstrated that inte rferon-alpha (IFN-alpha) inhibits MCMV replication by impairing the tr anscription of IE genes. To define the cis-acting elements and trans-a cting factors involved in this inhibition, permissive murine fibroblas ts were transfected with DNA constructs containing the chloramphenicol acetyl transferase reporter gene and portions of the IE enhancer. The region spanning -1185 to -259 relative to the IE1-3 promoter was suff icient to allow IFN-alpha-induced inhibition. Since this segment conta ins several NF-kappa B sites, cells were transfected with a construct containing three copies of NF-kappa B element in front of the homologo us minimal IE1-3 promoter. Upon IFN-alpha treatment the reporter gene activity was strongly reduced, indicating that NF-kappa B binding site is sufficient to confer inhibition. The specificity of this inhibitio n was demonstrated by the lack of a significant effect on the activity of DNA constructs containing either a mutated NF-kappa B trimer or an ATF/CRE trimer. Gel shift assays with NF-kappa B probes revealed that MCMV infection activated NF-kappa B proteins, whereas IFN-alpha treat ment significantly reduced their ability to bind NF-kappa B sites. In cotransfection experiments using various NF-kappa B subunit expression vectors and a reporter driven by three copies of an NF-kappa B elemen t, activation of NF-kappa B-dependent transcription was observed with expression of p65 or combinations of p50-p65. Taken as a whole, these results suggest that IFN-alpha inhibits MCMV IE gene enhancer activity by mechanisms that decrease the availability of virus-induced NF-kapp a B transcriptionally active in the nuclei of infected cells. (C) 1995 Academic Press, Inc.