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) genes 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 interferon-alpha (IFN-alpha) inhibits MCMV replication by impairing the transcription of IE genes. To define the cis-acting elements and trans-acting factors involved in this inhibition, permissive murine fibroblasts were transferred with DNA constructs containing the chloramphenicol acetyl transferase reporter gene and portions of the IE enhanced. The region spanning -1185 to -259 relative to the IE1-3 promoter was sufficient to allow IFN-alpha-induced inhibition. Since this segment contains several NF-kappa B sites, cells were transfected with a construct containing three copies of NF-kappa B element in front of the homologous 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 inhibition 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 treatment 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 element, 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-kappa B transcriptionally active in the nuclei of infected cells.
Gribaudo
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G
Institute of Microbiology, University of Torino, Italy.
Ravaglia
S
S
Gaboli
M
M
Gariglio
M
M
Cavallo
R
R
Landolfo
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S
D016475:3T3 Cells; D000818:Animals; D002467:Cell Nucleus / Q000378:metabolism; D015500:Chloramphenicol O-Acetyltransferase / Q000032:analysis / Q000096:biosynthesis; D003587:Cytomegalovirus / Q000187:drug effects / Q000235:genetics / Q000502:physiology*; D004268:DNA-Binding Proteins / Q000096:biosynthesis / Q000187:drug effects; D004742:Enhancer Elements, Genetic / Q000187:drug effects; D015870:Gene Expression / Q000187:drug effects / Q000502:physiology; D015967:Gene Expression Regulation, Viral / Q000187:drug effects*; D017781:Genes, Immediate-Early / Q000187:drug effects*; D016679:Genome, Viral; D006801:Humans; D007370:Interferon Type I / Q000494:pharmacology*; D051379:Mice; D008807:Mice, Inbred BALB C; D008815:Mice, Inbred Strains; D016328:NF-kappa B / Q000378:metabolism*; D010957:Plasmids; D011994:Recombinant Proteins / Q000032:analysis / Q000096:biosynthesis; D016385:TATA Box; D015533:Transcriptional Activation / Q000187:drug effects; D014162:Transfection; D014779:Virus Replication / Q000187:drug effects*
D004268:DNA-Binding Proteins; D007370:Interferon Type I; D016328:NF-kappa B; D011994:Recombinant Proteins; D015500:Chloramphenicol O-Acetyltransferase