No abstract

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.

We report here the complete coding sequence of a 203 cDNA, a member of the interferon-inducible Ifi 200 gene family. By combining reverse-transcriptase PCR and rapid amplification of cDNA ends (RACE) techniques we have obtained a 3.8-kb cDNA corresponding to a 203 mRNA. When used as a probe in northern analysis, its 3' segment hybridized to a 3.8-kb interferon-inducible mRNA, whereas the S'-end additionally hybridized to a less abundant interferon-inducible 1 .8-kb mRNA. Nucleotide sequence analysis revealed that the two mRNAs share the 5'-untranslated region and the same open reading frame, which encodes a hydrophilic protein composed of 408 amino acids. The difference between them is due to a 3'-untranslated region extended by alternative polyadenylation site selection. Furthermore, 203 mRNA was found to be inducible by interferon-a in various murine cell lines. Using polyclonal antibodies raised against a segment specific for the 203 protein, we established that p203 protein levels increase on treatment with interferon-a in murine fibroblasts and that p203 is located in the nucleus. MATERIALS AND METHODSCells and culture conditions. Balb/c 3T3, NIH 3T3 and BLKS cells (murine fibroblasts) were grown as monolayers in Dulbecco's modified Eagle's medium (GibcoiBRL) supplemented with 10% calf serum (Gibco/BRL; Balb/c 3T3 and NIH 3T3 cells) or 10% fetal calf serum (Gibco/BRL; BLKS cells). L12R4 (murine T lymphoma cell line) and L1210 (murine pre-B lymphoma cell line) cells were grown in RPMI 1640 me-

No abstract