Interferons induce transcription of a diverse family of genes whose protein products are believed to mediate the inhibition of cell growth and viral replication, and regulation of cell differentiation and immune functions. A well conserved cis-acting IFN stimulated response element (ISRE), has been identified in the 5' flanking region of many interferon stimulated gene (ISG) promoters. IFN-alpha signals cells through tyrosine kinase mediated phosphorylation of a transcription factor complex (ISGF3) that binds to the ISRE. However, biochemical and genetic evidence suggests the existence of multiple intracellular signaling pathways for IFN-alpha. Our major goal is to understand the basic mechanisms underlying IFN-alpha signal transduction through alternate pathways. Such information should prove useful in designing new therapeutic approaches using IFNs and other cytokines. We have provided direct evidence for the existence of alternate signal transduction pathways for IFN-alpha using biochemical, viral inhibitor, and mutant cell line approaches. We propose to define the sets of genes utilizing the alternate pathways and characterizing at a biochemical and molecular level, the different components of these pathways. To achieve our goal, we propose the following specific aims: 1) To identify and characterize alternate pathways for IFN-alpha transduction we will use cell lines with defined mutations in ISGF3 dependent signaling. We will characterize the IFN-alpha response in cells where ISGF3-dependent signaling is inhibited by adenovirus 5 E1A production. We will study the IFN-induction of the IRF-1 gene in U2A cells which are defective in ISGF3-dependent signaling and use a PCR approach to identify and clone other mRNAs which are induced in U2A, 293 and E1A-expressing HeLa cells in response to IFN-alpha. To isolate and characterize mutant cell lines which are unresponsive to IFN-alpha, we will generate new mutant cell lines by selecting for IFN mediated expression of drug selectable markers transcribed from the ISGF3- independent IRF-1 promoter. 2) To define at a biochemical level the action of unique components which may be involved in both ISGF3 -dependent and -independent signaling we will identify components that may interact with the IFN-alpha receptor using affinity chromatography and co-immunoprecipitation of extracts prepared from normal and mutant cell lines. To define the mode of action of the ISRE and IR element binding protein, IBF-1, we will use transfection assays, cell-free transcription reactions and the isolation of genetic suppressor elements.
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