The OVERALL OBJECTIVE of the proposed investigation is to elucidate the role of the interferon-inducible RNA-specific adenosine deaminase (ADAR) in the actions which natural and recombinant interferons mediate on viral and host functions.
The SPECIFIC AIMS of our proposed continuation investigation of the mechanism of interferon (IFN) action are as follows: (1) To characterize the structural and functional organization of the 5'- flanking region of the human Adar gene. To further delineate the structure of the 5'-flanking regions of the Adar gene required for transcriptional activity, including the Pi promoter responsible for IFN-inducible expression of p150 ADAR1 and the Pc promoter responsible for constitutive expression of p110 ADAR. To characterize protein binding by DNase I footprint analysis and gel mobility shift assays. To elucidate the importance of potential protein binding sites in promoter activity through mutagenesis and transient transfection analyses. (2) To further characterize the biochemical and biophysical properties of the RNA-specific adenosine deaminase, ADAR. To characterize the substrate specificity of the ADAR-a. -2 and-c alternative splice-site variants, and to examine ability of adenovirus VA1 and vaccinia virus E3L gene products to antagonize the ADAR deaminase activity. TO study, through in vitro RNA selection approaches, the RNA structures capable of modulating deaminase activity. To examine the tissue and cell- type specificity of ADAR expression, and to characterize the structural basis and functional significance of ADAR protein-protein associations in uninfected and virus-infected cells. (3) To further characterize the expression of wild-type and mutant forms of ADAR deaminase cDNA in cells in culture, and to examine the roles of ADAR deaminase in the induction and action of IFN. To examine the ADAR expressing cell lines for their ability to support virus replication and protein synthesis, and for their phenotype and growth properties. To generate a targeted deletion of the mouse ADAR1 genomic locus, and to determine the effect of the mADAR1 mutation in viral infections and the interferon response. The health relatedness of the proposed research stems from the likelihood that the work may contribute to a better understanding of regulatory mechanisms involving RNA editing by adenosine deamination possibly operative in normal cells as well as virus-infected cells. Furthermore, the elucidation of the actions of interferon at the molecular level is of immediate importance in view of the potential applications of IFN in the clinic.
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