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.
|Tan, Meng How; Li, Qin; Shanmugam, Raghuvaran et al. (2017) Dynamic landscape and regulation of RNA editing in mammals. Nature 550:249-254|
|George, Cyril X; Ramaswami, Gokul; Li, Jin Billy et al. (2016) Editing of Cellular Self-RNAs by Adenosine Deaminase ADAR1 Suppresses Innate Immune Stress Responses. J Biol Chem 291:6158-68|
|George, Cyril X; Samuel, Charles E (2015) STAT2-dependent induction of RNA adenosine deaminase ADAR1 by type I interferon differs between mouse and human cells in the requirement for STAT1. Virology 485:363-70|
|Pfaller, Christian K; Mastorakos, George M; Matchett, William E et al. (2015) Measles Virus Defective Interfering RNAs Are Generated Frequently and Early in the Absence of C Protein and Can Be Destabilized by Adenosine Deaminase Acting on RNA-1-Like Hypermutations. J Virol 89:7735-47|
|Pfaller, Christian K; Radeke, Monte J; Cattaneo, Roberto et al. (2014) Measles virus C protein impairs production of defective copyback double-stranded viral RNA and activation of protein kinase R. J Virol 88:456-68|
|John, Lijo; Samuel, Charles E (2014) Induction of stress granules by interferon and down-regulation by the cellular RNA adenosine deaminase ADAR1. Virology 454-455:299-310|
|Okonski, Kristina M; Samuel, Charles E (2013) Stress granule formation induced by measles virus is protein kinase PKR dependent and impaired by RNA adenosine deaminase ADAR1. J Virol 87:756-66|
|Taghavi, Nora; Samuel, Charles E (2013) RNA-dependent protein kinase PKR and the Z-DNA binding orthologue PKZ differ in their capacity to mediate initiation factor eIF2?-dependent inhibition of protein synthesis and virus-induced stress granule formation. Virology 443:48-58|
|Ruggieri, Alessia; Dazert, Eva; Metz, Philippe et al. (2012) Dynamic oscillation of translation and stress granule formation mark the cellular response to virus infection. Cell Host Microbe 12:71-85|
|Taghavi, Nora; Samuel, Charles E (2012) Protein kinase PKR catalytic activity is required for the PKR-dependent activation of mitogen-activated protein kinases and amplification of interferon beta induction following virus infection. Virology 427:208-16|
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