The overall objective of the proposed investigation is to elucidate the role of the interferon-inducible double-stranded RNA-dependent protein kinase (PKR) in the actions which natural and recombinant interferons mediate on viral and host functions.
The SPECIFIC AIMS of our proposed continuation investigation of protein phosphorylation and interferon (IFN) action are as follows: (1) To continue our characterization of the structure and function of the 5'-flanking region of the human and mouse Pkr genes. To delineate the structure of the 5'-flanking region of the Pkr gene required for IFN-inducible as well as basal transcriptional activity. To attempt to map the major transcription sites by S1 nuclease protection and primer extension analyses, and 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. To examine Pkr regulation by cytokines other than interferons alpha and gamma. (2) To further characterize the biochemical and biophysical properties of the PKR kinase. To identify the sites of PKR phosphorylation associated with activation of kinase catalytic activity through the use of molecular genetic and chemical approaches. To study the RNA-structure(s) capable of modulating kinase activity (autophosphorylation; eIF-2alpha phosphorylation). To elucidate the mechanism of heparin-mediated activation of PKR, and to characterize the structural basis and functional significance of PKR protein-protein associations that occur in uninfected and virus- infected cells (3) to further characterize the expression of wild-type and mutant forms of PKR cDNA in cells in culture. To examine the PKR- expressing cell lines for phenotype and growth properties, and for their ability to support virus replication and protein synthesis relative to cell lines devoid of PKR. The health relatedness of the proposed research stems from the likelihood that the work may contribute to a better understanding of regulatory mechanisms involving phosphorylation possibly operative in normal cells as well as virus-infected cells. Furthermore, the elucidation of the actions of IFN at the molecular level is of immediate importance in view of the potential applications of IFN in the clinic.
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