The OVERALL OBJECTIVE of the proposed investigation is to elucidate the role of interferon-regulated protein kinase(s) 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 full-length CDNA of the IFN- regulated, RNA-dependent P1/eIF-2alpha protein kinase. To characterize the activity of the expressed recombinant kinase, both wild type P1 and catalytic and regulatory domain P1 mutants, and to determine the chromosome assignment of the kinase. To utilize the protein P1 KIN CDNA clone, and antibody prepared against the recombinant P1 protein, in studies on the regulation of P1 expression in IFN-treated and virus-infected animal cells: to attempt to obtain expression of P1 CDNA in eukaryotic cells as an approach to structural and functional analyses of P1. (2) To purify the P1 protein expressed from the CDNA clone, and then to characterize the recombinant P1 protein for its ability to catalyze the RNA-dependent autophosphorylation of protein P1 and the subsequent phosphorylation of the alpha subunit of protein synthesis initiation factor EIF-2. To continue our biochemical characterization of P1 with emphasis on the identification of the site(s) of autophosphorylation associated with activation of the kinase; the construction and characterization of mutants in which the putative ser/thr autophosphorylation site(s) have been converted to ala or to asp; the further characterization of the allosteric RNA binding site of protein P1 through the use of 8-azido double-stranded RNA (dsRNA) photoaffinity probes; the construction and characterization of protein P1 mutants in which the candidate RNA binding site domain has been mutated; the further definition of the reovirus s! MRNA structure capable of activating the kinase; and, to attempt to identify proteins present in uninfected or virus-infected human cells which may associate with protein P1, either as substrates or as regulatory proteins. (3) To attempt to stably express wild-type and mutant (ser to ala, ser to asp; lys to arg) forms of the P1 KIN CDNA in cells in culture, and then to examine the P1-expressing cell lines for phenotype and growth properties, and for their ability to support virus replication and protein synthesis. 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, and that 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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