The overall objective of the proposed investigation is to elucidate the role of the interferon (IFN)-induced protein kinase in the actions which cloned and natural IFNs mediate on viral and host functions.
The specific aims are as follows: (1) We plan to attempt to further purify by biochemical and immunochemical techniques the IFN-induced protein kinase which catalyzes the phosphorylation of ribosome-associated protein P1 and the Alpha subunit of protein synthesis initiation factor eIF-2; the kinase copurifies with P1. Starting material will include cells grown in culture and animal tissue. We plan to characterize the P1 kinase preparations using the following: affinity probes to identify ATP binding sites; reversible cross-linking agents to study the arrangement of P1 on the 40S ribosomal subunit; gradient centrifugation to study the apparent size of the kinase prior to and following activation; and inosine- and bromouri-dine-substituted reovirus mRNAs to study the role of mRNA secondary structure in kinase activation and translation inhibition. (2) We plan to quantitate by using 2-dimensional electrophoresis and immunoblot procedures the phosphorylation status of eIF-2Alpha and protein P1 in whole cells grown in culture, with and without IFN treatment and infection with reovirus. Three serotypes of reovirus will be studied (1, 2 & 3) which differ appreciably in their virulence, including their capacity to inhibit cellular protein synthesis. (3) We plan to prepare additional antisera directed against protein P1, and to attempt to prepare hybridomas secreting monoclonal antibodies against P1. We plan to characterize the antibodies with regard to their cross-reactivity with P1, both phosphorylated and dephosphorylated, from heterologous sources. We plan to utilize the antibodies to study the structural and functional properties of P1 in whole cells and cell-free systems. (4) As a long term objective, we plan to attempt to construct and characterize complementary DNA copies of the mRNA coding for protein P1; to utilize the cDNA clone(s) in studies on the regulation of P1 expression in IFN-treated animal cells; and, to attempt to obtain expression of P1 in prokaryotic and eukaryotic cells after insertion of the cloned cDNA into appropriate expression vectors.
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