Interferon synthesis and action is an important mechanism by which animals respond to and limit virus infection. Interferon treatment directly protects cells from virus infection and modulates the immune response to the infection. In addition, interferon inhibits the growth of some tumors in animals. Some of these responses to interferon treatment appear to be due to induction in cells of an eIF-2(alpha) protein kinase, which is activated by low but not high concentrations of dsRNA. Recently, several viruses have been characterized which code for inhibitors of the interferon-induced protein kinase. In addition it has recently been shown that histone is an inhibitor of this kinase. The goal of the proposed research is to purify the dsRNA-activated, interferon-induced protein kinase and to characterize the interaction of this kinase with its substrates and activators, and with several of its inhibitors, including EMC virus RNA and histone. The kinase will be purified, in its inactive form, from the tissue of animals which have been induced to synthesize interferon. The affinity of kinase, both in its inactive and preactivated forms, for its substrates eIF-2 and ATP will be characterized. A nitrocellulose filter binding assay will be developed and the purified kinase will be characterized in terms of its ability to bind to dsRNA and EMC/mengo virus RNA. The sequences on EMC/mengo virus RNA responsible for kinase inhibition and binding will be isolated and analyzed for the presence of secondary structure. The effects of any secondary structure found on the ability of EMC/mengo virus RNAs to interact with the kinase will be analyzed by site specific mutagenesis. The proposed research will lead to a better understanding of the activation and inhibition of this important protein kinase. As such it will increase our understanding of the molecular mechanism of interferon action. In addition, since strains of EMC virus which are relatively resistant to interferon treatment are diabetogenic in mice, and since other picornaviruses have been associated with juvenile onset diabetes in humans, this research may provide a better understanding of this important disease.
