The main objective of the proposed study is to determine the mechanisms by which double-stranded RNA (dsRNA) regulates metabolic processes in mammalian cells with emphasis on the control of translation. Though dsRNA is sparsely distributed in nature there is increasing evidence that several physiological functions are associated with dsRNA treatment including (i) increased cytotoxicity, (ii) suppression of tumor growth, (iii) enhancement of the immune response, and (iv) promotion of an antiviral state through the induction of interferon. Significantly, interferon treatment cause the induction of dsRNA-regulated activities which are potent translational inhibitors. One of these is a cAMP-independent protein kinase which inhibits protein synthesis by the phosphorylation of the Alpha-subunit of the protein chain initiation factor eIF-2. This activity is also variously found in tumorigenic cells, virus-infected cells, and is present in a variety of cells as a constitutive component, most notably in rabbit reticulocytes, which provides an exceptional laboratory model. The proposed study is designed to clarify the (a) physiological role and (bb) biochemical mechanism of the dsRNA-dependent eIF-2 Alpha kinase (dsI) in reticulocytes, and in normal, interferon-treated, and virus-infected cells. Since both the dsRNA-dependent and heme-regulated eIF-2Alpha kinases of reticulocytes display similar regulatory mechanisms, and since both are mediated by phosphorylation-dephosphorylation equilibria, a related goal is to broaden current studies on the comparative mechanisms of inhibition. Research objectives will focus on several related areas. These include: (a) Biochemical, physiological, and molecular properties of constitutive dsI and interferon-induced dsI. (b) The regulation of protein chain initiation by dsI and related factors, with emphasis on the roles of reversing factor and dsI phosphatase. (c) The application of polyclonal anti dsI antibodies as a probe to examine the distribution and physiology of constitutive and induced dsI. (d) Characterization of the dsI encoding gene to study the structural and regulatory domains of the dsI gene. (e) Biological function and molecular nature of reticulocyte terminal uridylyl transferase in the initiation of poliovirus RNA. (f) Studies of the GTP-binding/GDP-exchange translational factors (eIF-2, EF-1, EF-2, RF) as models to examine the GTP/GDP regulatory system.
