The 2-5A dependent RNase (RNase L), a ubiquitous enzyme in mammalian cells, functions in the antiviral mechanism of interferon action. The requirement of RNase L for allosteric effectors, consisting of a type of 2' to 5, linked oligoadenylate known as 2-5A, provides RNase L with a level of control that is absent from other nucleases. The goals of this project are to understand how RNase L functions at the molecular level and to determine the fundamental role of RNase L in the control of viral infections. To probe the structure and function of RNase L, mutagenesis, biochemical and biophysical studies will be performed. We will localize and study the functions of the catalytic domain, the 2-5A and RNA binding sites, the protein/protein interaction domains, and the protein kinase homology regions in RNase L. Structural studies on RNase L that focus on the 2-5A binding domain will be performed. To study the involvement of RNase L in the control of viral growth and cell death, we will regulate expression in mammalian cells of wild type and mutant forms of RNase L and of proteins that interact with RNase L. We will determine the effect of suppressing RNase L activity with a dominant negative RNase L mutant on reovirus- and vaccinia virus-replication and on viral-induced apoptosis. To determine the function of RNase L in an animal model, we will disrupt by homologous recombination the RNase L gene in mice. Viral replication and virus-induced apoptosis will be studied in cell lines derived from mouse embryos lacking RNase L. The effects of the RNase L gene disruption on growth, development, tumorigenesis and virus infections in the mice in the presence and absence of interferon will be determined. The ability of RNase L to be regulated by a small molecule, 2-5A, provides a unique opportunity to control viral infections at the level of RNA turnover. This project may lead to the discovery and development of novel therapeutic agents for viral infections, cancer and other diseases.
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