lnterferons are a family of cytokine with a wide variety of biological activities. They are components of the innate immune system protecting hosts against viral and parasitic infections and certain types of malignancies. Interferons affect cell proliferation and cellular differentiation as well. These pleiotropic cellular effects of interferons are mediated by a large number of interferon-inducible proteins. Some of these proteins are also induced directly by virus infection or double-stranded RNA. In this application, we propose to investigate the antiviral and cellular functions and the structure-function relationships of two families of such proteins: the 2-5 (A) synthetases and the P56 family of proteins. 2-5 (A) synthetases are a family of enzymes that, when activated by double-stranded RNA, can synthesize 2'-5' linked oligoadenylates that, in turn, activate the latent ribonuclease, RNase L. In addition, a specific isozyme of this family, E17, causes cellular apoptosis by interacting with proteins of the Bcl-2 family. Recently, several essential sites of the synthetase proteins have been identified by us by genetic, biochemical and crystallographic analyses. Continuation of these studies to identify the double-stranded RNA-binding sites is proposed. The cellular and antiviral functions of the P69 isozyme will be investigated using dominant-negative mutants and by gene disruption in mice. In addition, the mechanism of the apoptotic function of the E17 isozyme will be delineated by genetic and biochemical studies. The members of the P56 family of proteins function by interacting with specific cellular and viral proteins. The structural basis of this specificity will be explored. Several P56-interacting proteins have been identified including the mammalian Int-6 protein that is a component of the eukaryotic translation initiation factor 3. As a consequence of this interaction, P56 inhibits initiation of protein synthesis. The detailed mechanisms of this inhibition will be investigated. Because Int-6 also regulates cell division, the effects of P56 on that process will be investigated as well. Similar investigations will be extended to the functions of other members of the human and murine P56 families. A viral protein, to which P56 binds, is the human papilloma virus E1 protein. This interaction causes translocation of E1 from the nucleus to the cytoplasm and inhibition of HPV DNA synthesis. Potential interactions between other members of the P56 family and E1 proteins of several HPV strains will be investigated and the mechanism of the observed P56-mediated inhibition of HPV DNA replication will be delineated.
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