The research proposed focusses on the biochemical characterization of nucleotide-protein interactions required for maintenance of the 2-5A synthetase/RNase L pathway, a vital process in cellular antiviral defense. RNase L is distinctive among nucleases in its dependence on 2- 5A for activation and cleavage specificity at uridylate residues. The molecular mechanism of 2-5A in the binding and activation of RNase L will be examined. To this end, The Pi chemically synthesized a new class of metabolically stable agonists and antagonists of 2-5A to study the biochemical processes for RNA hydrolysis in vitro and in vivo. Transmembrane passage of the 2', 5`-phosphorothioates into virus infected cells will be accomplished by poly(L-lysine) conjugation and the effect of these probes on the stability of viral and cellular mRNA will be measured. In addition, experiments are designed to examine (i) the order of addition of the substrate and allosteric activator to RNase L needed for productive complex formation, (ii) the requirement for complementary base pairing between 2-5A and RNA for RNase L activation, and (iii) the contribution of each 2', 5'- phosphodiester bond in 2-5A to the binding and activation processes of RNase L. Finally, mono- and bifunctional nucleotide site-directed affinity labels have been synthesized to study the acceptor and 2' -adenylation sites of recombinant 40 kDA, homogeneously pure 100 kDa and 110 kDa 2-5A synthetases, the ATP binding site of protein kinase, and the 2-5A binding site of RNase L.
