[31] Chromatographic assay on DEAE-cellulose for (2′-5′)-oligo(A) synthesis in cell extracts

Treatment of animal cells with interferon results in the induction of synthesis of new proteins. One of the induced proteins is an enzyme that, in the presence of double-stranded RNA (dsRNA), polymerizes ATP into a series of nucleotides linked by the unusual 2',5'-phosphodiester bond. The chapter explores that these nucleotides (2'-5')-oligo(A) reflect both the unusual phosphodiester linkage and the oligomeric nature of the series. Correspondingly, the enzymic activity that synthesizes the oligonucleotides is designated (2'-5')-oligo(A) polymerase. The (2'-5')-oligo(A) inhibits in vitro protein synthesis at subnanomolar concentrations through the activation of a latent endonuclease. The (2'-5')-oligo(A) inhibits protein synthesis and causes RNA degradation also when introduced in intact cells. Formation of (2'-5')-oligo(A) may inhibit the replication of RNA viruses in interferon-treated cells and detectable levels of these oligonucleotides have indeed been observed in interferon-treated L cells infected with encephalomyocarditis virus. To measure the levels of (2'-5')-oligo(A) polymerase in extracts of animal cells and to study the activation of this enzyme by dsRNA, an assay for (2'-5')-oligo(A) synthesis is developed. This assay is based on the conversion of [3H]ATP into labeled (2'-5')-oligo(A) that is separated from less charged nucleotides by DEAE-cellulose chromatography. The enzymic activity can be measured from the rate of conversion of ATP into (2'-5')-oligo(A).