Synthesis of 2'5'-oligo(A) in extracts of interferon-treated HeLa cells.

An enzymatic activity which, upon addition of double-stranded RNA (dsRNA), polymerizes ATP into pppA(2’pYA), or 2’5’-oligo(A), is increased in interferon-treated cells. An assay for this enzymatic activity, designated 2’5’-oligo(A) polymerase, is described here. The assay uses DEAE-cellulose chromatography for a direct measurement of 2’5’-oligo(A) synthesized by cell extracts. The optimal conditions for 2’5’-oligo(A) synthesis have been determined in extracts of interferontreated HeLa cells. The reaction shows a high Mg” optimum and its rate is proportional to the ATP concentration. The requirement for dsRNA has been examined by using synthetic homopolymers. Singlestranded and triple-stranded polyribonucleotides and double-stranded hybrid homopolymers formed with complementary polydeoxyribonucleotides do not activate synthesis of 2’5’-oligo(A). Synthetic dsRNAs are good activators. The 2’5’-oligo(A) formed in the assay described here has been characterized by chromatography on DEAEcellulose and polyethyleneimine-cellulose plates, before and after digestion with nucleases. The reaction products are identical with 2’5’-oligo(A) previously described for other interferon-treated cell types. The high rate of formation of 2’8-oligo(A) cannot be explained by an inhibition of degradation of these oligonucleotides due to the high Mg2+ concentration used in the assay. The assay has been used to measure 2’5’-oligo(A) polymerase activity in HeLa cells treated with different doses of interferon or treated for different times with a standard dose. The polymerase activity increases with dose and time of treatment. Conversely, after removal of interferon, the polymerase activity declines with the increase in cell mass.