2'5'OLIGO(A) POLYMERASE-ACTIVITY AND INHIBITION OF VIRAL-RNA SYNTHESIS IN INTERFERON-TREATED HELA-CELLS

A quantitative assay for 2'5'oligo (A) polymerase, based on the adsorption of cell extract to poly (I)-poly(C)-agarose and incubation with [3H]ATP, has been developed. The [3H]2'5'oligo (A) synthesized is resolved from ATP by chromatography on DEAE-cellulose. The 2'5'oligo(A) polymerase activity has been measured in extracts of control HeLa cells and cells incubated with different concentrations of human fibroblast interferon or for different lengths of time with 100 units/mL of interferon. An increased enzymatic activity can be detected in cells treated with 12.5 units/mL or higher concentrations for 17 h and in cells treated with 100 units/mL longer than 3 h. The synthesis of encephalomyo-carditis virus (EMC) RNA is inhibited in these cells in parallel with the increase in polymerase activity. Treatment of HeLa cells with interferon followed 2 h later by actinomycin D prevents the increase in 2'5'oligo (A) polymerase and the inhibition of viral RNA synthesis. After 4-h treatment with interferon, actinomycin D does not show this effect, whereas cycloheximide prevents the increase in polymerase activity. Active RNA synthesis 2 h after the start of interferon treatment and active protein synthesis 4 h afterward is necessary for the increase in 2'5'oligo (A) polymerase activity. Another enzymatic activity, previously reported to be increased in interferon-treated cells, is a double-stranded RNA activated protein kinase, which phosphorylates two polypeptides associated with ribosomes. Assays of this protein kinase have shown an increase in activity in cells treated with 12.5 units/mL or higher interferon concentrations. However, the increase in kinase activity can only be detected in cells treated for at least 10 h. In particular, HeLa cells treated for 7 h with 100 units/mL of interferon do not show increased protein kinase activity; upon such treatment, EMC RNA synthesis is already significantly inhibited. Therefore, an increased protein kinase activity may not be required for an inhibition of EMC RNA synthesis. © 1979, American Chemical Society. All rights reserved.