Induction of 2′5′-oligoadenylate synthetase activity and a new protein by chick interferon

Abstract

Abstract Treatment of primary cultures of chick embryo cells with homologous interferon increased the activity of 2′5′-oligoadenylate synthetase in their cytoplasmic extracts. This enzyme is activated by double-stranded RNA to synthesize the novel trinucleotide pppA 2′ p 5′ A 2′ p 5′ A (and higher oligomers) which is a potent inhibitor of cell-free protein synthesis. The increase in enzyme activity was dependent upon the concentration of interferon and the time of treatment. For example, 1 unit of interferon elicited a 10-fold increase in 20 hr, whereas 100 units or more produced a 2000- to 10,000-fold increase. The kinetics of enhancement showed an initial lag of about 1 hr, followed by a 4- to 5-hr period during which the activity increased exponentially. Maximum levels were reached about 10 hr after interferon treatment. Neither an increase in enzyme activity, nor the establishment of a virus-resistant state was observed in cells treated with chick interferon in the presence of actinomycin D or p -fluorophenylalanine, or in cells treated with heterologous (mouse) interferon. Analysis of the proteins labeled by incorporation of [ 35 S]methionine after interferon treatment revealed the appearance of a new polypeptide in the postribosomal supernatants of interferon-treated cells. Upon gel filtration, this polypeptide coeluted with the peak of oligoadenylate synthetase activity, at 50,000–60,000 MW. It had an apparent molecular weight of 56,000 when analyzed by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. Like the 2′5′-oligoadenylate synthetase activity, the 56,000-dalton polypeptide bound to polyriboinosinic acid -polyribocytidylic acid covalently attached to agarose and could be partially purified in this manner. The appearance of the polypeptide was dependent on the concentration of interferon and the time of treatment employed and was blocked by the presence of actinomycin D. Pulse-labeling studies showed that its rate of synthesis increased sharply about 2 hr after interferon treatment but decreased again about 8 hr later despite the continued presence of interferon in the medium. The possibility that the 56,000-dalton polypeptide is the oligoadenylate synthetase enzyme is being studied. Whatever the outcome, these data suggest that the enzyme and the polypeptide are each the product of a gene whose function is controlled by interferon treatment.