MECHANISM OF INTERFERON ACTION - SYNTHESIS AND ACTIVITY OF THE INTERFERON-MEDIATED LOW-MOLECULAR-WEIGHT OLIGONUCLEOTIDE FROM MURINE AND HUMAN-CELLS

The interferon-mediated synthesis of the low-molecular-weight 2′,5′-oligoadenylate effectors (LMWE) of endoribonuclease activity and the interaction of the LMWE with components present in extracts prepared from mouse fibroblast and human amnion cells was investigated. The following results were obtained: (1) The LMWE was synthesized by cell-sap fractions prepared from mouse and human cells treated with their homologous interferons. Preincubation of interferon-treated cell-free S-10 extracts increased by about 50-fold the apparent concentration of the LMWE oligonucleotide synthetase observed in the S-100 cell-sap fractions. Mouse and human LMWEs enhanced ribonuclease activity in their heterologous human and mouse cell-free extracts prepared from untreated cells. (2) Degredation of the reovirus [3H]mRNA by the LMWE-activated endonuclease was quantitated both by sucrose density gradient centrifugation and by formation of acid-soluble oligonucleotides. (3) Free LMWE was rapidly inactivated (t 1 2 < 10 min) by cell-free extracts prepared from untreated L929 cells. The inactivation of LMWE appeared to involve the cleavage of a 2′,5′-phosphodiester bond of the oligonucleotide rather than removal of the 5′-terminal phosphate residue. (4) In addition to the effect of LMWE on ribonuclease activity, the phosphorylation of a 110,000-dalton protein present in S-10 extracts prepared both from untreated and interferon-treated cells was observed in the presence of [γ-32P]-LMWE. Increased phosphorylation of a protein of comparable molecular weight was also observed in ribosomal salt-wash fractions that contained an interferon-activated nuclease. The LMWE did not, however, substitute for RNA with double-stranded character in the phosphorylation of either the 38,000-dalton subunit of eIF-2 or the 67,000-dalton ribosome-associated protein P1. © 1979.