Inhibition of protein synthesis by 2′–5′ linked adenine oligonucleotides in intact cells

Abstract

A SERIES of 2–5 linked oligoadenylic acid triphosphate (2–5A) inhibitors of cell-free protein synthesis are formed from ATP by an enzyme (2–5A synthetase) activated in interferon-treated cell extracts or rabbit reticulocyte lysates by double-stranded RNA^1–4. The major active species is the trimer, pppA2′p5′A2′p5′A. Cell-free protein synthesis is inhibited by subnanomolar concentrations of 2–5A and this inhibition seems to be mediated at least in part, by a nuclease which degrades mRNA⁵. However, 2–5A, the nuclease and the inhibition of protein synthesis are all unstable in cell-free systems. 2–5A is rapidly degraded in such systems prepared from control (or interferon-treated) cells, nuclease activity is transient and in the absence of a 2–5A regenerating system protein synthesis resumes if fresh mRNA is added^6,7. Extracts derived from cells that have never been treated with interferon, therefore, have mechanisms for the synthesis of 2–5A (rabbit reticulocytes) and for responding to and degrading 2–5A. Thus, the 2–5A system, in addition to any role it has in the antiviral action of interferon, may also be involved in the regulation of normal cell growth and development. The extraordinary potency of 2–5A and its instability in cell-free systems makes the task of detecting 2–5A in intact cells very difficult. As an alternative we have studied the effect of exogenous 2–5A on protein synthesis in intact cells, using a recently described method for making animal cells reversibly permeable to small molecules⁸. Here we report that pppA2′p5′A2′p5′A and related 2′–5′ linked oligonucleotides inhibit protein synthesis in hypertonically treated BHK–21 cells.