Inhibition of protein synthesis by 2'-5' linked adenine oligonucleotides in intact cells [23]

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.