Synthesis of low molecular weight inhibitor of protein synthesis with enzyme from interferon-treated cells.

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PROTEIN synthesis in cell-free systems from mouse L cells pretreated with the antiviral agent interferon1 shows an enhanced sensitivity to inhibition by double-stranded RNA (dsRNA) (refs 2–4). The inhibition of protein synthesis is dependent on incubation with ATP as well as dsRNA and we and others have reported a dsRNA-dependent protein kinase activity(s) in extracts from interferon-treated cells5–8. The possible involvement of a viral dsRNA-mediated inhibition of protein synthesis in the sequence of events following virus infection in intact, interferon-treated cells has been discussed previously2,4. The situation with extracts from interferon-treated cells5–8 is reminiscent of that in rabbit reticulocyte lysates in which phosphorylation of an initiation factor by a protein kinase has been implicated in the inhibition of protein synthesis in a variety of conditions including the presence of dsRNA9–12. In the interferon-treated L-cell system, however, in addition to the kinase, a heat-stable, low molecular weight inhibitor (LMW inhibitor) of protein synthesis is formed on incubation with ATP and dsRNA (ref. 6). It remained possible from our previous work6, that the LMW inhibitor might be a small phosphorylated peptide product of the dsRNA-dependent kinase. We show here that this is not the case. In addition, we show that the enzyme responsible for the synthesis of the LMW inhibitor will bind to a column of dsRNA attached to a solid support. It can be eluted in high salt but is relatively unstable in this form. We have used the highly-purified enzyme in its stable column-bound state to synthesise and radioactively label the LMW inhibitor. This synthesis, the partial purification of the inhibitor and its activity in the inhibition of protein synthesis in cell-free systems from L cells and rabbit reticulocytes, are reported here. A more detailed characterisation of the inhibitor is described in the accompanying paper13.