Interferon-induced inhibition of protein synthesis in L-cell extracts: an ATP-dependent step in the activation of an inhibitor by double-stranded RNA.

Abstract

ABSIRACT The translation of encephalomyocarditis virionRNA in extracts from interferon-treated L-cells is inhibited by the addition of double-stranded RNA (dsRNA) at 400 ng/ml. A similar inhibition in response to dsRNA is seen in control cell extracts supplemented with small amounts of a postribosomal supernatant fraction from interferon-treated cels (interferon cell sap): Neither interferon cell sap nor dsRNA alone is inhibitory in control systems. The inhibition is much reduced if translation is carried out at low ATP concentrations. Conversely, the inhibitory capacity of the interferon cell sap is increased 100-fold if it is preincubated with dsRNA and ATP prior to its addition to the protein-synthesizing system. After this preincubation all detectable dsRNA can be removed without any diminution of the inhibitory activity of the ceU sap. These results are compatible with a two-step model for the inhibition in which a pre-inhibitor is activated by dsRNA, the activated inhibitor then interacting with the protein synthesis system to inhibit translation. Double-stranded RNA (dsRNA) inhibits protein synthesis in animal cells and cell-free systems (1-4). Interferon treatment of cells renders them more sensitive to the toxic effects of dsRNA (5); and with extracts from interferon-treated L-cells, the translation of encephalomyocarditis virion RNA (EMC RNA) in the cell-free system shows an enhanced sensitivity to inhibition by dsRNA (6). The latter inhibition is specific for dsRNA, and the interferon dose-response curve and heat-inactivation studies show a close correlation between the antiviral activity of interferon and the enhanced sensitivity to dsRNA observed in the cell-free system (6).Recently we have found that the addition of small amounts of a postribosomal supernatant fraction from interferon-treated cells (interferon cell sap) renders cell-free protein-synthesizing systems from control cells sensitive to inhibition by dsRNA*. This has permitted the quantitation of a putative dsRNAdependent inhibitor in the interferon cell sap. Here (10). Postmitochondrial supernatant fractions (S10) were prepared from L-cells (9) and preincubated and dialyzed as described (11). Interferon and control cell saps were prepared from S10 that had not been preincubated. These were centrifuged at 100,000 X g for 2 hr at 40, and the supernatant cell saps were dialyzed as above.Amino Acid Incorporation Assays. Preincubated and dialyzed S10 from control cells provided the ribosomes, enzymes, factors, and tRNA for the amino acid incorporation assays. Interferon and control cell saps and dsRNA were added with and without preincubation, as indicated in the individual experiments. The conditions used in the assay of the incorporation of a mixture of fourteen "4C-labeled amino acids (54 mCi/ milliatom of carbon, The Radiochemical Centre, Amersham, England) were as described, with the omission of the reticulocyte initiation factors (11). Incubations were for 120 min at 300 unless otherwise stated, and 10-,sl aliquots were assayed for ...

A scientific journey through the 2-5A/RNase L system

“…I came to the Kerr lab because of my interest in unusual nucleotide regulators and my fascination with Ian Kerr's remarkable discovery of 2-5A [9,38]. The discovery of 2-5A followed Ian Kerr's observation of an IFN-induced increase in the sensitivity of protein synthesis to inhibition by dsRNA [39][40][41]. My transition was an easy one in that I was immediately able to go to work because all of the cell-free system components were in place in Eppendorf tubes in the −70C° freezer.…”

Inhibition of interferon-mediated induction of indoleamine 2,3-dioxygenase in mouse lung by inhibitors of prostaglandin biosynthesis.

“…Numerous studies have been carried out concerning the mechanisms of these interferon actions. Four enzymes (2'-5')oligoadenylate synthetase (19), protein kinase (20), phosphodiesterase (21), and IDOase (3), have been reported to date to be induced by interferon. The first two have been suggested as mediators in the establishment of the antiviral state by interferon (22).…”

Multiple forms of PKR present in the nuclei of acute leukemia cells represent an active kinase that is responsive to stress

“…1,2 PKR is activated in response to diverse stress signals. In addition to phosphorylating eIF2a, PKR has been demonstrated to phosphorylate and regulate p53, inhibitor kB kinase, the B56a regulatory subunit of protein phosphatase 2A, RNA helicase, insulin receptor substrate-1 and cell division cycle-2.…”