Isolation of two interferon-induced translational inhibitors: a protein kinase and an oligo-isoadenylate synthetase.

Abstract

Large-scale purification of translational inhibitors present in interferon-treated mouse L cells, but not in untreated cells, led to the isolation of two interferon-induced activities. One is a protein kinase system that is activatable by double-stranded RNA and ATP and that phosphorylates a Mr 67,000 protein and the smallest subunit of eukaryotic initiation factor-2. The purified protein kinase is a strong translational inhibitor. The second activity is an enzyme that, with doublestranded RNA, slowly polymerizes ATP into oligoadenylate with a 2'-5' phosphodiester linkage. The oligo-isoadenylate in turn activates a potent inhibitor of mRNA translation. The translational inhibition induced by interferon treatment can be demonstrated in intact virus-infected cells (1, 2) and in cell-free systems (3-5). The mechanism of translational inhibition is complex: both elongation and initiation of polypeptide chains are affected (6,7). Addition of certain minor tRNA species has been shown to relieve the elongation block (8, 9), and activities that affect the function of tRNA have been reported (10, 11). On the other hand, the partial translational inhibition seen in many interferon-treated cell extracts can be potentiated by addition of double-stranded RNA (ds RNA) (6, 12) which, as in reticulocyte lysates, primarily blocks initiation (13). Ds RNA itself appears to have several effects on interferon-treated extracts: in the presence of ATP, it induces phosphorylation of certain ribosome-associated proteins (14-17); it induces the synthesis of a nucleotide inhibitor of translation (16,(18)(19)(20)(21); and it activates a nuclease activity (22)(23)(24). Changes in the methylation of mRNA were observed with reovirus (25) and simian virus 40 (unpublished data).To establish which of these effects are related to the inhibition of protein synthesis, we have undertaken the large-scale isolation, from cell extracts, of the dominant interferon-induced translational inhibitors. We reported earlier (21) that several fractions from interferon-treated cells contained translational inhibitors, some of which were activated by ds RNA/ATP. In this work we describe the isolation of two such inhibitors, an eukaryotic initiation factor 2 (eIF-2) kinase system and an oligo-isoadenylate synthetase. MATERIALS AND METHODSInterferon-Treated and Control L Cell Fractions. Crude extracts (S10) from 25 liters of CCL1 cell suspension cultures treated 24 hr with purified mouse L cell interferon at 200 units/ml (26) and from the same amount of untreated cultures were prepared as described (14). Cell sap (150,000 X g supernatant) and 0.5 M KCl ribosomal extracts (RWF) were prepared (14) at 40 and stored in liquid air. Each cell sap (360 mg of protein; 5 mg/ml) was dialyzed against 20 mM N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (Hepes), pH 7.5/5 mM Mg acetate/i mM dithiothreitol/10% glycerol (buffer D) with 25 mM KCI and was loaded (1 ml/min) on a 28 X 2.6 cm DEAE-cellulose column. Effluent (DE25) (60 mg of protein; 1 mg/ml) was applied ...

Biological Activities and Receptor Binding of Two Human Recombinant Interferons and their Hybrids

“…Interferons exhibit a number of biological activities including establishment of an antiviral state, inhibition of cell multiplication and enhancement of natural killer cell activity (Masucci et al, 1980). At the biochemical level, IFN has been shown to induce the synthesis of HLA antigens and fie microglobulin (Heron et al, 1978;FeUous et al, 1979) and to enhance a number of pre-existing enzyme activities including (2'-5')oligoadenylate synthetase (Kerr & Brown, 1978), protein kinase (Zilberstein et al, 1978), and 2'-phosphodiesterase (Schmidt et al, 1979).…”

A cytotoxic substance (CTS-51) produced by human buffy coat cultures stimulated by Staphylococcal enterotoxin B: Further characterizations and combined action with interferon.

“…4). These phenomena seem to imply that the development mechanism of the anticellular action of CTS-51 inside the target cells should be independent from that of the anticellular effect of IFN-a (Le), which is well-known to be promoted by some intracellular proteins inside the target cells (Zilberstein et al 1978).…”

Inhibition of vaccinia mRNA methylation by 2',5'-linked oligo(adenylic acid) triphosphate.

“…It inhibits cell-free protein synthesis at nanomolar concentrations. The inhibition apparently involves the activation of an RNase that degrades mRNA (9)(10)(11)(12)(13). The enzymatic activity, 2,5-oligo(adenylate) synthetase, which synthesizes pppApApA from ATP in the presence of double-stranded RNA has also been found in extracts of interferon-treated chicken embryo, 3T6, and HeLa cells (11,14,15).…”

“…S-Adenosyl[methyl-3H]methionine (specific activity [10][11][12][13][14][15] Ci/ mmol; 1 Ci = 3.7 x 1010 becquerels) was purchased from New England Nuclear. Nucleosides and mono-and triphosphates were from Sigma.…”