Influence of preincubation on the development of the inhibition of protein synthesis in extracts from interferon-treated mouse L cells. Action on tRNA.
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Biomedical subjects
Publications and source records attributed to R Falcoff.
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Phytohemagglutinin is able to trigger interferon synthesis in spleen cell cultures from nude (nu/nu) mice as effectively as in splenic cell cultures from haired, control (nu/+), thymus-bearing mice. A minor theta-bearing cell population present in the spleen of nude mice appears essential to phytohemagglutinin interferon production, although cooperating cells are also required. The properties of nude mouse phytohemagglutinin interferon are indistinguishable from those displayed by the interferon induced in thymus-bearing mouse spleen cell cultures. Both interferons are unstable at pH 2 and cannot be neutralized by an antiviral interferon serum; hence, their characteristics correspond to those described for type T interferon. As in the case of viral interferon, pretreatment of L cells with nude phytohemagglutinin interferon induced specific enhanced phosphorylation of a 67,000-molecular-weight protein in vitro when cell extracts were incubated with double-stranded RNA and gamma-[32P]ATP.
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Complementary DNA (cDNA) from Mengo virus RNA has been synthesized and used as a probe to measure the synthesis and accumulation of viral RNA in Mengo infected L cell cultures, treated or untreated with interferon. Under experimental conditions used (200 units interferon/ml and 50 virus plaque-forming units/cell) results show that there is some synthesis of Mengo virus RNA in cells treated with interferon. One hour after infection, treated cells contain three times less viral RNA than untreated cells; five hours after infection, this difference has increased to ten fold. As in the control, no fragmented Mengo virus RNA molecules were found in interferon treated cells. The smaller recovery of infectious particles from interferon treated cells as compared to RNA accumulation suggests that not only RNA accumulation is inhibited but also a step posterior in viral maturation.
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Murine interferons induced by viruses and by non viral substances such as lectins, tilorone, lipopolysaccharide and Brucella have been analysed by affinity chromatography on a column of Sepharose coupled to anti-bodies directed against viral interferon. B cell stimulants induced interferons which are antigenically related to viral interferon and T cell dependent mitogens induced an interferon species which appears to be antigenically and physicochemically different. Despite their antigenic differences, both kinds of interferon seem to follow similar pathways for the induction of the antiviral state (sensitivity to antimetabolites, and in vitro phosphorylation of a 67 000 MW protein).
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Mengo virus double-stranded ribonucleic acid (dsRNA) was obtained on a semi-industrial scale from infected cultures of BHK-21 cells grown in suspension. Yield of the extraction and purification operations was small (about 22 mg from 10(11) cells in a 100-liter culture). Physicochemical characterization of this dsRNA gave an estimated molecular weight close to 4 x 10(6), a density of 1.59 (similar to that of the poliovirus dsRNA), and a thermal transition midpoint of 94 C. This product was a little more toxic for the mouse, by the intravenous route, than polyriboinosinic . polyribocytidylic acid (poly I:C) and strictly comparable in this respect to poliovirus dsRNA. The interferon-inducing capacity in the mouse and the antiviral activities in the mouse (infected with encephalomyocarditis, Semliki Forest, influenza, foot-and-mouth disease, and murine hepatitis viruses) and in the rabbit (Shope fibroma virus) of the ultraviolet light-inactivated product were practically identical, on a quantitative basis, with those of poly I:C. In vitro and in vivo experiments showed the dsRNA from Mengo virus to be slightly but significantly more resistant than poly I:C to the inactivating effect of human serum.
When noninfected L-cell suspension cultures are treated with interferon (specific activities superior to 10(6) reference units per mg of protein), the cell-free cytoplasmic extracts obtained are inactive for the translation of exogenous natural mRNAs. The dose-response curve shows that comparable amounts of interferon are required to produce a 50% reduction of Mengo virus multiplication in vivo and Mengo RNA translation in vitro. With higher doses of interferon, Mengo RNA translation is completely abolished, while poly U translation and endogenous protein synthesis are only slightly affected. The inactivation of Mengo RNA translation is reversible; after removal of interferon, normal translation activity is regained together with the ability to support Mengo virus multiplication. Fractionation of the cell-free extracts shows that the effect is localized in the fraction which can be washed off the ribosomes by high salt. These results establish that interferon induces a block in genetic translation in noninfected L cells.
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