Mechanism of the interferon-induced block of mRNA translation in mouse L cells: Reversal of the block by transfer RNA (original) (raw)
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Virology, 1974
The effect of treating Krebs 11 ascites tumor cells with interferon on the ability of extracts prepared from them to catalyze the translation of a variety of messenger RNAs was investigated. The following results were obtained : (1) Extracts of untreated cells translated endogenous mRNA, as well as exogenously added synthetic mRNA [poly(U)l, cellular mRNA (Krebs cell and L cell), and viral mRNA (reovirus and vaceinia virus). By contrast, extracts of Krebs cells treated in the ascitic form with interferon translated endogenous mRNA, exogenously added cellular mRNA and poly(U) as efficiently as extracts of untreated cells, but they translated viral mlLNAs very poorly (less than 10% as efficiently as extracts of untreated cells). Thus, the ability to discriminate between cellular and viral mRNAs that is characteristic of whole cells was also exhibited by these cell-free extracts. (2) Virus infection was not required for the development of the interferon-induced inhibition of viral mRNA translation. (3) Mixing experiments indicated that the inability of extracts of interferon-treated cells to cat alyze the translation of viral mRNAs was due to the presence of an inhibitory factor(q) rather than to the absence of a required factor(s). (4) The inhibitory factor(s) was associated with ribosomes, and could be dissociated from them by washing with KC1. (5) Polyacrylamide gel electrophoresis revealed the presence of a 48,000 dalton polypeptide in the 0 .3-0 .6 M KCl wash fraction of ribosmmes prepared from interferon-treated cells that was not detectable in the corresponding wash of ribosomes prepared from untreated cells. This fraction inhibited the translation of viral mRNAs in cell-free extracts of untreated cells more than any other salt wash fraction. These results suggest that the antiviral activity of interferon is mediated, at least in part, by a ribosome-associated polypeptide that permits discrimination between cellular and viral mRNAs .
Journal of interferon research, 1982
Human lymphocytes obtained by cytapheresis were stimulated in spinner culture conditions by nonpurified PHA in order to study the production of 7 interferon, and the characterization of IFN-7 mRNA. Titers of interferon prepared in 0.6 to 4 1 batches, varied in 20 preparations from 8,000 to 32,000 units/ml. This interferon was unstable at pH 2: the residual antiviral activity after 20 h treatment was less than 3%. Antibodies raised against 7 interferon from Con A and SEA-stimulated lymphocytes neutralized the interferon induced by PHA, indicating that all three preparations are antigenically related. Poly(A)RNA from control, noncultivated lymphocytes and from lymphocytes stimulated by PHA for 18 h were translated in reticulocyte lysates and analysed by polyacrylamide gel electrophoresis. The pattern of synthesized polypeptides was different suggesting modifications in the population of mRNA. When total poly(A)RNA was inoculated into Xenopus Laevis oocytes, interferon activity was found with both, control and stimulated mRNAs although only at low levels in the control. After sucrose gradient fractionation of poly(A)RNA, each fraction was inoculated into oocytes and interferon activity measured in the oocyte bathing medium. A low level was synthesized by the RNA fractions around 28 S from control as well as from stimulated lymphocytes. These interferons were not neutralized by anti-IFN-a or anti-IFN-7 sera but they were neutralized by anti-IFN-0 serum. Only the 16 S RNA fraction from PHA-stimulated lymphocytes induced high levels of interferon in occytes. This interferon has been characterized as 7 interferon. Each fraction obtained from sucrose gradients of poly(A)RNA from control and PHAstimulated lymphocytes was translated in reticulocyte lysate. Gel analysis of the products showed stricking differences when the same fraction of both RNAs were compared. Concerning particularly the 16 S RNA from PHA-stimulated lymphocytes, where 7 interferon mRNA was present, polypeptides ranged from 15 to 55 K with a bulk around 45 K, indicating heterogeneous RNA molecules.
European Journal of Biochemistry, 1975
Synthesis of polypeptide chains coded by exogenous messenger RNAs is inhibited in cell-free extracts from interferon-treated mouse L cells, due to a "deficiency" in some specific tRNA species.' A detailed analysis shows that polypeptide chain elongation is blocked and incomplete chains are formed. After a few minutes, however, initiation of new polypeptide chains is also blocked. Messenger RNA still binds to ribosomes but initiator Met-tRNAfMet binding is inhibited. The block in initiation appears to be secondary to the block in elongation.
European Journal of Biochemistry, 1981
A mouse cell line, N I H 3T3, does not respond to some of the activities of interferon. Even after treatment with high concentrations of interferon the replication of lytic viruses, such as encephalomyocarditis virus (EMCV) and vesicular stomatitis virus (VSV) is not inhibited in these cells. In contrast, interferon treatment of these same cells results in the inhibition of Moloney murine leukemia virus (MMuLV) production. We have analyzed enzymatic pathways which are induced by interferon in these cells. After interferon treatment, the level of the (2'-5')oligoadenylate [(2'-5')A,] synthetase activity and the phosphorylation of the 67000dalton protein (PI) are enhanced in NIH 3T3 cells to approximately the same level as interferon-sensitive mouse L-cells. Moreover, NIH 3T3 and L-cells contain approximately the same levels of enzymcs which inactivate (2'-5')A,. Both exogenously added (2'-5')Aj or double-stranded RNA (ds RNA) failed to inhibit protein synthesis in NIH 3T3 extracts even though they were potent inhibitors of L-cell extract-directed protein synthesis. Direct measurements of the (2'-S')A,-dependent ribonuclease F (RNase F) failed to detect such activity in NIH 3T3 cells. Our results, therefore, suggest that the presence of RNase F activity is necessary for the interferon-induced antiviral activity against EMCV and against VSV. The induction of protein kinase activity by interferon treatment of NIH 3T3 cells appears to have no direct effect on EMCV and VSV replication.