Interferon messenger RNA content of human fibroblasts during induction, shutoff, and superinduction of interferon production

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Abstract
Translation of injected mRNA in oocytes of Xenopus laevis was used as a highly sensitive and quantitative assay for interferon mRNA. Injection into oocytes of polyadenylylated RNA extracted from poly(I)-poly(C)-induced human diploid fibroblasts (FS-4) leads to the synthesis of biologically active human fibroblast interferon over a period of 24-32 h. There is a linear relationship between the amount of mRNA injected and the interferon yield obtained over a range of 1-20 ng of injected RNA. Injection of 40-80 ng of mRNA into each of 15 oocytes, homogenized in 0.3 ml of incubation medium, gave a titer of 128-256 interferon reference units[IRU]/ml of homogenate. FS-4 cells at the peak of interferon production, i.e., approximately 2.5 h after the beginning and induction with poly(I)-poly(C), gave mRNA that yielded 24-48 IRU/ml in the oocyte assay (30 ng of RNA injected/oocyte). An equivalent amount of mRNA from FS-4 cells in the shutoff phase, approximately 6 h after induction, gave .ltoreq. 4 IRU/ml. In contrast, mRNA extracted from FS-4 cells that were induced and maintained in the presence of 40 .mu.M 5,6-dichloro-1-.beta.-D-ribofuranosylbenzimidazole for 6 h produced 64-128 IRU/ml. Polyadenylylated RNA obtained from uninduced FS-4 cells did not lead to detectable interferon synthesis (<4 IRU/ml). The shutoff of interferon production in FS-4 cells probably involves a regulatory event leading to the posttranscriptional inactivation or degradation of interferon mRNA. Because the inactivating mechanism is sensitive to inhibition by 5,6-dichloro-1-.beta.-D-ribofuranosylbenzimidazole, a selective inhibitor of nuclear heterogeneous RNA and mRNA synthesis, synthesis of an RNA molecule is probably necessary for the shutoff of interferon production.