Biosynthesis of mouse interferon by translation of its messenger RNA in a cell-free system.

Abstract

A fraction of mouse RNA containing messenger RNA coding for mouse interferon was translated with high efficiency in a wheat germ system into a fully active product. This product fulfills the criteria for mouse interferon, namely: (1) it was active against vesicular stomatitis virus and herpes simplex virus in mouse cells; (2) its antiviral activity was species specific; (3) its activity was completely neutralized by mouse anti-interferon serum. The synthesis of interferon in this cell-free system requires the presence of spermine.Interferon is a species-specific protein with high antiviral activity that is induced in most vertebrate cells by either virus or double-stranded RNAs. Using metabolic inhibitors, it was shown that interferon mRNA appears very soon after induction (1). We have previously described a biological assay of interferon mRNA using heterologous intact cells (2).We report herewith that the mouse interferon message can be efficiently translated in vitro by wheat germ extracts. The product thus synthesized possesses both antigenic and antiviral properties of mouse interferon as well as its species specificity. MATERIALS AND METHODSInduction of Cells for mRNA Extraction. Mouse C-243 cells, a line transformed by murine sarcoma virus (3), were induced with poly(I)-poly(C) in large petri dishes (150 mm diameter); they received 10 ml of MEM (Eagle's minimum essential medium) without serum containing 30 ,ug/ml poly(I)-poly(C) and 100 ,g/ml of DEAE-dextran. The inducer was left for 2 hr on the cells and then removed; the cells were washed once with warm MEM and 30 ml of MEM containing 3% calf serum were then added. Nine hours after the onset of induction, the cells were harvested and frozen at -70°until the time of RNA extraction.RNA Extraction was performed according to the two-step technique previously described (2). A typical extraction, in which cells harvested from 15 confluent dish cultures were utilized, yielded 3550 .g of RNA in the A phase (RNA solubilized in aqueous layer after sodium dodecyl sulfate and phenol extraction), and 2360,ug in the I phase (RNA solubilized only after Pronase treatment). The I phase was further fractionated by sedimentation over a 10-20% sucrose gradient for 30 min at 30,000 rpm in a SW 41 rotor. The bottom half of the gradient contains rapidly labeled nuclear RNA in an aggregated form, whereas the upper half (called 13) contains nuclear RNA as well as cytoplasmic poly(A)-containing mRNA (L. Montagnier and H. Collandre, in preparation). Since this latter fraction was found to be the most active, using the mRNA assay in intact Vero (African green monkey) cells, it was exclusively utilized for the in vitro translation experiments.Preparation of Wheat Germ Extractsk. Commercial wheat germ, mechanically extracted and not toasted, was supplied by Grands Moulins de Paris, Paris (France). Before use, the wheat germ was pre-washed with the grinding buffer: 20 mM N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (Hepes) (pH 7.6); 100 mM potassium chloride; 1 mM magn...

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

“…premature to conclude that the poly(A)-lacking fraction does not contain any interferon mRNA (24)(25)(26). DISCUSSION In previous reports, we have systematically evaluated several different hypotheses that might explain superinduction of human interferon production (for reviews see refs.…”

“…It has been discovered recently that polyadenylylated RNA extracted from interferon-producing human or mouse cells can be translated in wheat-germ, mouse Ehrlich ascites, Krebs-II ascites, and rabbit reticulocyte cell-free translation systems and in the Xenopus laets* oocyte, with the production of biologically active and specific interferon (24)(25)(26).…”

Synthesis of human interferon by Xenopus laevis oocytes: two structural genes for interferons in human cells.

“…The oocyte product resulting from injection of Namalva mRNA exhibited a similar molecular weight distribution with the peak of activity corresponding to 16,000 daltons. DISCUSSION Recent studies have shown that mRNA extracted from cells actively producing interferon can direct interferon synthesis in some heterologous intact animal cells (32)(33)(34) or in cell-free systems (18,35,36). In this study, we used X. Iaevis oocytes for the translation of human interferon mRNA isolated from two different cellular sources.…”

The Effects of some Different Metabolic Inhibitors on Interferon Superinduction

“…This scheme was originally based on results obtained with a limited number of metabolic inhibitors, notably actinomycin D and cycloheximide, although recently much more direct evidence has been obtained for the production of a new interferon messenger RNA after treatment of cells with an interferon inducer (De Maeyer-Guignard et al 197z;Kronenberg & Friedman, 1975;Pestka et al 1975;Reynolds et al 1975;Thang et al 1975). A second gene system, which represses the interferon gene, has also been invoked from the results of experiments in which actinomycin D and cycloheximide were added after interferon production had started.…”