Identification of the Translation Products of Human Fibroblast Interferon mRNA in Reticulocyte Lysates

Abstract

Messenger RNA was purified from human foreskin fibroblasts FS11, a high interferon-producer line, after induction with synthetic double-stranded RNA. The mRNA was translated in a cell-free protein-synthesis system from rabbit reticulocytes. The translation products, containing biologically active human interferon, were immunoprecipitated by a serum from rabbits immunized against partially purified interferon. Analysis of the immunoprecipitate by polyacrylamide gel electrophoresis in dodecylsulfate shows that the product of human fibroblast interferon mRNA is a 23000-M, polypeptide. Methods are described for the synthesis and rapid identification of this polypeptide, which should be useful for structural analysis of interferon and isolation of its mRNA.Interferon mRNA from human cells has been translated in heterologous intact cells [l,21, in Xenopus luevis oocytes [3, and in a variety of cell-free systems [3-51. In all these cases, as found also for mouse interferon mRNA [9-141, biologically active interferons, showing the correct species specificity and immunological properties, were obtained. The translation of human fibroblast interferon mRNA was reported to be low in cell-free protein-synthesis systems as compared to oocytes [5]. Use of cell-free extracts, such as nuclease-treated reticulocyte lysates [15], would however be advantageous to study the proteins synthesized, because of the high efficiency and low background of this translation system. This work describes the identification by immunoprecipitation and polyacrylamide gel electrophoresis, of a highly labeled 23 000-M, polypeptide which appears as the specific product of human fibroblast interferon mRNA translation in reticulocyte lysates. This method should allow the structural analysis of this polypeptide and facilitate the purification of its mRNA. MATERIALS A N D METHODS Growth oj'FS1 I Cells and Induction of' InterferonHuman foreskin fibroblast F S l l cell cultures were established in our laboratory by Dr D. GurariRotman. These diploid cells, grown from foreskin Abbreviations. Poly(r1) . poly(rC), poly(inosate) . poly(cytidy1-ate) double-stranded RNA; NaCI/Pi, phosphate-buffered saline; Hepes, 4-(2-hydroxyethyl)-l-piperdzineethdne sulfonic acid ; tricine, A'-tris(hydroxymethy1)-methyl-glycine; p.f.u., plaque-forming units; vsv, vesicular stomatis virus; CM, carboxymethyl. ~~ explants taken 8 days after birth, were selected among 15 individual isolates for their capacity to produce high titers of interferon. The cells were grown in Eagle's minimum essential medium with 10 o/, fetal calf serum in 5 % CO;?, 95 % air at 37 "C and maintained by subculture at 1 : 5, in 90-mm plastic dishes. For interferon production [16], five such plates of cells were seeded in 4-pint (2.27-1) roller bottles with 80 ml minimum essential medium containing 10% fetal calf serum. Three days after confluency (8th day) the cultures were exposed to 100 pg/ml poly(r1) . poly(rC) and 50 pg/ml cycloheximide in 25 ml medium without serum. After 3.5-4 h actinomycin D was added...

Two interferon mRNAs in human fibroblasts: in vitro translation and Escherichia coli cloning studies.

“…Poly(rI-rC) superinduction of human FS11 fibroblasts, preparation of poly(A)+ mRNA, sucrose gradient purification, and mRNA translation in micrococcal nuclease-treated rabbit reticulocyte lysates were described in detail (5). Antibodies to human fibroblast interferon were produced and used for immunoprecipitation of the [35S]methionine-labeled translation products with staphylococcal protein A-Sepharose, as before (5). Interferon mRNA injection into Xenopus laevis oocytes was carried out according to Raj and Pitha (6).…”

Selection and Characterization of an Interferon-responsive Clonal Cell Line of HeLa Cells

“…Yet, in general, F-H12 is not particularly responsive to I F N since wild-type mengovirus, wild-type and thymidine kinase-negative strains of herpes simplex virus type 1 and vesicular stomatitis virus (which is generally highly sensitive to IFN), grow no differently in this line than in the unselected N O R line. Furthermore, even using is-l, the F-H12 line is no more responsive to I F N than the standard human diploid fibroblast line, FS11 (Weissenbach et al, 1979). Nevertheless, it is apparent that some aspect of the IFN-mediated antiviral state is altered in the F-H12 line since there are marked differences in the cell survival assay (Fig.…”

Isolation of Daudi Cells with Reduced Sensitivity to Interferon.: I. Characterization

“…Human interferon-c~ was prepared from the human lymphoid cell line Namalwa as previously described (Mogensen & Cantell, 1977) and purified by affinity chromatography (Mogensen & Cantell, 1979) to a specific activity of 2 × 108 international units/rag protein. Electrophoretically pure human interferon-fl, prepared from human FS11 diploid fibroblasts (Weissenbach et al, 1979) and purified by high-pressure liquid chromatography to a specific activity of > 2 × 108 units/mg protein, was a gift from Dr M. Rubinstein. Human interferon-fl, purified by controlled-pore glass and zinc-chelate chromatography to a specific activity /> 2 × 10 s units/rag protein, was a gift from Dr A. Billiau.…”