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Relationship between interferon production and interferon messenger RNA synthesis in human fibroblasts
Abstract:
Poly(A) containing mRNA prepared from poly(rI)-poly(rC)-induced human fibroblasts stimulated [14C]leucine incorporation into protein in wheat germ cell-free extracts. For the translation of interferon mRNA into a biologically active product, the presence of spermine was essential. The protein synthesized in vitro fulfilled the criteria for human interferon--namely, its antiviral activity was species specific, and its activity was completely neutralized by antiserum to human fibroblast interferon. The amount of interferon synthesized in human fibroblasts induced by poly(rI)-poly(rC) (normal induction) and poly(rI)-poly(rC) in the presence of cycloheximide (superinduction) was compared to the amount of translatable interferon mRNA both in the wheat germ cell-free system and the Xenopus oöcyte system. Although the production of interferon after the termination of transcription by actinomycin D was markedly increased in superinduced cells, the measurable amount of interferon mRNA as assayed in the oöcyte system was only slightly higher in superinduced cells than in cells induced with poly(rI)-poly(rC) alone. When compared in the wheat germ cell-free system, however, the translational product of mRNA preparation from cells induced with poly(rI)-poly(rC) alone was inactive while that from superinduced cells was active.
Insights
Superinduction of human fibroblasts with poly(I:C) and cycloheximide enhances interferon mRNA translation. This process, essential for producing biologically active interferon, was confirmed using wheat germ cell-free and Xenopus oöcyte systems.
Area of Science:
- Molecular Biology
- Virology
- Biochemistry
Background:
- Interferon production is crucial for antiviral defense.
- Poly(I:C) is a known inducer of interferon synthesis.
- Superinduction techniques can enhance the yield of specific proteins.
Purpose of the Study:
- To compare the amount of translatable interferon mRNA in human fibroblasts under normal induction versus superinduction conditions.
- To assess the biological activity of interferon synthesized in vitro.
- To investigate the role of spermine in interferon translation.
Main Methods:
- Extraction of poly(A)-containing mRNA from human fibroblasts.
- Wheat germ cell-free protein synthesis system.
- Xenopus oöcyte translation system.
- Assay of antiviral activity and neutralization by antiserum.
Main Results:
- Spermine is essential for the translation of biologically active interferon mRNA.
- Superinduction significantly increased interferon production compared to normal induction.
- While oöcyte systems showed only a slight increase in mRNA, the wheat germ system demonstrated a marked increase in active interferon translation from superinduced cells.
Conclusions:
- Superinduction enhances the translational efficiency of interferon mRNA.
- The wheat germ cell-free system is effective for assaying translatable interferon mRNA and producing active interferon.
- Interferon translation requires specific conditions, including the presence of spermine.