Inhibition of 2',5'-oligo(A)-dependent endoribonuclease by 2',5'-oligo(A) degradation products.
Addition of extract of interferon (IFN)-treated HeLa cells to transcription reactions containing activated reovirion cores decreases the yield of viral mRNA (C. Baglioni, A. De Benedetti, and G. J. Williams, 1984, J. Virol. 52, 865-871). The 2'5'-oligo(A) (2-5A)-dependent endonuclease (RNase L) cleaves specifically viral mRNA, but little 5'-triphosphate 2-5A is recovered from these reactions by DEAE-cellulose chromatography. However, in the present study we detected microM concentrations of 2-5A derivatives by binding to RNase L. Similar results were obtained when the synthetic double-stranded RNA poly(I) X poly(C) was incubated with extract from IFN-treated cells: microM concentrations of 2-5A were detected by the binding assay, but little rRNA was degraded by RNase L. 2-5A derivatives which inhibited the activation of RNase L by authentic 2-5A were eluted from DEAE-cellulose with 90 mM KCl. These inhibitors were also formed by incubating purified 2-5A with HeLa cell extract. These results indicated that 2-5A was synthesized in the incubations with reovirion cores or poly(I) X poly(C), but that it was in large part degraded to compounds inhibitory for RNase L. IFN-treated HeLa cells were incubated with poly(I X C), but little rRNA degradation was detected in spite of the presence of high concentrations of 2-5A in these cells. Most of this 2-5A was eluted with 90 mM KCl from DEAE-cellulose and was inhibitory for RNase L. This indicated that 2-5A was degraded to inhibitory derivatives also in intact cells. The structure of the degradation products of 2-5A has not been established, but they contain free terminal phosphate(s), since their binding to RNase L and the inhibition of this enzyme is abolished by digestion with phosphatase.
G J
GJ
Williams
A
A
De Benedetti
C
C
Baglioni
D000227:Adenine Nucleotides / Q000378:metabolism* / Q000494:pharmacology; D004722:Endoribonucleases / Q000037:antagonists & inhibitors* / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D007372:Interferons / Q000494:pharmacology; D012089:Mammalian orthoreovirus 3 / Q000235:genetics / Q000378:metabolism*; D009843:Oligoribonucleotides / Q000378:metabolism* / Q000494:pharmacology; D011070:Poly I-C / Q000494:pharmacology; D012333:RNA, Messenger / Q000378:metabolism; D012335:RNA, Ribosomal / Q000378:metabolism; D012367:RNA, Viral / Q000378:metabolism; D012087:Reoviridae / Q000378:metabolism*; D014158:Transcription, Genetic
D000227:Adenine Nucleotides; D009843:Oligoribonucleotides; D012333:RNA, Messenger; D012335:RNA, Ribosomal; D012367:RNA, Viral; C023505:2',5'-oligoadenylate; D007372:Interferons; D004722:Endoribonucleases; C031956:2-5A-dependent ribonuclease; D011070:Poly I-C