Nuclease activation by double-stranded RNA and by 2′,5′-oligoadenylate in extracts of interferon-treated chick cells

Abstract

Cell-free extracts were prepared from primary chick embryo cells that had been pretreated overnight with chick interferon or with mock preparations of interferon. After the addition of double-stranded RNA (dsRNA) to these extracts, the synthesis of vesicular stomatitis virus (VSV) messenger RNA and its corresponding proteins was measured under conditions of coupled transcription and translation directed by purified virus. The addition of dsRNA to extracts of mock interferon-treated cells did not affect VSV-directed transcription or translation. However, in extracts of interferon-treated cells, the rate of RNA accumulation was inhibited by about 50% and that of the viral proteins by about 95% relative to the rates in control extracts. Furthermore, no intact VSV mRNA was detectable among the transcription products made in extracts of interferon-treated cells under these conditions. These results contrasted with those obtained in the absence of dsRNA, where the interferon-mediated inhibition of protein synthesis was less extreme, and where both sets of transcription products were intact and fully functional. The differences could be ascribed, at least in part, to a dsRNA-mediated increase in the rate of mRNA degradation that occurred only in extracts of interferon-treated cells. Enhancement of mRNA breakdown was also observed when protein synthesis was inhibited by the addition of pppA 2′p 5′A 2′p 5′A and higher oligomers (2′,5′-oligoadenylate) to extracts of either interferon-treated or mock interferon-treated cells. (These novel oligonucleotides are synthesized in interferon-treated cell extracts by an enzyme that requires dsRNA for activation.) Under a variety of conditions, enhanced mRNA degradation correlated with the inhibition of protein synthesis mediated by 2′,5′-oligoadenylate but not by other inhibitors. Thus, the data suggest that the inhibition of protein synthesis by dsRNA in extracts of interferon-treated chick cells is due, at least in part, to mRNA breakdown catalyzed by a nuclease that is activated by 2′,5′-oligoadenylate.