Interferon, double-stranded RNA, and protein phosphorylation (original) (raw)
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Double-stranded RNA inhibits a phosphoprotein phosphatase present in interferon-treated cells
Proceedings of the National Academy of Sciences, 1980
In accord with previous studies, (I)n . (C)n, a potent inhibitor of the cell-free protein-synthesizing system of interferon-treated L cells, stimulates incorporation of 32P from [gamma-32P]ATP into the 67,000-dalton protein, P1. The double-stranded RNA (I)n . (br5C)n, which is inactive as an inhibitory of protein synthesis, does not stimulate phosphorylation of P1 under conditions approximating those of protein synthesis. However, we have found conditions under which (I)n . (br5C)n is approximately as effective as (I)n . (C)n in stimulating incorporation of label from [gamma-32P]ATP into 67,000-dalton protein. Upon transfer of labeled P1 from these conditions to those compatible with protein synthesis, there is a time-dependent decrease in label in the 67,000-dalton protein. This decrease is more rapid in the presence of (I)n . (br5C)n than in the presence of (I)n . (C)n. This differential decrease is also observed when 32P-labeled extracts are diluted into buffer containing 10 mM A...
Interferon, Double-Stranded RNA and RNA Degradation. Characteristics of an Endonuclease Activity
European Journal of Biochemistry, 1977
Extracts from interferon-treated Ehrlich ascites tumor cells differ in various biochemical characteristics from extracts from control cells. Thus, as reported earlier, double-stranded RNA (dsRNA) promotes the phosphorylation by ATP of at least two proteins in extracts from interferon-treated cells,but not,or to only a lesser extent, in extracts from control cells. Moreover reovirus mRNAs are degraded faster in reaction mixtures containing extracts from interferon-treated cells than in those containing extracts from control cells but only if the reaction mixtures are supplemented with dsRNA and ATP. The faster RNA degradation in extracts from interferon-treated cells is due to enhanced endonuclease action. We designated the agent(s) catalyzing this as endonuclease1NT. There is some enhancement of nuclease activity by dsRNA and ATP also in extracts from control cells. The extent of this enhancement is, however, much smaller than in extracts from interferon-treated cells. We report now that the promotion of mRNA cleavage in extracts from interferon-treated cells by dsRNA and ATP is apparently not a consequence of a possible impairment in the attachment of the mRNA to ribosomes since (a) the protein synthesis inhibitors (sparsomycin and edeine) do not affect the degradation and (b) dsRNA and ATP enhance RNA degradation also in the 200000 x g supernatant fraction from extracts of interferon-treated cells and this fraction is essentially free of ribosomes.
Molecular and Cellular Biochemistry, 1983
The effects of double-stranded RNA (dsRNA) on interferon (IFN)-induced antiviral and anticellular activities was investigated by introducing poly(I)-poly(C) into mouse L-cells. Coprecipitation ofdsRNA with calcium phosphate enabled its efficient penetration into cells in culture. Rate of cellular protein synthesis was inhibited by dsRNA only in cultures pretreated with IFN. Moreover, the anticellular effect of IFN, as measured by the inhibition of cell DNA synthesis, was also enhanced by dsRNA. The kinetics of dsRNA-mediated inhibition of protein synthesis were relatively slow as compared with the inhibitory effect of 2'-5'oligoadenylic acid (2'5'A), which was also introduced into cells by the calcium phosphate coprecipitation technique. To analyze the effects of dsRNA on the antiviral state induced by IFN, vesicular stomatitis virus (VSV) and encephalomyocarditis virus (EMC), replications were followed by measuring viral-specific RNA synthesis in the cell. Introduction of dsRNA after the infection had no effect on VSV and EMC replication in control cells, and it enhanced, to a small extent, the antiviral state of cells pretreated with IFN. In contrast, introduction of 2'5'A into virus-infected cells inhibited VSV and EMC replications regardless of IFN pretreatment. This work demonstrated that the role of dsRNA in regulating the antiviral and anticellular activities of IFN could be studied by introducing exogenous dsRNA into cells in culture by the calcium phosphate coprecipitation technique. Abbreviations 2'5'A = triphosphoadenyl (2'5') adenylyl (2'-5') adenosine; Pi = the interferon-induced 67 000 dalton protein; RNase F = the 2'5'A-dependent ribonuclease; Hepes = 4-(2-hydroxy)-l-piperazineethane sulfonic acid.
Journal of General Virology, 1977
Extracts from mouse interferon-treated mouse cells (Ehrlich ascites tumour and L929) were reported earlier to differ in several characteristics from extracts of untreated ceils. We report now that the effect of treatment with human interferon of cells of a human line (HeLa 3)issimilarlymanifestedinthecellextract.Acomparisonofextractsfrominterferon−treatedHeLa3) is similarly manifested in the cell extract. A comparison of extracts from interferon-treated HeLa 3)issimilarlymanifestedinthecellextract.Acomparisonofextractsfrominterferon−treatedHeLa3 cells (S3o~r) with that from untreated cells ($3oc) revealed: (a) an impairment in S3OrNT of the methylation of (unmethylated) reovirus mRNAs by the cellular and virus enzymes; (b) a faster degradation of reovirus mRNAs in S3o~T but only in reaction mixtures supplemented with double-stranded RNA and ATP ;(c) an increased phosphorylation by ATP of one (or two), proteins in S3Or~T but only in reaction mixtures supplemented with double-stranded RNA and (d) a more pronounced inhibition of translation by double-stranded RNA in S3o~T. No such effects were observed in extracts prepared from HeLa cells treated with mouse interferon.
Double-Stranded Rna and the Enzymology of Interferon Action
Annals of the New York Academy of Sciences, 1980
Interferons were discovered in 1957 as antiviral agents.' Investigations in the last 22 years, however, revealed their involvement in the regulation of a large variety of seemingly diverse physiological phenomena and processes. These include for example cell growth, delayed hypersensitivity, graft rejection, histocompatibility antigen expression, natural killer-cell recruitment and macrophage activation. It appears to be in line with this multiplicity of effects that the biochemistry of interferon (IF) action is also complex. Much of our knowledge of this biochemistry is based on the comparison of enzyme activities in extracts from IF-treated and control cells and has been gained in the last five In this communication we will summarize briefly our studies of two enzyme systems controlled by IF. The action of one results in the accelerated cleavage of single-stranded RNA, that of the other in the impairment of peptide chain initiation. The two enzyme systems are distinct, though both require doublestranded (ds) RNA for activation' (see also references 4 and 6-9). Our studies concerning other effects of IF treatment of cells on enzyme reactions ( e g . an impairment of mRNA cap methylation in vitro and in vivoto-'' and the acceleration of tRNA inactivation in vitroI3) have been summarized e 1 s e~h e r e . l~