Interferon-induced biochemical changes in cell membranes: possible role of cellular enzyme superoxide dismutase (original) (raw)
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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.
Role of Interferon-Induced Enzymes in the Antiviral and Antimitogenic Effects of Interferon
Annals of the New York Academy of Sciences, 1980
Exposure of human or animal cells to their respective interferons (IFs) produces, within a few hours, an increase in several enzymes, whose function appears to be mainly related to regulation of protein biosynthesis. FIGURE 1 summarizes what is known of the mechanism of action of three such enzymes: protein kinase PK-i, (2'-5') oligo A synthetase E, and phosphodiesterase 2'-PDi. (For a complete review and bibliography see ref. 1). Induction of these enzymes was seen in our laboratory in human fibroblasts and Namalva cells, in monkey BSC-1 cells, in bovine MDBK cells and in mouse L cells and splenic lymphocytes. Using a microassay that allows the determination of the enzyme levels with about 25,000 cells (one well of a 96-hole microplate], the kinetics of induction were determined.'A typical lag period of 2-4 hr is observed after IF addition and before the enzyme levels begin to rise. Maximum increase is reached after 18-24 hr. With actinomycin D, it was possible to show that in mouse L cells, the transcription step necessary for induction of the protein kinase and of the (2'-5')
Journal of Biological Chemistry, 1985
The double-stranded RNA (dsRNA)-dependent protein kinase which catalyzes the phosphorylation of ribosome-associated protein Pl and the a subunit of eukaryotic protein synthesis initiation factor 2 (eIF-2) was purified and characterized from mouse fibroblast L929 cells treated with either natural or recombinant interferon and from untreated cells. The dsRNA-dependent Pl/eIF-2a kinase was purified at least 1,500fold from interferon-treated cells; the kinase activity that catalyzed the phosphorylation of eIF-2a copurified with protein PI. The yield of Pl/eIF-2a protein kinase activity obtained following purification from cells treated with interferon was about 5-10 times greater than the yield from an equivalent number of untreated cells. The purified protein kinase remained dsRNA dependent. When PI kinase was activated by dsRNA, a major phosphopeptide designated Xa. was phosphorylated; X& was not phosphorylated from PI which had not been activated by dsRNA. The apparent native molecular weight of the purified mouse L929 dsRNAdependent kinase as determined by sedimentation analysis was about 62,000, comparable to the molecular weight of 67,000 determined for denatured L929 phosphoprotein PI by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The purified protein kinase was highly selective for the a subunit of protein synthesis initiation factor eIF-2 and endogenous protein P1. Kinase activity was dependent upon Mg2+, and the K, for ATP was determined to be 5 x lo-' M. Histones (HI, Ha, and H4) and protein synthesis initiation factors other than eIF-2 (eIF-3, eIF-4A, eIF-4B, and eIF-5) were not substrates or were very poor substrates for the purified dsRNA-dependent protein kinase. N-Ethylmaleimide, ethylenediaminetetraacetic acid, AMP, pyrophosphate, spermine, spermidine, and high concentrations of potassium inhibited both PI and eIF-Za phosphorylation by the purified kinase, whereas ethylene glycol bis(8-aminoethyl ether)-NtNtN',N'-tetraacetic acid and phenanthroline did not significantly affect the phosphorylation of either protein PI or eIF-2a. Interferons are a family of regulatory proteins that can profoundly affect a variety of functions in animal cells includ
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~
Studies on the Mechanism of Interferon Action
The Journal of General Physiology, 1970
Interferon does not inactivate viruses or viral RNA. Virus growth is inhibited in interferon-treated cells, but apart from conferring resistance to virus growth, no other effect of interferon on cells has been definitely shown to take place. Interferon binds to cells even in the cold, but a period of incubation at 37°C is required for development of antiviral activity. Cytoplasmic uptake of interferon has not been unequivocally demonstrated. Studies with antimetabolites indicate that the antiviral action of interferon requires host RNA and protein synthesis. Experiments with 2-mercapto-1(ß-4-pyridethyl) benzimidazole (MPB) suggest that an additional step is required between the binding and the synthesis of macromolecules. Interferon does not affect the adsorption, penetration, or uncoating of RNA or DNA viruses, but viral RNA synthesis is inhibited in cells infected with RNA viruses. The main action of interferon appears to be the inhibition of the translation of virus genetic infor...
Interferon, double-stranded RNA, and protein phosphorylation
Proceedings of the National Academy of Sciences, 1976
We reported earlier that the addition of double-stranded RNA and ATP increases the endonuclease activity more in an extract of Ehrlich ascites tumor cells which have been treated with an interferon preparation than in a comparable extract from control cells. We repo here that the addition of double-stranded RNA to an extract from Ehrlich ascites tumor cells which have been treated with an interferon preparation [or with the interferon inducer poly(I).poly(C)J promotes the phosphorylation by ['y-32PJATP of at least two proteins: P1 (molecular weight of 64,000) and P2 (molecular weight of 37,000) Double-stranded RNA also promotes the phosphorylation of at least one (i.e., PI) of these two proteins in an extract from cells
Antiviral Actions of Interferons
Clinical Microbiology Reviews, 2001
SUMMARY Tremendous progress has been made in understanding the molecular basis of the antiviral actions of interferons (IFNs), as well as strategies evolved by viruses to antagonize the actions of IFNs. Furthermore, advances made while elucidating the IFN system have contributed significantly to our understanding in multiple areas of virology and molecular cell biology, ranging from pathways of signal transduction to the biochemical mechanisms of transcriptional and translational control to the molecular basis of viral pathogenesis. IFNs are approved therapeutics and have moved from the basic research laboratory to the clinic. Among the IFN-induced proteins important in the antiviral actions of IFNs are the RNA-dependent protein kinase (PKR), the 2′,5′-oligoadenylate synthetase (OAS) and RNase L, and the Mx protein GTPases. Double-stranded RNA plays a central role in modulating protein phosphorylation and RNA degradation catalyzed by the IFN-inducible PKR kinase and the 2′-5′-oligoa...