Affinity labelling and characterization of the ppp(A2'p)nA-dependent endoribonuclease from different mammalian sources

The ppp(A2'p)nA-dependent endoribonucleases from a number of different mammalian sources have been investigated. The enzyme from reticulocyte lysates shows optimal activity of 50-150 mM KCl and requires the presence of Mg2+. Whilst the enzyme is inactivated after passage of reticulocyte lysates through Sephadex columns in the absence of ATP, it retains full activity provided ATP is included in the column buffer. The activity of the partially purified nuclease was unaffected by the addition of reticulocyte RNase inhibitor, which, in contrast, effectively inhibited other endogenous endonucleases. The ppp(A2'p)nA-dependent Rnase co-purified with a ppp(A2'p)nA-binding protein and with a protein which could be specifically covalently labelled with an oxidised radioactive analogue of ppp(A2'p)nA. This covalent labelling could be carried out either with the partially purified RNase or in crude extracts from rabbit reticulocytes, mouse Krebs and Ehrlich ascites tumour cells and human lymphoblastoid (Daudi) or HeLa cells. In each case the affinity labelled protein migrated to a position corresponding to a apparent molecular weight of about 85 000 on electrophoresis on dodecylsulphate/polyacrylamide gels. In all cases labelling could be prevented by the addition of an excess of unlabelled ppp(A2'p)nA but not, for example, by a similar excess of the biologically inactive dimer ppp(A2'p)'A. It is concluded that the RNase and ppp(A2'p)nA binding activities are likely to reside in the same molecule.     

Intracellular metabolism of the interferon mediator, 2-5A, using permeabilized cells

2-5A synthetase and 2'-phosphodiesterase, the enzymatic activities which respectively synthesize and degrade the interferon mediator 2-5A (ppp(A2'p)nA), were studied in digitonin-permeabilized cells. 2-5A synthetase was higher in permeabilized than in lysed Daudi cells. Mouse L cells appeared to contain two different 2-5A synthetase activities, one of which could be separated from 2'-phosphodiesterase activity, which was only cytosolic. Permeabilization techniques offer opportunities to investigate (2',5')-oligoadenylate intracellular metabolism, which remains incompletely known.     

Double-stranded RNA-dependent protein kinase and 2-5A system are both activated in interferon-treated, encephalomyocarditis virus-infected HeLa cells.

Activation of the interferon-inducible, double-stranded RNA-dependent protein kinase was monitored in monolayer cultures of control and interferon-treated HeLa cells infected with encephalomyocarditis virus. The extent of phosphorylation in the intact cell of the alpha-subunit of eucaryotic protein synthesis initiation factor eIF2 by the kinase was determined for the first time in this type of system, using a two-dimensional immunoblot technique. Virus protein synthesis and the kinetics of activation of the ppp(A2'p)nA (n greater than or equal to 2) system were analyzed in parallel. Enhanced phosphorylation of eIF2-alpha was obvious at 9 h and increased by 12 h postinfection. ppp(A2'p)nA and ppp(A2'p)nA-mediated rRNA cleavage were observed from 6 h. No viral protein synthesis was detected in cells in which a general inhibition of protein synthesis developed with time. It can be concluded that both the kinase and ppp(A2'p)nA system are active in interferon-treated, encephalomyocarditis virus-infected HeLa cells.     

Regulation of the 2',5'-oligoadenylate level in mouse L cells

A study of pH dependence for ppp5'A2'p5'A2'p5'A hydrolysis in interferon treated and untreated mouse L-cells extracts led to the detection of two types of the 2'-phosphodiesterase activities: interferon dependent and interferon resistant. Several pH-optima were observed for hydrolysis of ppp5'A2'p5'A2'p5'A in cell extracts after their treatment with non-ionic detergent NP-40 or their differential centrifugation. The 2'-phosphodiesterase activity was found in the membrane fraction as well as in the cytoplasmic one. The presence of several pH-optima for 2'-phosphodiesterase activity in L-cells and changes of the level of this activity depending on the growth stage of cells and time of their interferon treatment indicate the complicated character of the regulation of 2'-5'-oligoadenylate's concentration and localization. The results obtained suggest that in mouse L-cells several 2'-phosphodiesterases or one enzyme in different forms may be present.     

Activation of the ppp(A2'p)nA system in interferon-treated, herpes simplex virus-infected cells and evidence for novel inhibitors of the ppp(A2'p)nA-dependent RNase

High doses (100-1000 reference units/ml) of alpha or beta interferons are required to inhibit the growth of herpes simplex virus types I and II (HSV-I and HSV-II) in human Chang cells. In contrast, much lower doses (10-100 reference units/ml) of interferon inhibit replication of encephalomyocarditis virus (EMCV) in these cells. In the HSV-infected cells these high doses did not prevent the virus-induced shut off of host protein synthesis. The interferons were more effective in reducing the virus yield of HSV-I than of HSV-II. At the above concentrations they inhibited HSV-I protein synthesis but had little apparent effect on that of HSV-II. Similar amounts of (2'-5')oligo(adenylate)s were synthesised in response to HSV-I, HSV-II and EMCV infection of Chang cells after treatment with alpha or beta interferons. No (i.e. less than 1 nM) (2'-5')oligo(adenylate)s were found in control cells or on virus infection alone. Only low levels of ppp(A2'p)nA-specific rRNA cleavage were observed in the interferon-treated HSV-infected cells. In contrast, high levels were found in response to EMCV, despite the fact that ppp(A2'p)nA accumulated to similar levels with each of the three viruses in these cells. High-performance liquid chromatographic analysis of material from interferon-treated Chang cells 18 h after infection with HSV-I or HSV-II, combined with radiobinding, radioimmune and rRNA cleavage assays, confirmed the presence of ppp(A2'p)2A and ppp(A2'p)3A at greater than nanomolar concentration. In addition, apparently equivalent amounts of two other putative (2'-5')oligo(adenylate) derivatives which compete in the radiobinding and radioimmune assays, were present. These compounds were only weak activators of the ppp(A2'p)nA-dependent RNase and under appropriate conditions were capable of inhibiting the activation of this RNase by authentic ppp(A2'p)nA. The presence of these potentially inhibitory compounds provides a possible explanation for the relatively low levels of activation of the ppp(A2'p)nA-dependent RNase in interferon-treated, HSV-infected Chang cells.     

Oligo(2'-5')adenylate synthetase in human lymphoblastoid cells

The enzyme oligo(2′–5′)adenylate synthetase, when activated by double-stranded RNA, polymerizes ATP into the novel oligonucleotide (2′–5′)ppp(Ap)_nA. We describe conditions for assay of this enzyme in crude extracts of a human lymphoblastoid cell line, Namalwa. The production of (2′–5′)ppp(Ap)_nA by Namalwa extracts was 3–5 times greater than the production by extracts of interferon pretreated mouse L cells, and 700 fold higher than the production by extracts of untreated mouse L cells. The relatively high level of oligo(2′–5′)adenylate synthetase in Namalwa cells was not attributable solely to their constitutive secretion of low levels of interferon. Analysis of the size distribution of the oligomers formed at different times suggested that the enzyme can add ATP to a free pppApA. Infection by Newcastle disease virus or treatment with interferon raised the apparent synthetase levels only marginally. Experiments that employed antibody to interferon suggested that the interferon must be externalized from the NDV-infected cell to induce maximal synthetase levels.     

Biological activities of phosphodiester linkage isomers of 2-5A

To determine the relative importance of the 2',5'-phosphodiester bond of 2-5A in its binding to and activation of the 2-5A-dependent ribonuclease (RNase L, RNase F), a number of phosphodiester linkage isomers of 2-5A were prepared. These isomers were obtained either by lead ion-catalyzed polymerization of adenosine 5'-phosphorimidazolidate or by T4 polynucleotide kinase-catalyzed 5'-phosphorylation of adenylyl(3' leads to 5')adenylyl(3' leads to 5')adenosine followed by reaction of the corresponding phosphorimidazolidates with tri(n-butylammonium)pyrophosphate. The following 2-5A isomers thus were prepared: ppp5'A2'p5'A3'p5'A, ppp5'A3'p5'A2'p5'A, ppp5'A3'p5'A3'p5'A("3-5A"), ppp5'A2'p5'A3'p5'A2'p5'A,and ppp5'A3'p5'A2'p5'-A2'p5'A. The ability of these isomeric 2-5As to interact with the 2-5A-dependent endonuclease was ascertained by three different criteria: (i) ability to prevent the protein synthesis inhibitory effects of 2-5A, (ii) activity as an inhibitor of translation in encephalomyocarditis RNA-programmed L cell extracts, and (iii) ability to prevent binding of the radiolabeled probe, ppp5'A2'p5'A2'p5'A2'p5'A3'[32P]p5'Cp, to the endonuclease of L cell extracts. In certain experiments, degradation of oligonucleotide was minimized or eliminated by altering assay conditions, providing alternate phosphodiesterase substrates, or by using purified endoribonuclease of Ehrlich ascites cells. By all criteria, replacement of 2',5'-bond by a 3',5'-bond led to a substantial decrease in biological activity. Generally, replacement of just one 2',5'-phosphodiester bond with a 3',5'-linkage led to at least a one order of magnitude loss of activity. In accord with this trend, ppp5'A3'p5'A3'p5'A(3-5A) was greater than 10,000 less active than 2-5A in binding to the endonuclease or as an inhibitor of protein synthesis.     

Analogue inhibitor of 2-5A action: effect on the interferon-mediated inhibition of encephalomyocarditis virus replication.

Chemically synthesised CH3Sp(A2'p)2A2'pp3'OCH3 has been used to assess the importance of the ppp(A2'p)nA (n greater than or equal to 2: 2-5A) system in the antiviral action of interferon against encephalomyocarditis virus (EMC). It inhibits activation of the 2-5A-dependent RNase by 2-5A in intact mouse L929 cells and cell-free systems. In interferon-treated, EMC-infected L929 cells it inhibits 2-5A-mediated rRNA cleavage and partially restores EMC RNA synthesis and virus yield. Activation of the 2-5A-dependent RNase must, therefore, play some part in interferon action against the growth of EMC virus in such cells.     

Ribosomal RNA cleavage, nuclease activation and 2-5A(ppp(A2''p)nA) in interferon-treated cells.

Ribosomal RNA (rRNA) in intact ribosomes is cleaved into discrete products on incubation of reticulocyte lysates or L-cell extracts with ppp(A2'p)3A. Cleavage of rRNA may, therefore, provide a useful assay for 2-5A (ppp)A2'p)nA; n = 2 to 4) or for the presence of a 2-5A-dependent nuclease. The results with reticulocyte lysates differed from those obtained in the L-cell-free system in that (a) a different RNA cleavage pattern was produced (with added L-cell ribosomes) and (b) cleavage was fully activated by the analogue ppp(A2'p)3A3'pCp. As might be expected from the relatively high levels of 2-5A present in interferon-treated, encephalomyocarditis virus (EMC)-infected L-cells, rRNA extracted from these cells was also cleaved. The cleavage pattern observed overlapped with that obtained on incubation of an L-cell-free system with 2-5A. Thus, not only is 2-5A present, but the 2-5A-dependent nuclease also appears to be active, in interferon-treated, EMC-infected L-cells.     

Use of a 2-5A analogue probe for detecting RNA ligase and RNA ligase substrates in mammalian cell extracts

The compound ppp(A2'p)3A3'[32P]pCp is a commercially available radioactive analogue of the 2',5' oligoadenylate series ppp(A2'p)nA, n greater than or equal to 2, commonly referred to as 2-5A. It is used as a probe for measuring concentrations in competition radiobinding and radioimmune assays. We have found that incubation of the probe with extracts from HeLa, CV1, or neuroblastoma cells results in its covalent attachment to two size classes of RNA: the first includes a major species with a molecular weight of approximately 350,000, the second is much smaller (40 +/- 5 nucleotides in length) and could represent tRNA half-molecules. Ligation is to the 3' end of the probe molecule with formation of a 3',5'-phosphodiester bond. Thus, probe ligation provides a sensitive and convenient assay for the detection not only of RNA ligase(s) but also of ligatable RNAs (such as the putative tRNA half-molecules) in mammalian cell extracts.     

Novel 2'',5''-oligoadenylates synthesized in interferon-treated, vaccinia virus-infected cells.

2-5A[ppp(A2'p)nA] and related materials accumulate to greater than micromolar concentrations in vaccinia virus-infected HeLa cells and in interferon-treated, vaccinia-infected HeLa, CV1, and L929 cells even when virus replication is not inhibited. A variably complex mixture of authentic 2-5A (n = 2 to 4), nonphosphorylated cores [(A2'p)nA; n = 2 to 5], and additional compounds of unknown structure was observed.     

Phosphorylation of 2-5A core 5'-diphosphate to 2-5A in mouse L cell extracts

When added to extracts of mouse L cells containing ATP and an energy regenerating system, the 5'-diphosphate of 2-5A core, pp5'A2'p5'A2'p5'A, as well as a bromoadenylate analog, pp5' (br8A)2'p5'(br8A)2'p5'(br8A), can be phosphorylated to the corresponding 5'-triphosphate, ppp5'A2'p5'A2'p5'A and ppp5'(br8A)2'p5'(br8A)2'p5(br8A), respectively. The extent of this conversion was about 0.5% when the concentration of 5'-diphosphate was about 10(-4) M. Thus, although previous studies have shown that the 5'diphosphate, pp5'A2'p5'A2'p5'A, can activate the 2-5A-dependent endonuclease, this may be related to a phosphorylation reaction in the crude cell extracts employed in these studies and may not represent a true ability of such a 5'-diphosphate to activate directly the endonuclease.     

Respective role of each of the purine N-6 amino groups of 5'-O-triphosphoryladenylyl(2'----5')adenylyl(2----5')adenosine in binding to and activation of RNase L

We have synthesized a series of 2-5A (ppp5'-A2'p5'A2'p5'A) analogs in which each adenosine residue has been sequentially replaced by inosine: viz., ppp5'I2'p5'A2'p5'A, ppp5'A2'p5'I2'p5'A, and ppp5'A2'p5'A2'p5'I. These transformations enabled us to delineate the role of each of the three purine N-6 amino groups of 2-5A in determining oligonucleotide binding to and activation of the 2-5A-dependent endoribonuclease, RNase L. With the RNase L activity of both mouse L cells and human Daudi lymphoblastoid cells, we found that the N-6 amino group of the first adenosine nucleotide residue (from the 5'-terminus) is of crucial importance in determining binding to the endonuclease; however, removal of the N-6 amino moieties of the second or third adenosine nucleotide residues resulted in only a minimal decrease in binding to the endonuclease. On the other hand, conversion of the third adenosine residue to inosine effected a dramatic (10,000-fold compared to 2-5A) loss in ability to activate the nuclease; however, execution of the same N-6 amino group conversion at either the first or second adenosine residue did not cause a major change in nuclease activation ability when the accompanying decreased endonuclease binding was considered. These results clearly demonstrate that the N-6 amino group of the first adenosine residue of 2-5A is critical in RNase L binding whereas the N-6 amino function of the third adenosine residue of 2-5A is crucial for the activation of RNase L.     

Interferon- and sarcolectin-dependent cellular regulatory interactions

Interferons, via specific membrane-bound receptors, induce various cellular functions of which antiviral protection is the most extensively studied. We have previously reported the existence of interferon antagonists (referred to as sarcolectins) in various tissue extracts from placental blood, cartilage, brain, muscle, or from sarcomas. These sarcolectins have been fully characterized and purified to homogeneity. In interferon-treated cells, they restore virus sensitivity 4-6 h after the establishment of antiviral protection. In the present study we investigate the effect of sarcolectins on the steady state levels of two double-stranded RNA dependent enzymes, 2-5A (p chi (A2'p)nA) synthetase and protein kinase. Several authors have previously emphasized the role of these enzymes in the mechanism of interferon's antiviral action. Interferon promotes a 4-8 fold increase in protein kinase and 2-5A synthetase in cells. Addition of sarcolectin 5 h after interferon results in a dramatic reduction in the steady state levels of both these enzymes, as shown by their decreased activity and yield observed in Western blot assays. The degradation of the antiviral response in sarcolectin-treated cells might therefore be at least partially attributed to a reduced synthesis of protein kinase and 2-5A synthetase. Since there are no direct interactions between sarcolectins and interferon or its receptors, it can be postulated that sarcolectins exert their effect through these interferon-dependent proteins. We postulate that the opposing biological effects of interferon and sarcolectins strike a balance which may, however, be modified in one direction or the other, depending on their respective concentrations.     

Antiviral effects of 2',5'oligoadenylates (2-5As), and related compounds

Dephosphorylated "core" of 2',5'-oligoadenylate (2-5A) dimer (A2'p5'A), exogenously added to nonpermeabilized FL cells, inhibited the multiplication of Sindbis virus and vesicular stomatitis virus (VSV). The compound was shown to inhibit viral protein synthesis. The addition of A2'p5'A at the early stage of viral replication was more effective than that at the late stage. In contrast with the core, phosphorylated 2-5A (p5'A2'p5'A and ppp5'A2'p5'A) and 2-5A analogs containing cordycepin (3'-deoxyadenosine) did not show such antiviral effects. The rate of uptake of [3H]ppp5'A 2'p5'A into acid-soluble and acid-insoluble fractions, especially into the acid-insoluble fraction, was faster than that of [3H]A2'p5'A. These results suggest that the difference of antiviral activity between A2'p5'A and ppp5'A2'p5'A does not result from the different rate of uptake by cells, but from the different rate of from acid-soluble to acid-insoluble fractions.     

Synthesis and biological activity of tubercidin analogues of ppp5'A2'p(5'A2'p)n5'A

A series of tubercidin (7-deazaadenosine) analogues of 2-5A of the general formula p5'(c7A)2'p[5'(c7A)-2'p]n5'(c7A) (n = 0-5) were prepared by lead ion catalyzed polymerization of the 5'-phosphoroimidazolidate of tubercidin. Through the corresponding imidazolidates, these oligonucleotide 5'-monophosphates were converted to the 5'-triphosphates. All reported structures were corroborated by enzyme digestion and 1H or 31P nuclear magnetic resonance. When evaluated for its ability to bind to the 2-5 A-dependent endonuclease of mouse L cells, the tubercidin analogue of trimeric 2-5A, namely, ppp5'(c7A)2'p5'(c7A)2'p5'(c7A), and the corresponding tetramer were bound as effectively as 2-5A itself; nonetheless, it and the corresponding tetramer, ppp5'-(c7A)2'p5'(c7A)2'p5'(c7A)2'p5'(c7A), failed to stimulate the 2-5A-dependent endonuclease as judged by its inability to inhibit translation in extracts of mouse L cells programmed with encephalomyocarditis virus RNA and to give rise to ribosomal RNA cleavage in the same cell system under conditions where 2-5A showed activity at 10(-9) M. The trimer, ppp5'(c7A)2'p5'(c7A)2'p5'(c7A), was an antagonist of 2-5A action in the L cell extract. In the lysed rabbit reticulocyte system, both the trimeric and tetrameric tubercidin 2-5A analogues were bound to the 2-5A-dependent endonuclease as well as 2-5A, but in this case, the tetramer triphosphate, ppp5'(c7A)2'p5'(c7A)2'p5'(c7A)2'p5'(c7A), was just as potent an inhibitor of translation as 2-5A tetramer triphosphate. Moreover, this inhibition was prevented by the established 2-5A antagonist p5'A2'p5'A2'p5'A.(ABSTRACT TRUNCATED AT 250 WORDS)     

Distribution of the ppp(A2'p)nA-binding protein and interferon-related enzymes in animals, plants, and lower organisms

A ppp(A2′p)_nA binding protein and synthetase, but no double-stranded RNA-dependent protein kinase, have been found in extracts from reptilian tissues. A binding protein is also present at low levels in amphibia. No evidence was obtained for the presence of any of these proteins or of ppp(A2′p)_nA in extracts from differently pretreated tobacco plant leaves with or without tobacco mosaic virus infection, despite reports (1,2) of the sensitivity of the latter to interferon and (A2′p)₂A. This is consistent with our inability to detect the ppp(A′p)_nA system in any of the lower eukaryotes or prokaryotes investigated.     

Cordycepin analogs of ppp5'A2'p5'A2'p5'A (2-5A) inhibit protein synthesis through activation of the 2-5A-dependent endonuclease

Analogs of the triphosphate 2'-5'-linked adenylate trimer (ppp5'A2'p5'A2'p5'A, called 2-5A) which contain 3'-deoxyadenosine (cordycepin) instead of adenosine either in positions one and two, or in all three positions, are 10-100-fold less potent than is parent 2-5A in inhibition of protein synthesis in intact cells, when utilizing calcium co-precipitation techniques to introduce the 5'-triphosphate oligonucleotides into the cells. That the inhibition of protein synthesis was a consequence of activation of the 2-5A-dependent endonuclease by the 3'-deoxyadenosine analogs of 2-5A was demonstrated in obtaining the ribosomal RNA cleavage pattern that is characteristic of endonuclease activation by parent 2-5A. Additional results (i.e. lack of activity by the dimer species ppp5'(3'dA)2'p5'-(3'dA) or the monomer 3'dA) as well as kinetic analysis both in intact cells and in cell-free extracts provided further evidence that the inhibition of protein synthesis observed with these 3'-deoxyadenosine 2-5A analogs was not due to their degradation to the antimetabolite monomer unit 3'-deoxyadenosine.     

Induction of interferon-specific enzymes in cultures of cells sensitive and resistant to interferon

Induction of 2'-5'-oligoadenylatesynthetase (2-5A synthetase) by interferons and theophylline by means of activation of cAMP-system in interferon susceptible and resistant cell lines were studied. In interferon resistant cell lines the basal activity of 2-5A synthetase exceeded the level of the same enzyme in interferon susceptible cell lines. Activity of 2-5A synthetase is increased in interferon susceptible cell lines by interferon treatment, but the activity of the enzyme is not altered in interferon resistant cell lines. Among the studied cell lines the induction of 2-5A synthetase by theophylline was possible only in L929 cell line. The common mechanism for the absence of 2-5A synthetase induction by interferon and theophylline in interferon resistant cells is discussed.