rRNA cleavage as an index of ppp(A2''p)nA activity in interferon-treated encephalomyocarditis virus-infected cells.

In cell-free systems, 2-5A [ppp(A2'p)nA, n = 2 to greater than or equal to 4] activates a latent endoribonuclease, the 2-5A-dependent RNase, which cleaves rRNA in intact ribosomes into discrete and characteristic products (D. H. Wreschner et al., Nucleic Acids Res. 9:1571-1581, 1981). Here we present Northern blots which have identified the 18S or 28S origins of the cleaved products from rRNA. In addition, identical 3' termini were observed for fragments of 18S rRNA from a HeLa cell-free system incubated with 2-5A and from interferon-treated, encephalomyocarditis virus-infected HeLa cells. The previous assumption of identity of such fragments was based only on comigration on electrophoresis in agarose gels. We conclude that appropriate patterns of cleavage found in RNA isolated from intact cells are an indicator of prior 2-5A-dependent RNase activity. The assay of rRNA cleavage is relatively convenient and unambiguous. Accordingly, in the search for situations in which the 2-5A system may be active, it provides a useful alternative to the direct assay of 2-5A.     

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.     

Independent regulation of ppp(A2'p)nA-dependent RNase in NIH 3T3, clone 1 cells by growth arrest and interferon treatment

The regulation of ppp(A2'p)nA-(2-5A)-dependent RNase (RNase L or RNase F) was investigated in NIH 3T3, clone 1 cells using 2-5A-binding and nuclease activity assays. Minimal levels of 2-5A-dependent RNase were detected in actively dividing clone 1 cells; these levels were independently induced by growth arrest or interferon treatment. Accordingly, levels of the RNase were enhanced during growth arrest by confluency regardless of the presence or absence of interferon or antibody to interferon in the media. Measurement of 2-5A-dependent RNase was unaffected by the addition of any of six different proteinase inhibitors to the cells prior to extraction. The expression of 2-5A-dependent RNase in growth-arrested, interferon-treated cells was still relatively low (about one-third to one-half of that found in similarly treated murine Ehrlich ascites tumor cells). Although this amount of 2-5A-dependent RNase could not be detected by 2-5A-mediated ribosomal RNA cleavage, the activity was identified using a more sensitive novel assay for 2-5A-dependent RNase. In addition, introduction of 2-5A or poly(I) X poly(C) into growth-arrested, interferon-treated cells resulted in some inhibition of protein synthesis. The results indicated that the expression of 2-5A-dependent RNase in NIH 3T3, clone 1 cells is regulated under different physiological conditions and that low levels of 2-5A-dependent RNase were insufficient to significantly inhibit encephalomyocarditis virus replication.     

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)     

Modulation of ppp(A2''p)nA-dependent RNase by a temperature-sensitive mutant of vaccinia virus.

Activation of the ppp(A2'p)nA (2-5A)-dependent RNase was investigated during the abortive infection of BSC40 cells by a temperature-sensitive mutant of vaccinia virus, ts22. At the nonpermissive temperature, ts22 has an abortive late phenotype. At the onset of late-viral-gene expression, viral mRNA is degraded and rRNA is cleaved into discrete fragments in the absence of prior interferon treatment (R. F. Pacha and R. C. Condit, J. Virol. 56:395-403, 1985). Concomitant with rRNA cleavage, an increase in 2-5A occurred late during infection. Discrete 18S- and 28S-rRNA degradation products from BSC40 cells infected with ts22 at the nonpermissive temperature comigrated in denaturing agarose gels with rRNA cleaved fragments produced by the activation of 2-5A-dependent RNase in uninfected cells transfected with exogenous 2-5A. An increase in 2-5A levels and a similar discrete and characteristic degradation of rRNA were observed in BSC40 cells infected with wild-type vaccinia virus in the presence of isatin-beta-thiosemicarbazone. The results show that the ts22 lesion and the action of isatin-beta-thiosemicarbazone may affect the same pathway, leading to the activation of latent 2-5A-dependent RNase and resulting in indiscriminate RNA degradation and inhibition of viral replication.     

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.     

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.     

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.     

Release of ribosome-bound 5S rRNA upon cleavage of the phosphodiester bond between nucleotides A54 and A55 in 5S rRNA

Reticulocyte lysates contain ribosome-bound and free populations of 5S RNA. The free population is sensitive to nuclease cleavage in the internal loop B, at the phosphodiester bond connecting nucleotides A54 and A55. Similar cleavage sites were detected in 5S rRNA in 60S subunits and 80S ribosomes. However, 5S rRNA in reticulocyte polysomes is insensitive to cleavage unless ribosomes are salt-washed. This suggests that a translational factor protects the backbone surrounding A54 from cleavage in polysomes. Upon nuclease treatment of mouse 60S subunits or reticulocyte lysates a small population of ribosomes released its 5S rRNA together with ribosomal protein L5. Furthermore, rRNA sequences from 5.8S, 28S and 18S rRNA were released. In 18S rRNA the sequences mainly originate from the 630 loop and stem (helix 18) in the 5' domain, whereas in 28S rRNA a majority of fragments is derived from helices 47 and 81 in domains III and V, respectively. We speculate that this type of rRNA-fragmentation may mimic a ribosome degradation pathway.     

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.     

Virus-mediated inhibition of the ppp(A2′p)n A system and its prevention by interferon

Encephalomyocarditis virus infection of mouse Ehrlich ascites tumour or L-cells or human HeLa cells inhibits the activation of the ppp(A2′p)_n A-dependent nuclease and prevents the binding of a radioactive analogue of ppp(A2′p)_n A to the ppp(A2′p)_n A binding protein. This loss or inactivation of the ppp(A2′p)_n A-dependent nuclease in response to virus infection is prevented by interferon pretreatment of the cells.     

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.     

The ppp(A2'p)nA and protein kinase systems in wild-type and interferon-resistant Daudi cells

Daudi cells, a human lymphoblastoid line, are exceptionally sensitive to the growth inhibitory effects of interferon, 1 unit/ml being sufficient to inhibit cell growth. In addition, interferon treatment of these cells severely inhibits the incorporation of exogenous thymidine into DNA and causes cells to accumulate in the G1(G0) at the expense of the S phase of the cell cycle. The possible involvement of ppp(A2'p)nA(n = 2 to less than or equal to 4) in these effects has been investigated. No (less than 1 nM) ppp(A2'p)nA or (A2'p)nA or alternative products of the ppp(A2'p)nA synthetase [e.g. NAD (2'pA)2] were detected in interferon-treated cells. In addition no evidence was obtained for the occurrence of ppp(A2'p)nA-mediated ribosomal RNA cleavage in these cells even after several days of treatment with relatively high doses of interferon. A line of Daudi cells which is resistant to all three of the above effects of interferon was selected. The wild type and resistant lines were compared with respect to the ppp(A2'p)nA and interferon and double-stranded RNA (dsRNA)-mediated protein kinase systems. The resistant line was not receptor-negative as it responded to interferon by the production of elevated levels of the ppp(A2'p)nA synthetase similar to those observed in extracts from wild-type cells. There was no detectable difference between the lines in the levels of the (2'-5')phosphodiesterase responsible for the degradation of ppp(A2'p)nA. There was, however, about a twofold increase in the ppp(A2'p)nA-dependent endoribonuclease activity in response to interferon with extracts from the wild-type but not the resistant cells. In addition, although the dsRNA-dependent protein kinase activity increased in both types of cell there was a striking reduction in the level of protein phosphorylation in general in response to interferon with material from the wild-type but not the resistant cells.     

Degradation of messenger RNA, ribosomal RNA and 2-5A-dependent inhibition of protein biosynthesis

In rabbit reticulocyte lysates the addition of exogenous 2-5A leads after 10-20 minutes to the inhibition of protein synthesis. This inhibition can be blocked by rat antiserum to 2-5A. In intact ribosomes the ribosomal RNA is cleaved after 2-5A addition, but this cleavage is not in correlation with the protein synthesis shutoff. Ribosomal 5S RNA and 5,8S RNA are not cleaved even after several hours of incubation with 2-5A. The degradation of polysome associated mRNA correlates with the protein synthesis inhibition as revealed by Northern blot hybridization of globin mRNA with 32P-labelled p beta G plasmid. The addition of 2-5A antiserum to the rabbit reticulocyte lysate also inhibits the degradation of polysome bound globin mRNA.     

Photochemical crosslinking in oligonucleotide-protein complexes between a bromine-substituted 2-5A analog and 2-5A-dependent RNase by ultraviolet lamp or laser

2',5'-oligoadenylates known as 2-5A [px(A2'p)nA; chi = 2 or 3, n greater than or equal to 2] are produced in interferon-treated cells in response to double-stranded RNA. 2-5A binds with high affinity to a 2-5A-dependent RNase resulting in the cleavage of single-stranded RNA. An efficient, rapid, and extremely sensitive photoaffinity labeling method was developed to facilitate detection of 2-5A-dependent RNase. A bromine-substituted and radioactive derivative of 2-5A, the 5'-monophosphate, p(A2'p)2(br8A2'p)2A3'-[32P]Cp, was synthesized as probe for 2-5A-dependent RNase. Even though this bromine-substituted analog of 2-5A bore no 5'-terminal triphosphate or diphosphate, it bound to 2-5A-dependent RNase with the same high affinity as did 2-5A per se but it was a less effective activator of the RNase under the present assay conditions. The presence of bromine atoms in the 2-5A analog enhanced by more than 200-fold crosslinking to 2-5A-dependent RNase under a uv lamp; many additional polypeptides were also labeled but at much lower levels. Furthermore, using high-intensity uv laser irradiation (308 nm) covalent attachment of the bromine-substituted 2-5A analog to 2-5A-dependent RNase was readily achieved within 10(-6) s.     

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.     

Only one 3'-hydroxyl group of ppp5' A2'p5'A2'p5' A (2-5A) is required for activation of the 2-5A-dependent endonuclease

To investigate the relative importance of each of the ribose 3'-hydroxyl groups of 2-5A (ppp5' A2'p5'A2'-p5' A) in determining binding to and activation of the 2-5A-dependent endonuclease (RNase L), the 3'-hydroxyl functionality of each adenosine moiety of 2-5A trimer triphosphate was sequentially replaced by hydrogen. The analog in which the 5'-terminal adenosine was replaced by 3'-deoxyadenosine (viz. ppp5'(3'dA)-2'p5' A2'p5' A) was bound to RNase L as well as 2-5A itself and was only 3 times less potent than 2-5A as an activator of RNase L. On the other hand, when the second adenosine unit was replaced by 3'-deoxyadenosine (viz. ppp5' A2'p5'(3'dA)2'p5' A), binding to RNase L was decreased by a factor of eight relative to 2-5A trimer and, even more dramatically, there was a 500-1000-fold drop in ability to activate the 2-5A-dependent endonuclease. Finally, when the 3'-hydroxyl substituent was converted to hydrogen in the 2'-terminal residue of 2-5A, a significant increase in both binding and activation ability occurred. We conclude that only the 3'-hydroxyl group of the second (from the terminus) nucleotide residue of 2-5A is needed for effective activation of RNase L.     

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.     

n-Decyl-NHpppA2'p5'A2'p5'A A phosphatase-resistant, active pppA2'p5'A2'p5'A analog

n-Decyl-NHpppA2'p5'A2'p5'A, a gamma-substituted, phosphatase-resistant pppA2'p5'A2'p5'A analog, gives similar rRNA degradation pattern in interferon-treated HeLa cell extracts--even at a concentration of 10(-9)M--as the natural compound does.