the interferon renaissance: molecular aspects of induction and

MICROBIOLoGIcAL RzvIzws, June 1981, p. 244-266 Vol. 45, No. 2014&0749/81/020244-23$02.00/0

The Interferon Renaissance: Molecular Aspects of Inductionand Actiont

JULIAN GORDON* AND MICHAEL A. MINKS

Friedrich Miescher-Institut, CH-4002 Basel, Switzerland

INTRODUCTION ....................................... 244BIOLOGY........................ 244INDUCERS ........................................... 245INDUCTION PROCESS ....................................................... 247MODE OF ACTION ..................................................

.. .....248Ribonucleic Acid Methylation and Cap Structure ........................... 248Double-Stranded-Ribonucleic Acid-Activated Enzyme Systems. . 249

COMPARISON OF DOUBLE-TRANDED-RIBONUCLEIC ACID-DEPENDENTACTIVITIES ..........254.....................264

COMPARISON OF PLAUSIBLE LIS: INTERFERON OR DOUBLE-STRANDED RIBONUCLEIC ACID, PRODUCTION AND THERAPY. 254UPDATE. 256LITERATURECIED. 256

INTRODUCTIONInterferon was first described in 1957, in the

classic publication of Isaacs and Lindenmann(110). Since then there has emerged a wholefield of interferon study. There has also been astaggering proliferation of publications, and thegeneral reader is referred to books which coverthe subject comprehensively (17, 77, 222, 223).The original discovery of Isaacs and Linden-mann (110) was that treatment of naive cellswith the medium from virus-infected cells in-duced an antiviral state. This antiviral activityis the basis of the assay procedures used sincethen, as well as the motivation for continuingthe research, namely, the promise ofan antiviralelixir at the end of the road. This promise hasyet to be fulfilled, but hope has never beenextinguished. It has been further reinforcedmore recently by the promise of an anticancerelixir (28, 90, 129).For many years the field was discouraging for

a molecular biologist, versed in the importanceof exact science, because of the high degree ofimprecision of the experimental systems and thedifficulty in obtaining hard, reproducible data.The situation was first epitomized by the super-visor of one of us, when a graduate student:"They have been at it for five years and still notbeen able to purify it." It is now 23 years, andthey have been able to purify it (32, 60, 124, 193,262). The field is thus transformed from theZwischenferment stage to an exact science,hence the "renaissance" in the title. Further-

t This paper is dedicated to the memory of the late AlfredRudolf Schurch, who unfortunately did not live to see thefruition of projects which we initiated together.

more, the early recognition of double-strandedribonucleic acid (RNA) as an important inter-feron inducer (76) has a nascent appeal to mo-lecular biologists. This appeal is now supple-mented by the realization of the importance ofdouble-stranded RNA in triggering a system ofenzymes from interferon-treated cells (see Dou-ble-Stranded-Ribonucleic Acid-Activated En-zyme Systems).The purpose of this review is to summarize

the various levels at which double-strandedRNA has been implicated. We begin with acursory review of the biology of the interferonsystem, to provide the context and to convey asense of the general excitement. We continuewith a review of double-stranded RNA inducersand try to bring out the limits of chemical mod-ification which are innocuous for the interferon-inducing activity. We review the regulation ofinterferon induction insofar as the stability ofinterferon messenger RNA (mRNA) might beaffected by the products of its own translation.We review comprehensively the literature onprotein synthesis in extracts from interferon-treated cells and the involvement of double-stranded RNA. We compare data on structuralrequirements for double-stranded RNA in itsvarious activities. Finally, we make order-of-magnitude calculations on the plausible limits ofproduction of interferon or double-strandedRNA and the plausible limits of therapy withinterferon or double-stranded RNA.

BIOLOGYThe definition of interferon goes back to the

phenomenon on which its discovery (110) wasbased, namely, the antiviral effect. Interferon is

244

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capable of rendering cells resistant to viral infec-tion, and the assays have been based on reduc-tion of the yield of plaque-forming units, reduc-tion ofthe cytopathic effect of virus, or reductionof the synthesis of viral RNA (for literature onthese and following general aspects, see refer-ences 17, 77, 222, and 223).

Interferon is not in fact a single substance, butis rather a class of proteins with highly potentantiviral activity against a wide range of virusesand hosts. The different interferons are distin-guished according to the types of cells fromwhich they originate, the species, the inducingconditions, and the stabilities of the proteins.These are fibroblast, leukocyte, and immuneinterferons. Immune interferon is produced ex-clusively by cells of the immune system and isproduced in response to mitogens or antigens(see reference 222, p. 145-149). It has antiviralactivity against cells other than those of theimmune system. In addition to their antiviralactivities, the interferons are also characterizedby other cellular effects, namely, growth inhibi-tion, immune potentiation, or immune suppres-sion. In generaL the variety of effects and thepotency of the activity make the interferonsresemble hormones as elements in cellular com-munication.Immune interferons, though identified by an-

tiviral activity, have a high ratio of immune-modulatory activity to antiviral activity (217)and also a high ratio of growth-inhibitory activ-ity to antiviral activity (24), compared with theclassical interferons. One might suspect that theprimary function of this class of interferons isnot antiviral and that the antiviral activity isfortuitous. On one hand, it has been suggestedthat any agent which slows down cellular metab-olism might give the host a selective advantage(258). On the other hand, evidence has beenpresented that immune interferon induces thesame enzyme systems (as described in Double-Stranded-Ribonucleic Acid-Activated EnzymeSystems) as do fibroblast or leukocyte interfer-ons (105).

In addition to the immune-regulatory effectsalready mentioned, interferons exert dramaticeffects in activating cell types involved in cell-mediated immunity, which may then acquireanti-tumor activity (for a recent summary, seereference 95).Another interesting observation is that the

antiviral state, once established by interferonitself, can be transmitted from cell to cell via aninterferon-independent route (22, 23, 25), a factestablished by transfer ofthe antiviral state fromcells of one species to those of another whichwas insensitive to the added interferon. This

INTERFERON RENAISSANCE 245

gives an insight into how the protective effectcould rapidly extend throughout an entire organ.The mechanism of this phenomenon is un-known.Each interferon is characterized by its own

species range, with a trend for the highest po-tency on cells of the same species from whichthe interferon originated. Human leukocyte in-terferon is active on monkey and bovine cells, aswell as on homologous ones, whereas humanfibroblast interferon is only active on humancells. Species ranges of various interferons havebeen comprehensively summarized by Stewart(222, p. 134-145).The species range is presumably determined

by the presence of cellular receptors. There islittle structural information available concemingsuch receptors (see, for example, references 3and 20). Interferon itself must have some struc-tural feature which recognizes the putative re-ceptor. This might be determined by the pri-mary structure or by some carbohydrate modi-fication. Information is accumulating rapidly onthe sequence of the protein itself (125, 227, 261)and the deoxyribonucleic acid sequence of thecloned genes (170, 232). There is, however, onlylittle direct evidence concerning the presence ofa carbohydrate moiety (229), and the presenceof a carbohydrate moiety in human leukocyteinterferon has been questioned (166, 167). Inspite of the small amount of hard information,a crucial role has been proposed for a carbohy-drate moiety in determining species range (33).A direct lethal effect of interferon is the po-

tentiation of the toxicity of double-strandedRNA for cells (224-226). This phenomenonlooks attractive as a possible basis for a specificbut nonviral assay for interferon and is also awaring concerning the toxicity of double-stranded RNA inducers. However, this toxicitymay be of restricted significance, as it dependscritically on the cell type (64). The basis of thetoxicity is not understood (48, 49). Nevertheless,the phenomenon was important, as it led to thestudy of the action of double-stranded RNA oncell-free systems from interferon-treated cells(see Double-Stranded-Ribonucleic Acid-Acti-vated Enzyme Systems).

INDUCERSSubsequent to the original finding (110) of

induction of interferon by virus infection, animpressive range of substances, including poly-mers, small molecules, bacteria, immunogens,and mitogens, were found to be active in inter-feron induction (see reference 154 for a recentreview). We concentrate attention on double-

246 GORDON AND MINKS

stranded RNA, as introduction of a naked nu-

cleic acid molecule provides a basis for con-

structing reasonable models for what might bea uniquely viral feature which triggers the in-duction of interferon synthesis and facilitatesstructure-function studies. Field et al. proposed(75) that double-stranded RNA was an analogof a viral replicative intermediate. However, it isnot clear whether all viruses go through a dou-ble-stranded RNA stage, or why double-strand-edness should be a uniquely viral structure. Anearly search led to the finding of double-stranded RNA in deoxyribonucleic acid-virus-infected cells (42) and even in noninfected cells(59, 118), but as an extremely small fraction oftotal RNA. This double-stranded RNA was alsocapable of acting as an interferon inducer. Thenature of this double-stranded RNA is still notclear.A large amount of work went into the char-

acterization of the requirements for double-stranded RNA inducers. In broad outline, con-

clusions could be reached concerning limits ofmolecular weight, melting temperature, and nu-

clease resistance (summarized in reference 183).The goal of this work was a molecule with an

improved therapeutic index. Some positiveclaims have been made (34, 52, 55, 180, 186).However, few general conclusions concerningthe actual structural requirements could bemade, and no chemical modification with a con-

vincingly improved therapeutic index over theoriginally described synthetic double-strandedRNA, polyriboinosinate.polyribocytidylate-[poly(rI) .poly(rC)] was discovered. This pessi-mistic point of view was summarized by Stewart(222, p. 49). We want to emphasize those gross

chemical modifications where interferon-induc-ing activity is not destroyed. These are summa-

rized in Table 1. Although many effects of mod-ification might be very sensitive to the condi-tions of assay, we hope that changes which are

not deleterious represent a more solid core ofinformation. For a more comprehensive picture,the reader is referred to numerous earlier re-views (43, 51, 52, 98, 157, 159, 183).The contributions of the lengths of each of the

two complementary strands have also been in-vestigated (34, 44, 51, 52, 56, 239), but no firmconclusion can be reached concerning the mini-mal length which is still active, since the resultsdepend critically on the assay system used (44,57). However, where a difference was seen, allauthors agree that the length of the poly(rI)strand is more critical than that of the poly(rC)strand.Although the selection of non-deleterious

modifications has been made in order to estab-lish a hard core of information which is inde-

TABLE 1. Limitations of chemical modifications inpoly(rI) .poly(rC) which are not deleterious for

interferon-inducing activity

Category Actual Deg)ee In chainb Refer-M ~~~ence

Mismatch Substitute uridine 10 Poly(rI) (34, 55)Substitute uridine 14 Poly(rC) (34)

Base 5'-Bromo Poly(rC) (41)5'-Fluoro Poly(rC) (78)2'-Thio Poly(rC) (186)5'-Thio 10 Poly(rC) (179)Acetylamino 30 Both (182)Amino Poly(rI) (174)

Phosphate Thiophosphate Poly(rC) (21)

Ribose 2'-Azido Poly(rI) (58)2'-Chloro Poly(rI) (52)2'-O-Methyl 80 Poly(rI) (89, 158)2'-O-Methyl 40 Poly(rC) (89, 158)

a Limit of the degree of substitution which permits approx-imately full activity. Where not specified, the substitution wascomplete.

'Specifies which of the complementary homopolymers ismodified.

pendent of the particular biological system used,even this limited cross section of the literaturemay be misleading. Because of the primary mo-tivation of obtaining a compound with an im-proved therapeutic index, a clear-cut distinctionbetween quantitation ofinterferon induction andantiviral effects has not always been made: pol-ynucleotides may have antiviral effects otherthan those attributable to interferon. A critiquehas been published concerning this point (219).The activity of polyriboguanylate-poly(rC)

has long been controversial (54, 57). It has re-cently been claimed that polyriboguanylate.poly(rC) is just as active as poly(rI) .poly(rC),provided that care is taken with respect to mo-lecular weight, purity, and presence of counter-ions (174).Of the purely RNA inducers, poly(rI)-

poly(rC) is the most potent and is more potentthan some viral double-stranded RNAs. Reovi-rus RNA, for example, has sometimes beenfound to have low activity (44, 57; H. K. Hoch-keppel, A. R. Schurch, and J. Gordon, unpub-lished data). Reovirus RNA otherwise fits allthe obvious criteria of molecular weight, meltingtemperature, etc. (183), required for a potentinducer. There is evidence to suggest that somesingle-stranded structure must also be present:we found several double-stranded RNAs whoseinterferon-inducing activities could be inacti-vated by incubation with single-stranded-spe-cific nuclease Si (Hochkeppel, Mayr, Gross, Col-lins, Schurch, and Gordon, unpublished data).The absence (reovirus RNA [169] or presence(bacteriophage 46 genomic double-stranded

MICROBIOL. REV.


RNA [240] of single-stranded ends also corre-lates with the interferon-inducing activity. Apartly double-stranded, partly single-strandedstructure may be the signal which is recognizedand thus may be an analog of a viral replicativeof transcriptional intermediate. It does not fol-low from this that there is a simple requirementfor a mixed double-stranded and single-strandedstructure. Ribosomal RNAs in general havesome double-stranded structure (99), and this isespecially pronounced in eucaryotic 28S ribo-somal RNA (245). However, it has been knownfor a long time that ribosomal RNA does notinduce interferon. We have recently confirmedthis, and we have also found that no interferon-inducing activity is generated by digesting awaythe single-stranded regions with single-stranded-RNA-specific nuclease (Hochkeppel, Schurch,and Gordon, unpublished data).While naked viral double-stranded RNA is

often less potent than synthetic double-strandedRNA, some defective viral particles are out-standingly more potent than synthetic double-stranded RNA. From the data of Marcus andSekellick (151) it can be calculated that theirdefective vesicular stomatitis virus particle DI-011 is 105 times more potent than poly(rI).poly(rC). We calculated this from the concentra-tion of poly(rI) -poly(rC) required to obtain thesame interferon titer as that obtained with theirreported optimum of one particle per cell. Atfirst, it seemed that the key feature of this par-ticle was its RNA, which, when extracted, isfound in the form of a giant hairpin. We at-tempted to make an analog of this by introduc-ing interstrand cross-links in poly(rI).poly(rC)(100). The result was a slight reduction of inter-feron-inducing activity (200). Furthermore, Freyet al. (80) found a variety of defective particlessimilar to DI-011, but no relation between po-tency and the presence of giant-hairpin RNA.More recent work with a variety of defectiveparticles from vesicular stomatitis virus and withsimilar defective particles from Sindbis virus (83,84, 149, 150) led Marcus to conclude that, mini-mally, a viral replicase must be present or codedfor in order to generate a replicative intermedi-ate which is double stranded. Further, the defec-tive particles represent not naked RNA, butrather RNA which is encapsulated. The pack-aging may therefore be important for the 105-fold difference in potency noted above. Artificialways of encapsulating the double-stranded RNAmay then have the potential to improve theactivity greatly. The enclosure of synthetic dou-ble-stranded RNA in phospholipid vesicles in-creased the potency 10-fold, both in tissue cul-ture (147, 152) and in whole animals (155).

INDUCTION PROCESS

The induction of interferon is highly regu-lated, as cells need only be exposed to the in-ducer for a relatively short time to achieve max-imum induction (e.g., 1 h [222]). Also, the cellssubsequently become "hyporesponsive" to fur-ther induction. In addition, the response to aninducer can be potentiated by pretreatment ofthe cells with interferon, referred to as "priming"(reviewed in reference 222, p. 233-235). It is notour intent to review these regulations compre-hensively, but since we return later to the sub-ject of catabolism of mRNA in interferon-treated cells (see Double-Stranded-RibonucleicAcid-Activated Enzyme Systems), some sum-mary of the work on the synthesis and decay ofinterferon mRNA in cells induced to synthesizeinterferon is relevant. Interferon mRNA trans-lation has been easily assayable on account ofthe bioassay's sensitivity. It was in fact the firsteucaryotic protein with biological activity to besynthesized in a cell-free system (181, 187). Var-ious systems used and results obtained withthem up to 1978 have been reviewed (222, p. 91-92).An effect opposite to hyporesponsivity can be

achieved by the use of inhibitors of RNA orprotein synthesis. The phenomenon has beenreferred to as "superinduction". The observationled to the concept of a metabolically labile re-pressor whose continued presence was requiredfor the repression of interferon synthesis (222, p.97-103). Evidence has been given for both in-creased synthesis and increased metaboic stabil-ity of interferon mRNA during the superinduc-tion (204). This led to the suggestion of theexistence of multiple mechanisms (204). Sehgaland Gupta (203) have presented evidence infavor of the idea that the decay of interferonmRNA is not correlated with the catabolic path-way for RNA which is activated in interferon-treated cells (see Double-Stranded-RibonucleicAcid-Activated Enzyme Systems). They alsoshowed evidence that during priming, eventhough the conditions for activation of this cat-abolic pathway apparently are present, there isno effect on the stability of interferon mRNA(203). Furthermore, Fujita et al. (82) showed alarge increase in translatable interferon mRNAduring priming, whereas Abreu et al. (1) foundonly an earlier appearance, with no quantitativechange. The experimental systems appear to beindistinguishable. Lebleu et al. (133) showedthat interferon mRNA translatability was notselectively affected by double-stranded-RNA-dependent translational inhibition in reticulo-cyte lysates.

VOL. 45, 1981


Human lymphoblastoid cells (Namalva) re-spond poorly to double-stranded RNA inter-feron inducers and do not respond to the agentswhich give superinduction in other systems(244). They do, however, respond to butyrate,both when treated with viral inducers (2) andwhen treated with double-stranded RNA (H. J.Weideli, personal communication). The onlyknown biochemical action of butyrate in wholecells is inhibition of histone acetylation (188).The butyrate stimulation of the virally inducedinterferon in Namalva cells was apparently aconsequence of increased amounts of translata-ble mRNA, and not an effect on decay (168).The work on interferon mRNA in primed,

induced, and superinduced cells has all beenbased on measurements of translatable mRNA.It must therefore be regarded as tentative, as itis indirect. The situation should soon be clarifiedby the availability of probes of complementarydeoxyribonucleic acid cloned in Escherichia coli(61, 170, 232).A great deal of excitement has been generated

recently by the cloning of complementary de-oxyribonucleic acid made frommRNA from cellsinduced to produce interferon and the demon-stration of synthesis of interferon activity in therecombinant E. coli (170, 232). The genes forboth human leukocyte (170) and human fibro-blast (232) interferons have been cloned, and thesequences are being established (61, 101, 148,231). Although these two interferons are immu-nologically unrelated, enough sequence homol-ogy has been found to suggest a common ances-tor (230). Multiple genes for leukocyte interferonmay also exist, since there are discrepanciesbetween the deoxyribonucleic acid sequences ofthe cloned genes and the amino acid sequenceof interferon from Namalva cells (148).D. C. Burke (31a) has reviewed work on the

genetic organization of interferon genes and thecontroversy concerning the chromosomal local-ization of the interferon genes.

MODE OF ACTIONThe action of interferon has variously been

attributed to interference with virus attachment,uncoating, transcription, translation, matura-tion, or release (see reference 222, p. 207-220),depending on both the system and the condi-tions used. There has been no completely satis-factory explanation of the basis of the virus-versus-host selectivity or of the broad spectrumof antiviral action. The effects on protein-syn-thesizing systems from interferon-treated cellsare the best documented in terms of specificbiochemical parameters. We therefore concen-trate attention in this direction.

MICROBIOL. REV.

Ribonucleic Acid Methylation and CapStructure

Reductions of methylase activities in ex-tracts from interferon-treated cells have been re-ported (208, 209, 212). Experiments on the ef-fect of interferon on methylation of mRNA inintact cells yielded more detailed informationon the methylation affected. Only the cap struc-ture was affected. The cap structure is locatedon the 5'-terminal residue of most mRNA'sand consists of a 7'-methylguanosine linked tothe 5'-terminal residue by a 5',5'-triphosphatelinkage. Variants of this structure are addition-ally methylated at the preceding two positionsand are referred to as cap 0 (m7GpppXpYpZp),cap I (m7GpppXmpYpZp), and cap II(m7GpppXmpYmpZp), where X, Y, and Z rep-resent the three 5'-terminal bases. Kroath et al.(131, 132) have shown an impairnent of theconversion of cap 0 to cap I in vaccinia virus-infected chicken embryo fibroblasts, and Des-rosiers and Lengyel (62) have shown an inhibi-tion of the conversion to cap II in reovirus-infected mouse fibroblasts. The reason for theapparent discrepancy is not clear. Furthernore,Kroath et al. (131) failed to find any discrimi-nation in terms of viral mRNA versus hostmRNA. This does not then shed any light on apossible antiviral selectivity.There are a number of observations concern-

ing the cap in viral systems which, although theyhave not been investigated in interferon-treatedsystems, may point to possible targets for anti-viral action. The caps ofhost mRNA's have beenproposed as primers for the transcription of in-fluenza viral mRNA, resulting in a transfer ofthe host mRNA cap structures to viral mRNA(30). Poliovirus mRNA is not capped, and ex-tracts from poliovirus-infected cells have a trans-lational defect, which was overcome by the ad-dition of an initiation factor preparation (192).The restoring activity was subsequently shownto be due to the presence of the 24,000-dalton"cap binding protein" (236) isolated independ-ently by Sonenberg et al. (216) on the basis ofits binding to cap structure. The implication isthat poliovirus subverts the host cap bindingsystem, with consequent inactivation of hostprotein synthesis. Infection by Mengo virus, an-other picornavirus, also inhibits translation ofhost proteins (72). In the presence of interferon,the host cells recover from the viral action (72).

Double-Stranded-Ribonucleic Acid-Activated Enzyme Systems

In addition to its activity as an inducer ofinterferon synthesis, double-stranded RNA has


been intimately connected with the study ofeffects on translation in extracts from interferon-treated cells. Figure 1 is a comprehensive sum-mary of publications in this area. Each boxrepresents a conceptual group, and the boxes arearranged in approximately chronological order.It is hoped that the figure will provide a usefulguide to the literature concerning double-stranded-RNA-dependent reactions and theirproducts.The action of double-stranded RNA as a po-

tent translational inhibitor in reticulocyte ly-sates was shown in 1971 (63, 108). Furthermore,several systems were known where there was awell-defined effect of interferon on the specificinhibition of viral protein synthesis (26, 27, 86,142, 175, 248, 256, 257). Beginning in 1972, thelethal effect of double-stranded RNA on inter-feron-treated cells (224-226) and also sometrnslational defect in extracts from interferon-treated, virally infected cells (81, 117) were de-scribed. It was found that translation in extractsof interferon-treated cells was more sensitive toinactivation by preincubation than was transla-tion in control cells (69). This inactivation wasshown to be a result of inactivation of transferRNA (45, 46, 70, 71, 93, 156, 206) and could beovercome by the addition of a unique species oftransfer RNA (71).With the background of knowledge of the

synergistic effect of the toxicity of double-stranded RNA and interferon, and the descrip-tion of translational inhibition in reticulocytelysates by double-stranded RNA (63, 108), Kerret al. made the germinal observation that ex-tracts from interferon-treated L-cells were farmore sensitive to inhibition by double-strandedRNA than were extracts from control cells (114).The analogy with the activities found in reticu-locyte lysate protein synthesizing systems wascompelling: it was known that translational in-hibition resulted from heme deprivation, double-stranded RNA treatment, or oxidized glutathi-one treatment (reviewed in reference 137). Ac-tivity could be restored by addition of an initia-tion factor, eIF2. The inactivation appeared tobe a result of the activation of a kinase whichphosphorylates the a subunit of eIF2. However,difficulties were encountered in the demonstra-tion of eIF2 inactivation in a purified assaysystem (19, 237) and also in a heme-deprivedreticulocyte lysate; translational inactivationdoes not correlate invariably with eIF2a phos-phorylation (18, 137, 194).

In spite of this incomplete correlation, theanalogy with the interferon system was compel-ling, and smilar activities in extracts from inter-feron-treated cells were sought and found. An

adenosine 5'-triphosphate requirement wasfound for the inactivation of translation in ex-tracts from interferon-treated cells (189). Also,addition of extracts from interferon-treated cellsled to the translational inhibition in control ex-tracts (47, 115). Evidence then began to appearfor the existence of multiple mechanisms. Highnuclease levels were observed in extracts frominterferon-treated cells (152). Subsequently, anadenosine 5'-triphosphate-activated, double-stranded-RNA-dependent nuclease activity inextracts from interferon-treated cells was found(31, 146, 184, 207, 212).Furthermore, Kerr and co-workers found that

the inhibitory extracts from interferon-treatedcells contained both a protein kinase systemsimilar to that of the reticulocyte lysate andsome low-molecular-weight inhibitor (190). Twoproteins were found to be phosphorylated inthat system, a 34,000-dalton peptide with thesame molecular weight as the subunit of initia-tion factor eIF2a and a 67,000-dalton proteinwhich had properties similar to those of theauto-phosphorylating kinases of t4e rabbit retic-ulocyte system (see reference 137). Evidence wasgiven for the inactivation of eIF2 under theseconditions (112, 146, 177). The phosphorylationis on the same peptide in a proteolytic digest ofeIF2a both under the action of the interferon-induced kinase and with the heme-controlledkinase of the reticulocyte system (137, 196).

Since the results were all based on cell ex-tracts, attempts were made to demonstrate thesignificance of this phosphorylation in vivo.Phosphorylation of the 67,000-dalton proteinwas detected in intact cells treated with inter-feron and double-stranded RNA (92). However,no such phosphorylation of the eIF2a subunitwas detected. This may have been masked byhigh turnover of the eIF2a phosphate, which isknown to occur in the rabbit reticulocyte lysatesystem (194).

Analysis of the low-molecular-weight inhibi-tor (190) was facilitated by chromatographicseparation of the various activities on ion-ex-change columns (74, 121, 210, 260) or on double-stranded RNA affinity columns (102, 104). Theenzyme bound to the double-stranded RNA af-finity column was used as a solid-phase systemfor synthesis of the low-molecular-weight inhib-itor (102, 116). This solid-phase system provideda simple means of concentrating, purifying, andassaying the activity in one procedure. This per-mitted the demonstration of the synthesis of thesame compound in reticulocyte lysates and thebulk synthesis of the compound (103). The com-pound was identified by analysis of digestionproducts with specific nucleases and by compar-

VOL. 45, 1981


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MICROBIOL. REV.

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VOL. 45,1981 INTERFERON RENAISSANCE 251

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ison with known compounds. This was done forthe reticulocyte and mouse L-cell systems (113,199) and confirmed in HeLa (162, 163) andchicken embryo fibroblast (15) systems. Thestructure was finally confirmed definitively bynuclear magnetic resonance spectroscopy (153).It was shown to be an oligoadenylate series ofcompounds with 2',5'-ribose, phosphodiesterlinkages isomeric with the usual nucleic acid3',5'-linkage. The compound has a unique struc-ture (shown in detail in Fig. 2A), with no previ-ously known counterpart in any biological sys-tem. Another bizarre oligonucleotide-like struc-ture known for some years, polyadenosine 5'-diphosphate ribose (for a review, see reference94), is shown in Fig. 2B. This structure is in-cluded only for comparison: both compoundshave the common feature of 2' and 5' substitu-tions in a ribose moiety. They are otherwisedissimilar in structure, function, and origin. Thepoint is made here only to avoid future confu-sion.The identification of 2',5'-oligoadenylate

(2,5A) permitted clarification of the events lead-ing to the translational inhibition in extractsfrom interferon-treated cells. Interferon treat-

. i

2

NHo

O-p-o-p-02-0-H21000

ment leads to the de novo appearance of 2,5Asynthetase activity (13, 119); its product, in turn,activates a latent nuclease, thus inactivating themRNA (10, 16, 38, 39, 163, 199, 214, 241). Unlikethe protein kinase, which also appears de novoafter interferon addition with similar kinetics (inHeLa cells, the 2,5A synthetase appears slightlyearlier than the kinase [9, 246]; in L-cells, itappears slightly later [119]), the polymerase re-quires the continued presence of double-stranded RNA for activity (104, 161). The nu-clease also requires the continued presence of2,5A for activity (163, 214), whereas the 2,5Aitself turns over rapidly because of the presenceof a phosphodiesterase (119, 163, 198, 199, 253).The phosphodiesterase is not induced de novo,but it appears to be somewhat increased in theinterferon-treated L-cell system (119). The sys-tem is thus highly regulated, with the noveloligonucleotide 2,5A being both an extremelypotent translational inhibitor, active at nano-molar concentrations (15, 16, 102, 103, 163), andmetabolically quite labile.Schmidt et al. (198) carried out further puri-

fication ofthe phosphodiesterase and found thatthe preparation did not have absolute specificity

B

FIG. 2. (A) Structure of2,5A. (B) Structure ofpolyadenosine 5'-diphosphate ribose. For each compound, thetrimer is shown, and the repeat unit is enclosed in dotted lines.

MICROBIOL. REV.

VOL. 45, 1981

for the 2',5'-phosphodiester linkage. They found,amongst other things, that it would cleave theCCA terminus of transfer RNA: this provided arationalization for the earlier findings of transferRNA inactivation during incubation of extractsfrom interferon-treated cells.The substrate specificity of the 2,5A polym-

erase has been investigated. 2'- and 3'-deoxy-adenosine triphosphates are not incorporated(161), but a number of compounds appear to beable to take part in a 2'-adenylation reaction:reduced nicotinamide adenine dinucleotide(161), oxidized nicotinamide adenine dinucleo-tide (14), adenosine 5'-diphosphate-ribose (14),diadenosine 5'-triphosphate (161), and 1-N-6-ethenoadenosine 5'-triphosphate (161). Either2',5'- or 3',5'-diribonucleoside monophosphatescan function as acceptors, provided that there isadenosine at the 3' terminus (12). Furthermore,uridine, guanosine, cytidine, deoxyadenosine,deoxyribosylthymine, deoxyguanosine, or deox-ycytidine can be incorporated at the 2' position,but the chain cannot elongate any further (111).Although the 2,5A synthetase appeared to be

an interferon-induced activity, the questionarose as to whether the activity was unique forinterferon-treated cells and perhaps reticulo-cytes (38, 103) or whether it was of more generalsignificance. Surveys were therefore made of avariety of non-interferon-treated cells and tis-sues (120, 213, 218). Although many showedsignificant basal activities, none reached the ac-tivity ofinterferon-treated celLs, but a systematicincrease was noted in oviduct from estrogen-withdrawn chick (218). There was thus a strongimplication that the same system may be oper-ative in normal mRNA catabolism. Quantitativeinterpretation of such activities is difficult, how-ever, because of the simultaneous occurrence ofthe phosphodiesterase, as well as ofother uncon-trolled activities that might be present in crudeextracts. It is also not established whether it isin fact the same polymerase that is operative inthe noninterferon systems.Because of the findings of at least two totally

different enzyme systems induced by interferon,several authors went further to demonstrate theexistence of more than one pathway of putativeantiviral action (36, 74, 104, 146, 249, 260). Con-ditions were defined for activation of either orboth systems. The activation of a nuclease wasshown to correlate with apparent inhibition ofviral mRNA accumulation in vivo (8, 9, 16).These conclusions necessitate the assumptionthat the 2,5A synthetase activity was a measureof the intracellular 2,5A. Since the cellular con-centrations of this compound were so low, it wasonly possible to do this by demonstration of


material which behaved on high-pressure liquidchromatography as expected for 2,5A and whichcould be detected by translation-inhibitory ac-tivity (250).

Furthermore, attempts were made to demon-strate in vivo effects by the introduction of 2,5Ainto intact cells. This was done by the use ofhypotonically permeabilized cells (251, 252) in-troduction as a Ca2+ precipitate (106, 107) or, ipthe case oflymphocytes, by direct addition (120).Evidence was presented for translational inhi-bition (252) and activation of nuclease (106,251)after introduction of 2,5A into the cells. As inthe cell-free system (113), the dimer was foundto be inactive (252), but, unlike the cell-freesystem (113), there was no absolute requirementfor the terminal 5'-triphosphate (252). It is there-fore unclear whether one must postulate an ad-ditional enzyme system or whether there existsa mechanism for re-phosphorylation of the 2,5Acore structure in vivo. Hovanessian and Woodshowed some evidence for antiviral selectivitywhen 2,5A was introduced into intact cells (107).The endonuclease activity found in intact cells

after the introduction of 2,5A was not absolutelyselective: strand breaks in ribosomal RNA aswell as mRNA were apparent (106). This couldalso be responsible for translational shutdown inthose cells. Hovanessian and co-workers haveaccumulated evidence that the 2,5A and proteinkinase systems might not be sufficient for theantiviral effect of interferon: interferon treat-ment of embryonal carcinoma cells gave rise toignificant levels of 2,5A polymerase, but noantiviral effect (254). They showed that K/BALB cells (transformed) showed high endoge-nous 2,5A synthetase, no response of 2,5A syn-thetase to interferon, and a clear antiviral activ-ity of interferon (105a). NIH 3T3 cells showedan increase in 2,5A synthetase and no increasein kinase, but did show an antiviral effect (105a).Baglioni and co-workers have restored some or-der here: contrary to ideas previously held in thefield, they showed that embryonal carcinomacells do in fact respond to the antiviral effect ofinterferon, but that the effect is restricted to anarrower range of viruses (172). Those virusesshowing sensitivity to elevated 2,5A polymeraselevels also gave rise to measurable amounts ofdouble-stranded RNA (172).While all the work in cell-free systems showed

clear mechanisms operative in inhibition oftranslation, none could account for the selectiv-ity of the interferon action claimed in vivo. Nil-sen and Baglioni (171) addressed themselves tothe question of the possible selectivity of the2,5A system. They proposed the following modelfor selectivity in the action of 2,5A. Since 2,5A


is metabolically labile, its concentration will falloff rapidly with distance from the site of synthe-sis. Since it is synthesized by a double-stranded-RNA-requiring enzyme, it would be restricted tothe immediate neighborhood of the double-stranded RNA. It is an endonuclease, with spec-ificity for single-stranded RNA. It would thenselectively degrade partly double-stranded,partly single-stranded RNA. Nilsen and Baglioniin fact provided evidence for the preferentialdegradation of single-stranded regions of such astructure. This is, then, a model for a uniquelyviral structure, such as an RNA virus replicativeintermediate, which is required in the antiviralaction of interferon. In Inducers, above, we ar-rived independently at a strikingly similar modelfor a specific structural requirement in the in-duction of interferon by double-stranded RNA.This raises the question of what common fea-tures one might be able to recognize in themultiple double-stranded-RNA-dependent ac-tivities, i.e., in interferon induction, eIF2a kinaseactivation, and 2,5A polymerase activation.

COMPARISON OF DOUBLE-STRANDED-RIBONUCLEIC ACID-DEPENDENT

ACTIVITIES

It has been known for some time that "natu-ral" double-stranded RNAs are very potent in-hibitors of translation in the reticulocyte system(for example, the fungal virus RNA from Peni-cillium chrysogenum or reovirus RNA), withinhibition optima at levels of 0.01 ,ug/ml (44),whereas synthetic double-stranded RNA, suchas poly(rI) .poly(rC), is much less potent. This isthe inverse of the findings with interferon induc-tion. Furthermore, poly(rI) * poly-5-bromocyti-dylate is approximately equivalent to poly(rI)-poly(rC) as an interferon inducer (see Table 1)and is much worse as a translational inhibitor(44, 235). Translational inhibition by double-stranded RNA in the reticulocyte lysate systemalso displays the phenomenon of "high-concen-tration reversal," where high concentrations pre-vent the inhibition found with lower concentra-tions (37, 135). The high-concentration reversalphenomenon correlates with inhibition of phos-phorylation of the a subunit of eIF2 and of a67,000-dalton protein in both reticulocyte lysates(73, 138, 139, 249) and extracts from interferon-treated cells (121, 246). The only similar phe-nomenon which has been described for inter-feron induction is a clear optimum at one parti-cle per cell exhibited by defective viruses (151).There is no direct demonstration of such a phe-nomenon for the 2,5A synthetase. The 2,5A syn-thetase is activated by double-stranded RNA

MICROBIOL. REV.

concentrations which are above optimal for thekinase activation (249).The use of the poly(rI) poly(rC)-bound en-

zyme as the assay for 2,5A polymerase resultedin a convenient and sensitive assay system, butprevented the systematic analysis of the double-stranded RNA requirement. It is even conceiv-able that the activities described in noninter-feron systems (38, 103, 120, 213, 218) may reflectthe function of other enzymes with no double-stranded RNA requirement and which bind non-specifically to the double-stranded RNA col-umn. Minks et al. (162) optimized a liquid-stateassay system. This permitted the systematicevaluation of different double-stranded RNAsand comparison of the double-stranded RNArequirement for 2,5A synthetase with that ofother double-stranded-RNA-dependent activi-ties. Their results, compared with interferon in-duction in "optimal systems," are summarizedin Table 2. This work emphasizes the similaritiesbetween the requirements for interferon induc-tion and the two double-stranded-RNA-depend-ent enzymes, but the requirements are not iden-tical. Furthermore, from work mentioned earlier,there are clearly differences when natural versussynthetic double-stranded RNAs are comparedin interferon induction and translational inhibi-tion.

COMPARISON OF PLAUSIBLE LIMITS:INTERFERON OR DOUBLE-STRANDEDRIBONUCLEIC ACID, PRODUCTION

AND THERAPYThe future prospects of a pharmaceutical ap-

plication for interferons determine some of thecurrent directions in molecular biology, so theremainder of this review will deal with a criticalreassessment of the relative potentials of inter-feron therapy versus inducer therapy. The ac-tual merits of interferon therapy are not wellestablished, in spite of a great deal of advancepublicity. We will not discuss this point here(see reference 129 for a recent review), but willrestrict the discussion to a consideration of theequivalence of inducer and the interferon it in-duces: each approach has its own economic orpharnacological merits. In the case of the inter-feron genes expressed in E. coli (170, 232), thepublications coyly minimize mention ofthe com-mercial potential. However, optimistic calcula-tion of the maximum expectable yield of proteincan be made by comparison with other eucar-yotic proteins made from recombinant DNAcloned in bacteria, such as yields of insulin of 1mg/liter of culture (87). There is clearly someway to go from the yield of interferon of 1,000 U(ca. 2.5 x 10-6 mg) per liter of culture published


TABLE 2. Comparison of structural requirements for various double-stranded-RNA-dependent activities

Effecte on:Structure

Interferon induction Protein kinase 2,5A polymerase

Polyriboadenylate*polyribouridylatevs poly(rI) poly(rC). Equivalert (57) Equivalent (162)

Mismatched.Cutoff at 510% sub- Cutoff at 510% sub- Cutoff at 510% sub-stitutio i (34, 55) stitution (164) stitution (164)

Poly(rI) * (rC)n.Cutoff at a 430 (57) Cutoff at n < 30 Cutoff at n < 30 (164)(164)

2'-O-Methylribose in:Poly(rI).Cutoff at >40% (89, Cutoff at >40% Cutoff at >40% (165)

158) (165)Poly(rC).Cutoff at >40% (89, Cutoff at >40% Cutoff at >40% (165)

158) (165)Polydeoxyinosinate.poly(rC) Inactive (183) Inactive (162)Poly(rI) .polydeoxycytidylate Inactive (183) Inactive (183)

aEffects: equivalent, interferon induction and 2,5A polymerase activated equally well; cutoff, limits for whichsignificant activation is found.

at the time of this writing. It is realistic, there-fore, to expect a 4 x 105-fold higher yield. Thereare also unknown problems of recovery and pu-rification.

Double-stranded RNA as a means of inter-feron induction therapy generated a great dealof optimism 9 years ago (97), which has beendamped in the meantime by the reports of tox-icity (96) and ineffectiveness (79, 91, 96, 191).The toxicity found for poly(rl) .poly(rC) hasbeen generalized to all double-stranded RNAinducers, although it is not clear to what extentthe toxicity is due to the special character of thismaterial or to impurities in it. The safe limitswere established (50, 91, 215); Russian groupsclaim that poly(rI) .poly(rC) has approximately100 times the toxicity of the more natural poly-riboguanylate-poly(rC) and that they are bothequally potent as interferon inducers. There areclinical trials of polyriboguanylate *poly(rC) un-der way in the Soviet Union (reference 215 givesa summary of the relevant Russian literature).Clinical trials are also under way in Czechoslo-vakia, with bacteriophage R17 replicative inter-mediate double-stranded RNA (29). There is afear that double-stranded RNA will be immu-nogenic and trigger autoimmune disease. In fact,no causal connection between double-strandedRNA and autoimmune disease is known; anti-bodies to various nucleic acids are characteristicof some autoimmune diseases (5, 40, 85, 128, 136,201, 228). The presence of such antibodies issymptomatic, and not causal (127). Further-more, autoimmune symptoms in New ZealandBlack mice are enhanced equally by interferon(211) or inducers (220).Attempts have been made to reduce the tox-

icity of poly(rI).poly(rC) by making chemicalmodifications with greater nuclease sensitivities

and consequently shorter biological half-lives(180). This leads to the proposal that analogswith base-pairing interruptions have a highertherapeutic index (180). This proposal has notyet been solidly substantiated.A converse approach has been undertaken by

Levy and his co-workers, who have initiatedhuman clinical trials with a nuclease-resistantmodification of poly(rI) .poly(rC) (35, 140, 141).The nuclease-resistant modification was a com-plex of poly(rI) .poly(rC) with poly-L-lysine andcarboxymethyl cellulose (143). Although nativepoly(rI) .poly(rC) had excellent antiviral, anti-tumor activity and interferon-inducing activityin rodents, it was ineffective inhum (91, 191).The nuclease-resistant complex, in contrast, wasan effective inducer of interferon in primatesunder conditions where poly(rI) . poly(rC) itselfwas ineffective (143). The original deduction ofLevy and co-workers that the level of serumnuclease is much higher in primates than inrodents (173) has since been confirmed by Krish-nan and Baglioni (130) and by J. Gordon and A.R. Schurch (unpublished data). It is not clearwhy earlier descriptions ofribonuclease III (dou-ble-stranded RNA specific) in serum did notshow this marked species specificity (221). Acomplication might be a physiological regulationof nuclease levels. Ribonuclease II levels havebeen found to be increased in some tissues afterpoly(rI) *poly(rC) administration (160). Therehas been, to our knowledge, no systematic studyof physiological variations of ribonuclease III inserum. De Clercq (53) showed that the nucleasein human serum preferentially degraded thepoly(rC) strand of poly(rI) - poly(rC), but no spe-cies comparison was made at this level of anal-ysis.The data on doses of the nuclease-resistant

VOL. 45, 1981


poly(rI) poly(rC) complex and responses interms of serum interferon titers permits a cal-culation of the order of magnitude of the equiv-alence between an interferon inducer and theinterferon induced. We calculated from the datafor monkeys (143) and humans (35) that for each1 mg of double,.stranded RNA injected, 10 ,ug ofinterferon is induced in the entire circulation,assuming that the extracellular volume repre-sents 10% of the body weight and that pureinterferon has a specific activity of 5 x 108 U/mg. Thus, with the present state of the art, onewould need 1,000 times the mass of inducer toget the effect of one mass unit of interferon.Since a defective vesicular stomatitis virus wasmore potent than poly(rI) . poly(rC) by a factorof 105 (151) in a tissue culture system, it is notclear that the limits of potency of inducers inwhole animals have been reached.

It is also possible to make a calculation of theefficiency of production of double-strandedRNA by fermentation. There is an unusual bac-teriophage, 406, which has a lipid-containing coat(243) and a double-stranded RNA genome (205).The genome consists of 9.5 x 106 daltons ofRNA, and the phage can be grown up to titersof 5 x 10"//ml (243). This is then equivalent to8 mg of double-stranded RNA per liter of cul-ture. The realistic calculation for this phagegives a yield in terms ofmass which is then eighttimes the upper limit of the likely yield calcu-lated above for interferon. Furthermore, theRNA of this phage and its delipidated core hasbeen shown to be approximately equivalent inpotency to poly(rI)-poly(rC) in mice (123).

In conclusion, although interferon itself seemsto have economic advantages over double-stranded RNA inducers, inducers should not betotally eclipsed by the currently exciting devel-opments in the molecular technology of inter-feron production.

UPDATEBetween the completion of this review (Octo-

ber 1980) and the final preparations of the man-uscript for publication (February 1981), therehave been some important developments whichmerit attention. Our skeptical note concerningthe glycosylation of interferons and its relationto the observed heterogeneities is further sup-ported by newer findings, there are sequencedifferences between different subspecies of hu-man leukocyte-type interferon (4) and evidencefor heterogeneous gene products of fibroblasticinterferon (202). Our estimated value of the po-tential efficiency of production of interferon bybacteria carrying recombinant genes has nowbeen achieved in practice (88). Techniques have

MICROBIOL. REV.

been described for the determination of 2,5Aand its phosphatase core by binding to the 2,5A-dependent endonuclease and by antibody bind-ing, respectively (126). Furthermore, the speci-ficity of the 2,5A-dependent endonuclease hasbeen shown to be for U-A and U-U linkages(255).

ACKNOWLEDGMENTSWe acknowledge the help of E. Weir, K. Manches-

ter, and H. Weideli for their critical reading of themanuscript, and we thank those, too many to mention,who provided us with reprints and unpublished ma-terial.

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the interferon renaissance: molecular aspects of induction and

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