BACTERIOLOGICAL REVIEWSVol. 28, No. 4, p. 367-381 December, 1964Copyright © 1964 American Society for Microbiology

Printed in U.S.A.

IDENTIFICATION AND "INDUCTION" OF INTERFERON'

MONTO HO

Department of Epidemiology and Microbiology, Graduate School of Public Health, University of Pittsburgh,Pittsburgh, Pennsylvania

INTRODUCTION.................................................................................. 367INDUCTION OF INTERFERON IN CLASSICAL INTERFERON-PRODUCING SYSTEMS ...................... 368Biochemical Mechanism of "Induction".......................................................... 368Interferon Induction and Virus Virulence...................................................... 371

INTERFERON INDUCTION IN VIVO............................................................. 375IDENTITY OF VIRAL INHIBITORS ENDOGENOUS TO CELLS ........................................ 376SUMMARY...................................................................................... 378LITERATURE CITED............................................................. 379

INTRODUCTION

Interferon is a viral inhibitor which acts byinhibiting intracellular replication of virus withno direct inactivating effect on virus (reviewedin 25, 22, 77, 6, 35, 62). The precise site at whichit acts is unknown. Much evidence points tothe site being very early in the intracellularreplicative cycle (9, 10, 49). Since its discoveryin 1957 (36), it has been produced under a largevariety of conditions. These have usually in-volved a system consisting of cells in culture oran intact host, and a virus "inducer" of inter-feron. [The words "inducer" and "induction"are used here in the generic sense. They meanan agent which "brings about" interferon orthe phenomenon involving "bringing about"interferon. They are not used in the specialbiochemical sense (39).] The virus inducersused have been either infective virus, so thatvirus was produced along with interferon, orvirus partially or completely inactivated bysuch agents as heat or ultraviolet irradiation.An interferon induced in chick embryos byinfective type A (WS) influenza and isolatedfrom allantoic fluid has been highly purified(47). It is characterized as a small basic proteinwith a molecular weight of about 20,000 to34,000. It has high specific biological activity,since as little as 0.004 jug possessed detectableviral inhibitory effect. The physicochemicalaspects of this work have been confirmed by

1 A contribution to the Symposium on "CurrentProgress in Virus Diseases" presented as part ofthe program for the Centennial of the BostonCity Hospital, 1 June 1964, with Maxwell Finlandserving as Consultant Editor, and John H. Dingleand Herbert R. Morgan as moderators.

Kreuz and Levy (45) using CsCl gradient centrif-ugation, and by Merigan using Sephadex columnchromatography (53).Taken together, the source of interferon, its

mode and mechanism of action, and its physico-chemical properties should generally define whatinterferon is. The identifying properties ofinterferons, classified as either biological orphysicochemical, are as follows. Biologicalproperties: (i) produced by cells; (ii) does notdirectly inactivate virus; (iii) inhibits replicationof virus and infectious nucleic acid intracellu-larly; (iv) more effective in species of cells fromwhich it was produced, i.e., relative "speciesspecificity"; and (v) not activated by anti-bodies against virus. Physicochemical properties:(i) small nondialyzable protein molecule inac-tivated by proteolytic enzymes including tryp-sin; (ii) relatively stable to heat; (iii) stable inhigh and low pH; and (iv) no proteolytic ornuclease activities. These characteristics are notexclusive enough to predict authoritativelywhether a specific viral inhibitor which possessessome or most of the properties listed is or is notinterferon. This lack of precision in its definitionmay be illustrated by the fact that 31 scientistsinterested in problems of interferon met infor-mally to discuss this very problem at the 1964meeting of the American Society for Microbiologyin Washington, D.C. They could not and wouldnot produce an inclusive definition of what moreprecise conditions must be met for a substance tobe called interferon.

Extensive work on the physicochemical as-pects of interferon obtained from one particularinterferon-producing system also has its limita-tions. There may be physicochemical differences

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between different types of interferons obtainedfrom different sources. Indeed, crude interferonfrom primary mouse-cell cultures has been foundto be more heat-labile than chick interferon. Incontrast to other types of interferon, partialinactivation occurs at 56 C (43, 4, 25; Kono,Postic, and Ho, unpublished data); this has beenthe case when purified interferon made frommouse-cell cultures was compared with thatfrom chick-cell cultures (53). Other differenceshave been reported, such as ether sensitivity.Thus, interferons made in chick chorioallantoicmembrane (51) and in continuous mouse-celllines are reportedly inactivated (17), but onetype made in KB cell cultures induced by para-influenza type 3 (8) and another made in HeLacells induced by measles virus are not (12).Comparative studies of various types of inter-ferons have been reported (53, 18), but generallythese studies have not been numerous or com-plete. There is no a priori reason why an inter-feron will not be found which may have com-pletely different physicochemical properties fromthe ones described. Interferons may belong to arelatively homogeneous group of proteins suchas antibodies do, but they may also belong to avery heterogeneous group, and even the possi-bility that an interferon may be found that is non-proteinaceous cannot be excluded.The main biological characteristics, such as

those listed above, would appear to be morereliable. But each of the characteristics listed issubject to modification following advances inknowledge. For example, one of the most in-teresting properties of interferon, its "speciesspecificity," holds up well generally and is anextremely useful identifying property of inter-feron (73, 25, 18). But exceptions are readilyfound, and specificity is certainly not absolute(1, 66, 25). Another essential property of inter-feron would appear to be its lack of direct effecton viruses, and there are no accepted exceptionsto the necessity of meeting this criterion. How-ever, there are viral inhibitory substances whichare difficult to exclude on this basis, and thepossibility cannot be excluded in the future that,under appropriate conditions of production orby physicochemical alteration, the interferonmolecule will be found to inactivate virus par-ticles or viral nucleic acid (see last section).The problem of identifying interferon has

assumed some importance, because it has now

reportedly been produced under conditionsradically different from the classical cell-virussystems. The newer systems are distinguishedby the variety of nonviral inducers of interferon,including nucleic acid from animal tissues (34,63), rickettsiae (32), bacteria (80), and yeastsand plants (40, 67); and statolin, which is apolysaccharide (42). Interferon in these systemswas produced in cell cultures or in the intacthost. For example, it was induced in chickens bybrucella (80), in mice by yeast ribonucleic acid(RNA) (67), and in mice by pneumococci (W.D. Hann, unpublished data).Another important consideration in any final

definition of interferon, as well as in the under-standing of its significance, is the possibility thatinterferon may be present in untreated cellsin the absence of any known inducer. There arealready reports that interferon may be extractedfrom the chick embryo and from medium overlayof chick-embryo cell cultures (R. Z. Lockart,unpublished data).

In the absence of complete publications, itis difficult at this stage to evaluate some of thedata cited above. One conclusion seems war-ranted: perhaps it is not premature to excludefrom the definition of interferon any provisionthat it must be induced by a virus or a componentof virus (25). As to what these various inducersubstances have in common, if anything, andwhether there is endogenous interferon, it wouldbe profitless to speculate at this stage. In addi-tion, a certain lapse in time is desirable beforean overall analysis is attempted.What I would like to do in the following pages

is not to add to the many general reviews ofinterferon, but to review specifically the prob-lems of interferon induction and identity fromcertain arbitrarily selected points of view, bear-ing in mind some of the questions raised by newdata. First, I shall consider the nature of induc-tion of interferon in classical models of interferonformation in cell culture. Then I shall considersome problems of induction of interferon in theintact animal, and finally I shall consider viralinhibitors endogenous to cells as a specificproblem in the identification of interferon.

INDUCTION OF INTERFERON IN CLASSICALINTERFERON-PRODUCING SYSTEMS

Biochemical Mechanism of "Induction"

By "classical" interferon-producing systems,

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I mean those systems in which the production ofinterferon is induced by infectious or inactivatedvirus. Since the inception of studies on interferonin these systems (25, 22, 77, 6, 35), it has beenevident that interferon was different from viralparticles in terms of such properties as revealed bycentrifugation and antigenic characteristics (25).Therefore, the assumption has been that inter-feron is a cellular protein, the biosynthesis ofwhich may not be directly related to viral replica-tion. Until recently, proof of this on a biochemicalbasis was lacking; even now the relationship of thevirus inducer and intracellular events of virusinfection to interferon formation is still unclear.The most promising recent approach to this

problem has been to attempt to study the condi-tions under which virus replication may bedissociated from interferon production, andthereby to gain insight into the differing deter-minants of the biosynthesis of these two macro-molecules.There were scattered reports in the literature

prior to 1963 which purported to show the effectof various types of chemical inhibition on inter-feron formation. Thus, Burke and Isaacs men-tioned that proflavine did not inhibit interferonformation by influenza virus in chick chorioal-lantoic membranes (7). I also found this to bethe case, using proflavine in a chick-cell culturesystem with infective Sindbis virus as interferoninducer (Ho, unpublished data). I found, inaddition, that proflavine in nontoxic dosesfailed to inhibit Sindbis virus replication. In theabsence of further data, these results, includingmy own, are difficult to evaluate. Generally, instudies with antimetabolites, further data aredesirable. These should include controls whichdemonstrate that particular concentrations ofthe antimetabolite used are exerting the desiredantimetabolic effect on the one hand, and thatthey do not produce nonspecific toxicity andcell death on the other. The first type of controlmay be obtained in terms of a biochemicalmeasurement such as isotope incorporation, orin terms of inhibition cell or virus replication,depending on the antimetabolic effect in ques-tion. The second type of control demonstratingthe absence of nonspecific toxicity is more dif-ficult to establish, especially if both virus andinterferon formation are inhibited by an anti-metabolite. Reversibility of the antimetaboliceffect may be one method (68).

Another type of available information isrepresented by what has been reported on theinhibitory effect of steroids on interferon syn-thesis (41). Studies along this line have addedto the literature of the biology of steroids, andhave increased our understanding of the effectof steroids on the replication of viruses, but theyare of limited value in explaining the mechanismof interferon induction since the mode of actionof these steroids is not precisely known. Theseremarks apply also to studies on the effect ofcertain carcinogens, such as 20-methylcholan-threne, on interferon synthesis (12).Both of these objections have been overcome

by use of actinomycin D. In the case of thisantimetabolite, its mechanism of action isprecisely known, and adequate controls may bereadily established (61, 33). Wagner (76), usingEastern equine encephalomyelitis (EEE) virusin L cells, and Heller (20), using Chikungunyavirus in chick-embryo cell cultures, found (i)that interferon readily produced in the absenceof actinomycin is completely inhibited in itspresence, and (ii) that one possible result of inhi-bition of interferon is that virus replication insuch cultures is frequently enhanced. This isespecially dramatic in the case of EEE virusinfection of L cells, since ordinarily this virus doesnot replicate to high titers in these cells, presum-ably because of interferon formation in thissystem.Knowledge of the mechanism of action of

actinomycin D renders selective inhibition ofinterferon synthesis apart from virus formationby this agent highly significant. It is generallyaccepted that actinomycin D interferes directlywith the function of deoxyribonucleic acid(DNA) by combining with the guanine basis onthe DNA double helix (60). Hence, it inhibitssynthesis of messenger RNA and, therefore, cellgenome-directed protein. The replication ofRNA viruses and specifically of the above-mentioned arboviruses is not affected by thisantimetabolite (61), implying that these virusesreplicate without any participation of cellularDNA. The conclusion is then inescapable thatthe formation of interferon, unlike that of RNAvirus, is directly or indirectly controlled by thecell genome. This is the best evidence so farthat interferon synthesis is demonstrably distinctfrom that of viral protein synthesis (Fig. 3).Ho et al. (28) have also found that actinomy-

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en z

o w

c JiLLZc _Z

000

LL Z0 0

9ID2 AFz wWO

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0Ooo1100 Q3

90 a80 t

70 q

60 CL

50 .

40 Ww

30 E20 §

10 w

Ow

z

-I 0 2 4 6 8 12 16 20 24HOURS AFTER INOCULATION OF NDVuv

FIG. 1. Course of production of interferon afteradsorption of NDVuV in chick-cell cultures andperiods during which actinomycin D is effective ininhibiting interferon production. Symbols: *,course of extracellular interferon formation after a

2-hr period of adsorption of NDVu,; Q. inhibitionof interferon production. All cultures receivedNDVuV for 2 hr, but, at the times indicated, actino-mycin D (0.5 ,ig/ml) was added for 1 hr. Cultureswere all collected 18 hr after inoculation of NDVuV,and the interferon titers were obtained and comparedto that of fluid from a culture that was not treatedwith actinomycin D.

cin D inhibits interferon formation in chick-cellcultures, with ultraviolet-inactivated Newcastledisease virus (NDVuv) as an inducer. The meritof using NDVuv as inducer is that interferon isformed in the absence of significant viral replica-tion. This eliminates any complicating effectof infective virus replication on interferon forma-tion, including the uncontrolled increase ofinterferon inducer due to repeated cycles ofvirus formation. With this system, it is possibleto estimate the time of interferon messengerRNA synthesis following adsorption of NDVuv,and to test whether or not continuous messenger

RNA synthesis takes place prior to the release ofinterferon.These problems were approached in our labora-

tory by attempting to pinpoint the time, withrespect to the inoculation of inducer, at whichinterferon formation is inhibited by actinomycinD (28). Interferon becomes detectable afterabout 8 hr of incubation following a 2-hr adsorp-tion of NDVUV (Fig. 1). The titer reaches itsmaximum rapidly and does not appreciablyincrease in the course of the next 24 to 48 hr.If one tries to ascertain the time either before or

after adsorption of NDVU, when actinomycininhibition of interferon formation is effective,one finds that this is represented by a fairly short2-hr period after the adsorption of NDVUv. Itis also effective if added for 1 hr prior to theadsorption of NDVUv. The important point isthat at 3 hr after the adsorption of NDVUv thenecessary nuclear transcription for interferonsynthesis is already complete, and actinomycinis at this time, and thereafter, no longer effectivein inhibiting interferon formation. One explana-tion may be that messenger RNA necessary forinterferon production is not continuously synthe-sized after exposure of the cells to NDVUV. Suchsynthesis appears to be a "one-shot" affair.However, these experiments with actinomycin

D do not reveal the precise role of such mes-senger RNA. They do not answer the questionwhether interferon is a newly snythesized mole-cule or one that is slightly modified from aprecursor. Although I have no concrete evidenceon this point, there is evidence that new proteinsynthesis is necessary for effective induction ofinterferon. This has been shown by the demon-stration that puromycin, which is a specificinhibitor of protein synthesis (79), will preventproduction of interferon (28). Such a result isconsistent with the notion that interferon rep-resents newly snythesized protein, but it couldalso mean that the new protein is merely a neces-sary intermediary for interferon release.

Superficially, the notion that interferon for-mation resembles induced enzyme synthesisin which there is derepression of the expressi-bility of a genic function is very attractive (20,76). In both cases, a protein normally undetec-table in cells is released in response to an inducer.And, in both cases, this process is mediated bycell messenger RNA. Furthermore, the fact thatthe inducer for interferon is a virus, or perhapsviral nucleic acid, may be significantly related tothe fact that the target of interferon action is in-hibition of virus or virus nucleic acid synthesis.Beyond this, however, it would be imprudent tocarry the analogy very far.

Indeed, there are aspects of interferon induc-tion which still remind one more of the peculiari-ties of virus synthesis than of induced enzymesynthesis. In induced enzyme systems, synthesiscontinues in the presence of the inducer (39);in interferon induction, the presence of NDVuVinducer in cell cultures for 2 or 18 hr makeslittle difference in the total amount of interferon

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IDENTIFICATION OF INTERFERON

produced; it is about the same (Ho, unpublisheddata). This is similar to the nature of virus in-fection in that the amount of virus inoculatedor the length of cell exposure to virus does notdetermine the amount of virus replicated. Thecontinued presence, then, of inducer beyond acertain period is ineffective in further induction.Admittedly, this resemblance between virusand interferon synthesis may be only superficial,because the continued presence of NDVUV doesnot necessarily imply a constant rate of adsorp-tion and penetration of inducer and, therefore,an effective intracellular accumulation of in-ducer. However, alternatively, these data maysignify that a cell can only make one quantumof interferon irrespective of increased accumula-tion of inducer. In other words, either a cellmay make more interferon if more inducer couldget in the cell, or it is possible that even if theinducer penetrated the cell, no more interferoncould be made. Experiments testing these pointsare possible. These would include studies on theadsorption of both viral and nonviral inducers,or perhaps induction of interferon in a cell-freesystem in which the problems of adsorption ofinducer may be circumvented.There are some reported data on interferon

induction which bear on the relationship of doseof inducer to the amount of product, but theyare not quantitative or complete enough to beevaluated. For example, Burke and Isaacs (7)showed that a second crop of interferon may beinduced with heated influenza virus in fragmentsof chick chorioallantoic membrane. However,this may be due to cells that did not adsorbany inducer the first time. Attempts to makefurther crops of interferon production wouldhave been interesting. Heller (20) shows agraph representing "linear" accumulation ofinterferon in chick-cell cultures exposed to in-fectious Chikungunya virus. It is not clearwhether this increase represents more inductionwith increased exposure to inducer or whetherit may be due to progressive increase of infec-tious virus, and, hence, increasing numbers ofcells exposed to inducer. Continued measure-ment of interferon titers beyond the 24 hr chartedby Heller may reveal a sloping off of the"linear" accumulation of interferon; this wouldthen represent the point at which the maximalnumber of cells are infected or induced. Ingeneral, it would probably be better to use non-infectious inducers to test these points.

In summary, it is probably premature to de-scribe with any precision the mechanism ofinterferon induction. There are many modelswhich could suit interferon induction. Theseinclude examples of specific "inducers," such asantibody formation and induced enzyme syn-thesis. There are also nonspecific "stimuli"which increase cellular metabolic activity, asexemplified by increase in the aerobic andanaerobic glycolysis and other biochemicalparameters which attend phagocytic activityof white cells or their exposure to endotoxin (11).Possibly, interferon synthesis and release are aresponse to such "stimuli." [Recent evidence thatbacterial endotoxin indeed induces interferon-like viral inhibitors in chickens, mice (W. R.Stinebring and J. S. Youngner, Nature, in press),and in rabbits (M. Ho, Science, in press) sup-ports this possibility.]

Interferon Induction and Virus Virulence

The next problem I would like to consider iswhy certain viruses induce the production ofinterferon while others do not. I will not reviewhere all the possible factors involved but willconcentrate on the relationship of interferoninduction to virus virulence. There have beenattempts to correlate interferon productionnegatively with (i) virulence of the virus inducer(16), (ii) ability of the virus inducer to growoptimally at high temperatures (64, 65), and(iii) the growth of virus inducer at high pH (13).The details of these purported correlations havebeen reviewed recently (35, 77), and they holdonly in a very general way. However, it is prob-ably unwise to dismiss the validity of thesegeneralizations on the basis that they have notbeen tested in large enough groups of cell-virussystems, and that exceptions to at least someof them are readily found, because these ideasrecur as long as the significance of interferonformation remains undefined.

Let us consider the concept that avirulentviruses are better interferon inducers thanvirulent ones. First of all, the term "virulence"is ambiguous. Does it apply to manifestations ofhuman disease or does it apply to a specified cellor host system in question? There are exampleswhere either sense of the term could be em-ployed. Considering virulence in the anthropocen-tric sense, a strain of avirulent poliovirus (RMC)produces interferon in human amnion and

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kidney cells, and a virulent strain will not (27;Ho, unpublished data); an avirulent or vaccinestrain of measles virus produces more interferonthan the virulent parent strain (16). It is alsointeresting that arboviruses that are virulentfor humans, such as EEE or Japanese B encepha-litis viruses, produce less interferon in chick-cellcultures than do the avirulent or less virulentSindbis, Chikungunya, and Mayaro viruses (Hoand Mahdy, unpublished data; 21, 75, 25).Looking at virulence from the aspect of theparticular cell culture under study, one mightdefine a virulent virus as one which replicatesquickly and optimally with concomitant totaldestruction of cells. Under this definition, theexamples of polioviruses apply again, since RMCpoliovirus does not destroy human kidney cellsin culture and induces interferon formation,and wild strains are destructive and do notinduce interferon. Sindbis virus does not fitthis concept, because it replicates to high titersin chick-cell cultures and is cytopathic, whileinducing interferon. Isaacs also cites the ex-ample of Kumba virus which destroys cells andyet produces interferon (35, 65).An interesting model of the complex relation-

ship between properties of a virus and its capacityto produce interferon is Newcastle disease virus(NDV). In the infectious form, NDV in chick-cell cultures is a good example of a type ofvirus which is not an interferon inducer. It is"virulent" in chick-cell cultures. Parenthetically,

TABLE 1. Virus and interferon formation in chickembryos infected with NDV

Interferon titer*Inoculum Virus titer

1:1 1:4 1:16 1:64

24-hr harvests(embryos alive)

10-0 7 X 107 60 0 0 010- 1 X 108 0 0 0 010-5 7 X 107 0 0 0 0

48-hr harvests(embryos dead)

10-0 7 X 107 100100 75 400-3 3 X 108 100 100 91 5710-5 3 X 108 100 99 79 20

* Results are expressed as per cent inhibitionof 500 PFU of EEE virus.

-3uJ 2

co5I 76

137 I-

0 15 30 45 60 75 90 105 120SECONDS OF UV-IRRADIATION

100 CO

zi,Di

U-

cx

FIG. 2. Interferon-producing capacity of ultra-violet-irradiated NDV virus pellet. Pellet materialwas ultraviolet-irradiated with a 28.75-w lamp witha maximal emission of 2,537 A placed at 7.6 cm.

Residual virus was titrated by inoculating 10-folddilutions in duplicate chick cultures and observingfor plaque formation. Interferon was titrated inserial fourfold dilutions on duplicate chick cul-tures, challenged with EEE, and observed for plaquereduction. The values plotted are those of a 1:4 di-lution. Points shown are the means of duplicatevalues; where more than one point is shown, theserepresent values obtained in separate determinations.

it multiplies at low 02 tension (5) and requireshigh temperature for optimal replication (64).The behavior of NDV in mouse-embryo cellcultures is also consistent with the "virulence"theory. The virus does not produce lysis in thesecells, and replication is minimal. Hence, it maybe considered avirulent in this tissue. And, un-

like the situation in chick cells, NDV inducesinterferon in these cells (43, 65). On the otherhand, the situation is more complex in the em-

bryonated chick egg. NDV induces interferonin this host, for which it must be considered"virulent" (64). Virus replication begins in 2hr and reaches its maximum in 8 to 10 hr. Nointerferon is detectable at this time. But48 hr after inoculation the interferon titers be-come appreciable; at a time when the embryos are

dead (48; Ho, unpublished data, Table 1). This isan example of a lethal viral infection being associ-ated with interferon production. Similar systemsmay be gleaned from the literature (77).Although infectious NDV is not an inducer of

interferon in chick-cell cultures, it can be madeinto one if it is irradiated by ultraviolet light(Fig. 2). Irradiation for 30 to 60 sec under de-scribed conditions is optimal, but excessiveirradiation destroys this inductive capacity.

- 0-el 0

/0 * INTERFERON (1:4)- °--- RESIDUAL INFECTIVITY

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Inductive capacity appears to be a biologicalproperty of the virus particle which, along withothers such as hemagglutination or enzyme

activity, is susceptible to physicochemical inac-tivation.

It appears from these vagaries of NDV as an

interferon inducer as if the virus particle isbasically capable of inducing interferon, butthe reactions accompanying infectivity in cer-

tain cell systems counteract this inductioncapacity.

There is evidence from recent studies on thebiochemical effects of the virus infection whichbear on this point. These studies promise tosupply a biochemical definition of the wholeproblem of virulence, and may also provide a

reason why virulent viruses do not induce inter-feron. From the work on Mengo virus (2),poliovirus (30), and others (31), it is apparentthat certain virus infections are associated withsevere inhibition of cellular biosynthetic mecha-nisms. Since interferon synthesis depends on

such mechanisms, it too may be inhibited inthe course of virulent infection. Conversely, ifthe infective cycle does not take place, as whenNDVuv is used, the inductive function inherentin the virus particle may proceed undisturbed.These hypotheses may also explain the lateinduction of interferon by NDV in chick embryo.Possibly by 48 hr, at a time when the embryomay be moribund, certain interferon-producingcells may finally become exposed to NDV par-

ticles that have become inactivated by thistime, and may act as interferon inducers. Simi-larly, it is also possible that the inability ofother "virulent" viruses, such as wild polio-viruses, to induce interferon is related to theirinhibitory effect on cell synthetic mechanisms.The "inverse interference" of Lindenmann (50),where infectious virus added after the inducerconsisting of inactive virus inhibits interferonproduction, may also be explained by the cellinhibitory effect of infectious virus.

The parameters of classical interferon induc-tion unfortunately are not entirely exhausted interms of the NDV model. Certain problems are

brought out by another popular class of inducers,the arboviruses. Here, infective virus is more

effective than the inactivated (25). Usually, theinactivated virus induces no detectable interferon(for exception, see Gifford and Heller, quoted in35), but may render cells resistant to superinfec-

tion (26, 52). In addition, there is a phenomenoncalled "priming": if inactivated virus is added tocell cultures prior to infective virus, it will primeinterferon production. That is, interferon is pro-

duced where none is produced in the absence ofthe primer, or it may potentiate or increase inter-feron production when infective virus alone iseffective as an inducer (26, 52).

It appears then that, in the case of some vi-ruses, infective virus is the better inducer, andinactive virus may serve as a "primer." Thereare two possible explanations for infective virusbeing a more effective inducer. First, the induc-ing capacity of the virus may be as sensitive tophysicochemical inactivation as is infectivity,or more so. This would make it impossible todissociate the inducing property from the in-fective by inactivation. Second, viral replicationis associated with a large intracellular accumula-tion of virus; therefore, effective inducer may alsoaccumulate inside the cell. Infection may be a

way of getting a large amount of inducer insidecells, and a relatively ineffective inducer may notbe operative unless such accumulation followingcellular infection takes place. To account for the"priming" phenomenon, it is possible that inac-tive virus acting as a "primer" functions as an in-complete inducer by forming intracellular inter-feron which is not released, but which can actwithin the cell in which it is formed. This "directinhibitory effect" of the primer has the effectof slowing down the infective cycle of an addi-tional infective virus, and permitting interferonsynthesis and release to take place. The data

TABLE 2. Effect of virus infection (V.) on chick-cellcultures "primed" with inactivated EEE (Vi)

Direct inhibi-Interferon tion of infec-Infectious virus production tious virus

added (Va) tNousvirloNo Vi vi* -log V )

EEE ............. 9T 100 2.0Sindbis ............ 54 100 2.0VSV ............. 19 85 1.2NDV............. 24 36 0.0

* Cultures preincubated 5 hr with EEE inacti-vated by ultraviolet light and 37 C.

t V = titer of virus in cultures pretreated withVi . V, = titer in untreated cultures.

t Activity in per cent plaque inhibition of EEEvirus at 1:4 dilution of culture fluid in chick-cellcultures.

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NUCLEUS

MESSENGER RNA

RIBOSOMES

"INDUCER" Xa-:::'. *INTERFERON

z \/~~~///VIRAL NUCLEIC VIRUS

ACIDVIU

POTENTIATION.-- INHIBITION

FIG. 3. Induction of interferon and intracellularinterrelationships between virus replication andinterferon formation.

presented in Table 2 (Mahdy and Ho, unpub-lished data) may constitute indirect evidencethat the "primer" produces a forme fruste typeof intracellular interferon. The primer (Vi inTable 2) consisting of ultraviolet-inactivatedEEE virus can prime interferon formation byEEE, Sindbis, and vesicular stomatitis viruses.These three viruses are known to be susceptibleto interferon, and, hence, presumably to thehypothesized intracellular interferon formed bythe primer. On the other hand, the addition ofNDV does not result in production of interferonin the "primed" cells. This may be explainedby the well-known fact that NDV is not in-hibited by interferon in chick cells (38, 64).Hence, it is not inhibited by the primer, and itinfects and lyses primer-treated cells withoutsignificant interferon production. These dataare, of course, only suggestive and are not con-

clusive.The intracellular events leading from induction

to interferon formation, and the effect of virusand virus replication on these events, are dia-grammed in Fig. 3. These designated interrela-tionships explain the apparently conflictingeffects which may issue from superficially similarcircumstances, and constitute a complex intra-cellular dynamic situation. This situation may

be recapitulated as follows. (i) Infectious virusmay either inhibit or enhance interferon forma-tion: it inhibits by a disruptive action on cellularsynthetic mechanism; it augments by providinginducer. (ii) Intracellular interferon, presumably

formed by "primers," or incomplete interferoninducers consisting of inactivated virus, caninhibit viral infectivity, inhibit the cell-disrup-tive influence of infective virus, and therebyfurther interferon formation. However, if intra-cellular interferon is present in excess, it mayprevent interferon formation by preventingsuccessful viral infection, and thereby preventingaccumulation of sufficient viral inducer.The potential merit of these hypotheses is

that they suggest certain problems which maybe attacked experimentally. First, is there acorrelation between viral virulence, virus dis-ruptive effect on cell synthetic mechanisms,and effectiveness of the virus as an interferoninducer? This question is amenable to biochem-ical solution. Our model would predict thatthough some lytic or "virulent" infections, suchas poliovirus in primate cells, are totally disrup-tive to cell synthetic mechanisms, other infectionswhich are equally destructive morphologically,such as Sindbis virus in chick-cell cultures,would not be so disruptive biochemically, sinceinterferon is produced in them. Second, thesehypotheses postulate the existence of an intra-cellular interferon which is biologically active,but which is "incomplete" in that it is not re-leased from the cells. This type of interferon isformed by "primers." The basis for assumingthat unreleased interferon may be incompleteis that there is very little intracellular accumula-tion of interferon if one follows both extracellularand intracellular titers of interferon in chick-cellcultures induced by ultraviolet-irradiated NDV(28). At no time in such studies was intracellularinterferon found to be of higher titer than extra-cellular. The results suggest that interferon isreleased as soon as it is formed. On the otherhand, there is data showing that once interferonis adsorbed to chick cells, it is no longer elutable(75). The reason for this situation is unknown.It may be due to a lack of sufficient quantity ofinterferon to be detected, or, alternatively, inaccordance with our hypothesis, once interferonis adsorbed, it reverts to an "incomplete" formwhich is not readily released.These considerations are subject to experi-

mental scrutiny. First, if adsorbed interferonbecomes "incomplete," its release may be ef-fected by addition of a "complete inducer"consisting of an infectious virus, in a mannersimilar to releasing interferon after cells have

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been "primed." Second, the mechanism of"priming" may also be studied from variousvantage points. If intracellular "incomplete"interferon is formed, it too should be inhibitedby such agents as actinomycin D.

If the concepts of incomplete intracellularinterferon and incomplete inducers of interferonare accurate, certain obscure types of viralinterference may be explained. One of these maybe a type of "infection interference," a situationin which an infective virus may be the interferingagent in the absence of appreciable amounts ofinterferon (24).

INTERFERON INDUCTION IN VIVO

Baron and Buckler (4) described the appear-

ance of high titers (up to 1:300) of interferon1 to 4 hr after intravenous injection of largedoses of NDV and Sindbis virus in mice. Thismethod of inducing interferon is important, be-cause (i) interferon appears at a time before any

evidence of viral multiplication has occurred,(ii) it takes place in the intact animal, and (iii)the titers are of a higher range than those ob-tained from most other in vitro work (25). It isnot yet possible or desirable to review the fullsignificance of this phenomenon at this time,especially with respect to its significance as a

biological defense mechanism (3). I will, how-ever, review two problems with respect to inter-feron induction which this work brings out:(i) the difference between interferon formationin vivo and in vitro and (ii) the source of thisrapidly appearing interferon.The primary difference between interferon

formation in cell cultures and in animals afteran intravenous inoculation is one of speed. This

0

-

2

i

10.0_ 9.08.0 a-

7.0 o

6.0 -'

5.04.0 >

3.0 -2.0 i1.0 1

'1.02 3 4 5 6 7 24 48 72

HOURS AFTER INOCULATION

FIG. 4. Interferon and virus formation by Sindbisvirus in rabbit cells in vivo and in vitro.

TABLE 3. Interferon formation in rabbit-cellsuspensions

WhiteIncu- Spleen Liver blood Kidneybation Vrus (107.7 (107.0 cells (107.6time (log)6 cells)t cells) (101°l3 cells)

cells)

1 6.2 < 1:l10 <1:5 0 03 6.1 1:80 1:20 05 6.0 1:160 1:20 0 07 6.0 1:640 1:20 1:40

10 5.7 1:640 1:2023 5.0 > 1:640 1:40 >1:40 1:80

* PFU per 0.1 ml of splenic cell reaction mix-ture.

t Cells per milliliter.I Dilution at which 50% VSV plaque reduction

in rabbit-kidney cell cultures was observed.

is well demonstrated by contrasting interferonformation in rabbit-kidney cell cultures and inrabbits with large doses of Sindbis virus asinducer. After an intravenous dose of about 109plaque-forming units (PFU) of Sindbis virusin a 4-kg rabbit, circulating interferon is de-tectable in 1 hr, and reaches its peak in 7 hr(Fig. 4). On the other hand, in a cell culture ofabout 106 rabbit-kidney cells exposed to 107PFU of Sindbis virus, interferon formationdoes not start until 7 to 24 hr after infection,and virus replication is evident in 3 hr. Thelatter in vitro data is consistent with most ofthe data in which interferon formation has beenfound to lag behind virus replication (75).There may be exceptions. For example, Chik-ungunya virus seems to be able to form inter-feron in about 3 hr in chick-cell cultures (20).But what is of interest in Fig. 4 is the differencein speed between in vivo and in vitro interferonformation with one specified cell-virus system(rabbit-Sindbis). The circulating interferondetected in the intact rabbit does not seem tofollow any significant virus replication, andseems to be the primary effect of the inoculum.This rapidity of response suggests that thereare specialized cells in the intact animal whichare especially rapid interferon formers. Pos-sibly, this property of host cells may be de-pendent on cell differentiation and is lost whencells are cultured in monolayers.To test this hypothesis, the course of inter-

feron formation in suspensions of cells from

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various organs of a rabbit was tested (44).Sindbis virus was added to a concentration of2 X 107 PFU per milliliter, and the cell-virusmixtures were incubated under agitation at 37 C.Samples were titrated for interferon effect by inhi-bition of plaque formation of VSV on rabbit-kid-ney cell cultures. Interferon was detectable in 3 hrin cell suspensions derived from liver and spleen,but its appearance in kidney and white bloodcell suspensions was much later (Table 3). Thetime of appearance of interferon in kidney-tissue cells is suggestive of what occurs in rabbit-kidney primary explant monolayer cultures.These results suggest that there is a common

property of liver and spleen cell suspensions.

The most obvious carrier of this common prop-

erty is the reticuloendothelial cells present inboth tissues. Possibly, the rapidity with whichSindbis virus inducer is taken up is related tothe phagocytic function of the reticuloendo-thelial system (54). Experiments using thoratrastto blockade this system in rabbits suggest thatsome of this rapidly appearing interferon is in-hibited (44).Another possibility is that rapid interferon

formation represents release of preformed intra-cellular interferon. This is especially so in viewof the large variety, and therefore the possiblenonspecificity, of interferon inducers (see In-troduction). One can easily rule out a simplemechanism of release, since it is not possible torelease anything close to the amount of inhibi-tory material by simple mechanical lysis oftissue cells. In addition, experiments with ac-

tinomycin D in both mice and rabbits show thatrapidly appearing interferon can be inhibited,as it is in monolayer cultures of cells from theseanimals (28; Kono and Ho, unpublished data).This is indirect evidence that interferon forma-tion in vivo is no different than in cells culturedin vitro. It is, of course, entirely possible thatprecursors of interferon exist in liver and spleen,which are closer to the final intact interferonmolecule than those in monolayer cultures, thusaccounting for the rapidity of release.

Finally, the titers reached in the blood streamof the rabbit and in cultures of liver and spleencells are higher than those obtained in monolayercell cultures. This is also true with Sindbisvirus inducer in the mouse system. Circulatinginterferon is readily detectable after an intra-venous inoculation, but interferon is barely

found in mouse-cell cultures infected with Sind-bis virus (3; Ho and Postic, unpublished data).

It appears from the above that there may wellbe a difference in the capacity of cells to syn-thesize and release interferon. It would be ofgreat interest to identify the most efficient cellsin this regard, to study the underlying reasonsfor this difference, as well as to determine whetherthis capacity is in any way related to any otherfunction of host resistance, such as phagocytosisand antibody formation. With respect to theprecise biological significance of rapidly inducedinterferon in the host, very little that is definitecan be said at this time. One would expect suchinterferon to be more efficacious in preventingviral spread than interferon formed after virusreplication (77). Preliminary evidence withSindbis virus in mice suggests that, indeed, in-terferon may operate by preventing early viremicdeath (56).

IDENTITY OF VIRAL INHIBITORS ENDOGENOUSTO CELLS

The report that many different substancesstimulate interferon formation, as well as therapidity of in vivo interferon formation afterintravenous viral and bacterial (80) inoculation,suggest the obvious possibility that interferonis endogenous to cells. Such interferon may bepreformed without any inducing agent, or itmay be constantly formed in response to anoccult inducer of either viral or some otherunknown origin.The possibility that interferon-like inhibitors

may exist in uninfected cell systems has beenrecognized since early interferon studies. It hasbeen the experience of most workers in the fieldthat control fluids in interferon titrations derivedeither from uninfected cell culture overlay orfrom extracted cells are occasionally, but fre-quently erratically, inhibitory to viral action (see,for example, Table 9, 27; Gresser, private com-munication). This is true of fluid overlays of unin-fected continuous or primary cell cultures, chickallantoic fluid, and extracts of normal organs.Viral inhibitory activity of these fluids is usuallylow. It has undoubtedly been explained away invarious ways and dismissed as a vagary of labora-tory determinations.

Recently, however, Tsilinsky (69, 70, 71, 72),in a series of four papers, described in some detaila viral inhibitor found in the fluid overlay and

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cell extracts of five continuous human cell lines(HeLa, ERK, KB, Detroit-6, and FL), and onemonkey cell line (CMH). This inhibitor has acombination of properties suggestive of bothinterferon and cell receptors. It resembles in-terferon in that it is digestible by trypsin, it isfairly heat-stable (60 C, 2 hr), it can be adsorbedon cells, and it is not lost on washing. On theother hand, it resembles cell receptors in thatit is localized primarily in the cytoplasmicfraction and is sedimentable at 22,000 X g for 2hr, suggesting a "corpuscular diameter of 200-600 mju." When applied to monkey-kidney cellcultures, it produces an apparent increase inadsorption of ECHO virus from 55 to 76%. Ithas antigens of cellular protein, since it is neu-tralized by anticellular serum. Unfortunately, nodefinitive test for its direct viral-inactivatingcapacity was made, and no tests were performedon nonprimate cell cultures to test its speciesspecificity. Somewhat similar, though less com-plete, findings with various types of continuouscell lines have been reported by Pumper (57)and Pellegrini (55).

In a study of cell receptors, Quersin-Thiry(58, 59) also mentioned that "various receptormaterial can apparently leak out into nutrientfluid during the growth of normal, non-infectedHeLa cultures." Since these receptors are bydefinition direct inactivators of virus, a strongpossibility exists that her material and that de-scribed by Tsilinsky are identical, although datafor direct comparison are not available.There are also two considerations made in

her work on virus receptors of HeLa cells whichare especially relevant in any attempt to dis-tinguish between receptor and interferon. Bothincrease the difficulty of such a distinctionunder certain circumstances: (i) the kineticsof direct viral inactivation, which is the hall-mark of receptors, is reversible and accompaniedby "reactivation" of adsorbed virus especiallyupon dilution of the virus receptor mixture. Ofthe four viruses tested for this property [NDV,Western equine encephalitis (WEE), polio(Mahoney), and polio (MEF1)], this was trueof NDV and WEE. (ii) There are no physico-chemical properties common to all receptors(59). Hence, certain receptors for viruses areheat-labile (polio), and others are heat-stable(NDV); some are inactivated by trypsin (polio,WXEE), but others are not (WEE). Recent

evidence confirms the idea that there may bechemical differences between receptors of evenclosely associated viruses. It was found thatHeLa cell receptors to Coxsackie B3 and polio-virus Type 1 differed in their susceptibility tothe proteolytic enzymes, chymotrypsin andtrypsin. Chymotrypsin inhibited attachmentof B3 but not poliovirus, and trypsin inhibitedadsorption of poliovirus and not B3 (81). Ingeneral, it appears that studies of receptors todifferent viruses by use of different cells havenot reached the completeness with which recep-tors of primate tissues for poliovirus have beencharacterized (29, 31). It would, therefore, bepremature to generalize from these studies toother models.

In a recent attempt to detect endogenousinterferon in 11-day-old chick-embryo cell ho-mogenates, I found a virus inhibitor, similar tothat described by Tsilinsky and others, in the ab-sence of any inducer (Ho, unpublished data). Someof my results in studying this "endogenousinhibitor" may be summarized to further illus-trate the problems in distinguishing virus recep-tor from interferon (Table 4).

This inhibitor was similar to interferon, sinceit could adsorb on cell cultures and was not com-pletely removed by repeated washings. Further-more, it was resistant to 56 C and was not dia-lyzable. Studies on direct inactivation of EEEvirus were frequently equivocal, but there wasusually a reduction in virus titer. This may be areflection of the difficulty in doing adsorptionwork with arboviruses, the viruses most fre-quently used for testing interferon. Such difficultywas also brought out by Wagner (74). The inhibi-tor was found to be effective in mouse-cell cul-tures. It was apparent then that it had no speciesspecificity. Therefore, one of the hallmarks ofchick interferon was missing. In addition, thepartial sedimentability of the inhibitor suggestedthat it was not interferon, but probably an inhi-bitor with a high molecular weight or a smallermolecule adsorbed to some such large sediment-able material. Although I believe that this en-dogenous inhibitor is most likely virus receptorrather than interferon, in the absence of cleanseparation it is impossible to rule out the possi-bility that low titer interferon-like substancesmay be in this homogenate.The lesson from these experiments is that

viral receptors must be ruled out in any study

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TABLE 4. Summary of findings on an endogenous chick-cell viral inhibitor*

Procedure Result

Effect of washing treated chick cultures

Effect of heating (56 C, 30 min)

Effect of dialysis

Effect of dilution

Direct inactivation of EEE

Effect on heterologous mouse-embryo cultures

Effect of fractions of inhibitor homogenate

No wash: 93% plaque reduction1 wash: 85% plaque reduction3 washes: 78%70 plaque reduction

No effect

No effect

50% cell homogenate, effective up to 1:64

23 to 75% reduction of virus titer

92% VSV plaque reduction

Ribosomalt supernatant fluid, 32% plaque reduc-tion

Ribosomal sediment, 76% plaque reduction

* Inhibitory effect was measured by adsorbing 0.3 mnl of inhibitor for 3 hr at 37 C on duplicate chick-cell cultures, washing off inhibitor with 5 ml of Hanks' solution, challenging with 100 to 200 PFU ofEEE virus, and calculating reduction of plaque formation as compared with similarly treated controlculture.

t Obtained after 22,500 rev/min for 30 min (#40 rotor, model L Spinco centrifuge).

purporting to demonstrate induction of interferonor the presence of interferon. This is especiallytrue if the process of induction involves pro-longed incubation and cell injury (40) suchthat more opportunities exist for the "leakingout" of cell receptors. However, since little ap-pears to be known about the complex propertiesof different cell receptors for different viruses,the process of distinguishing between these twoviral inhibitors may be more difficult than isapparent at first sight.One final point should be considered: a pos-

sible relationship between interferon and cellreceptors. Is it possible that receptors are in-complete interferons or vice versa? One mightstill maintain that interferon is an intracellularinactivator of virus, except for the fact that itinhibits the infectivity of infectious RNA (23,15, 19). Though the notion that interferon actsby binding infectious nucleic acid is still plausible(78), the idea that it binds whole virus directlyeither extracellularly or intracellularly is un-tenable. Therefore, there is as yet no obviousbridge to connect the different mechanisms ofaction of interferon and cell receptors of virus.Nevertheless, there are other similarities betweenthese two cellular products. It is possible that

the production of cell receptors under certaincircumstances requires "inducers." It is wellestablished that the amount of cell receptorsvaries with types of tissue (29) and with age(46), and increases upon in vitro cultivation(29, 58). Further studies on both interferon andcell receptors are required before definitiveformulation of the situation is possible.

SUMMARY

Certain problems regarding the identity andinduction of interferon have been discussed andreviewed. Many of these arise because of thelarge number of heterogeneous substances re-ported to be inducers of interferon. A few, especi-ally intracellular microorganisms, appear wellestablished.

Interferon formation seems metabolically tobe a cellular, as distinguished from a viral,process. It is mediated by cell messenger RNAand requires new protein synthesis. This is welldemonstrated by work with antimetabolites.However, relatively little is known about thecellular control of this mechanism, and the roleof the "inducer." The resemblance to inducedenzyme synthesis is attractive but unproven.

Induction of interferon in experimental ani-

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mals by intravenous inoculation of virus is awell-established phenomenon. There is suggestiveevidence that there are specialized host cellswhich make more interferon and release itfaster than others.There are no precise criteria for the recognition

of interferon, only general ones. Attention iscalled to cellular substances which may simulateinterferon, especially if inhibitory activity is oflow potency. One such substance is cell receptorsfor viruses.

ACKNOWLEDGMENT

Portions of this work were supported by PublicHealth Service grant AI-01953 from the NationalInstitutes of Health.

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