BAcTzmuoLoGIcAL Ruvizws, Sept. 1977, p. 543-567Copyright 0 1977 American Society for Microbiology

Vol. 41, No. 3Printed in U.S.A.

Antiviral Activity of InterferonsROBERT M, FRIEDMAN

Laboratory ofExperimental Pathology, National Institute ofArthritis, Metabolism, and Digestive Diseases,Bethesda, Maryland 20014

INTRODUCTION.............................................................. 543ESTABLISHMENT OF THE ANTIVIRAL STATE ........... .................... 544

Interferon Binding ................... ............................. 544Development of Antiviral Activity................................. 546

LOCUS OF THE INTERFERON-INDUCED INHIBITION OF VIRUS GROWTH. . 547Evidence that Interferon Treatment Inhibits Virus Uncoating ....... ........... 547Evidence that Interferon Treatment Inhibits Transcription of the Viral Genome. 547Evidence that Interferon Treatment Inhibits Viral Protein Synthesis ..... ...... 550

Observations in virus-infected cells ........................... 551Observations in cell-free systems ....... .................... 552

Evidence that Interferon Treatment Inhibits Terminal Events in the ReplicationCycle of Murine Leukemia Viruses ........ ................. 557

INTERFERON TREATMENT IS INEFFECTIVE IN SYSTEMS IN WHICH THESV40 GENOME IS INTEGRATED INTO AN INTERFERON-RESISTANTVIRUS OR A HOST GENOME ........................... 559

DISCUSSION ............................. 560LITERATURE CITED........................... 562

Principles should not be unnecessarily multiplied.- William of Ockham, about 1320

I beseech you, in the bowels of Christ, think itpossible you may be mistaken.

-Oliver Cromwell, 1650

INTRODUCTION

Interferons are proteins, the production ofwhich can be induced in animal cells by a vari-ety of stimulating substances; interferons in-hibit a wide range of viruses by inducing anintracellular antiviral state, yet most interfer-ons are animal species specific in their range ofantiviral activity. Interferon was discovered in1957 (85), but to the present time there has beenno entirely statisfactory explanation of its po-tent antiviral effect. Part of the reason for thislies in the impressive biological activity of in-terferons. A recent estimate ofthe specific anti-viral activity ofhuman interferon was at least 2x 108 reference units per mg of protein, and itmay be 10 to 100 times more potent (71). With amolecular weight of about 25,000, this wouldmean that 1 active unit per ml is present in (atmost) a 10-12 M interferon solution. Choleraand diphtheria toxins are marginally active at10-9 M; therefore, on a molar basis interferonexceeds the specific activity of some of the mostpotent biologically active substances. As a con-sequence of this, interferon preparations con-taining very large amounts of antiviral activityturn out to have extremely small amounts ofinterferon. It is, therefore, very difficult to pu-rify interferons completely, and only recently

has there been any great optimism that thiscan be accomplished (71).

Interferon assays have added to this diffi-culty because they have been exclusively bio-logical and are based on the ability of a prepa-ration to inhibit the production ofa virus or of aviral product in infected cells. They are time-consuming and relatively inaccurate. Cellsmust be treated with interferon for severalhours before virus infection until antiviral ac-tivity develops. Because of the inherent inac-curacies of the biological assays, a two- tothreefold inhibition is considered just signifi-cant; therefore, a level ofuncertainty is presentthat is usually considered intolerable in a bio-chemical or biophysical system.One other problem has been the multiple

substances that bear the name interferon inany one species. There are several proteins ofdiffering molecular weights that have an anti-viral effect and are produced in differing cellsystems. Human buffy coat interferon containsthree antiviral proteins, two with molecularweights of about 20,000 and one of molecularweight 15,000. Interferon produced in humandiploid fibroblasts antigenically resembles oneof the proteins with a molecular weight of20,000 but is distinct from the other buffy coatinterferons. Finally, humanT lymphocytes pro-duce an "immune" interferon in response toantigens and mitogens; this is a distinct inter-feron species. All of these act to produce anintracellular antiviral state, but it is unclearwhether they have the same mechanism of ac-tion (107).

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Finally, results of studies on the antiviralactivity of interferons have often been confus-ing and contradictory. In some cases it is uncer-tain whether the reported effect was due to aninterferon, because the concentration of impur-ities in interferon preparations far exceeded theconcentration of interferon. Also, with distress-ing frequency, studies on the mechanism ofaction of interferon which appeared to herald areal breakthrough in our understanding haveseemed to lack that most fundamental require-ment of science, reproducibility.

This review will concern itself with the anti-viral activity of interferons. There is very con-vincing evidence that interferon also has othereffects on cells, such as inhibition of cell andtumor growth (72, 77, 106, 132), alteration inlevels of cellular enzymes (100), and regulationof the immune system (63). Although publica-tions on some of these observations are appear-ing with increasing frequency, there are stilltoo few reports to warrant a detailed review ofthese at this time.

ESTABLISHMENT OF THE ANTIVIRALSTATE

When cells are treated with an appropriateinterferon, they develop an antiviral state, anda wide variety of viruses grow poorly in suchcells. In the absence of virus infection, how-ever, it is usually difficult to show whether ornot a cell has been treated with interferon. Onecan arbitrarily look at two aspects of the anti-viral effect of interferon cells: first, how inter-feron treatment alters the uninfected cell toproduce an antiviral state; and second, the fateof viruses in an interferon-treated cell. In thissection I will discuss what is known about theinduction of the antiviral state. Until recently,most studies on this phenomenon have para-doxically had to involve virus infection at onepoint or another in order to test the degree ofantiviral activity developed.

Interferon BindingIn spite of the very low concentrations of

interferon that are required to induce an anti-viral state, there is strong evidence that only asmall portion of the interferon in a solutioninteracts directly with cells to induce antiviralactivity. No detectable interferon was removedfrom a preparation that was repeatedly used toinduce antiviral activity (21). There is, how-ever, some evidence that interferon is bound tothe surface of cells in which an antiviral state islater induced. When cells were treated withinterferon at 40C, no antiviral activity waspresent if the cells were washed and then im-

mediately infected at 370C with a rapidly repli-cating virus. If, however, the cells were allowedto incubate at 370C for even 1 or 2 h, antiviralactivity was evident. There is, therefore, analteration in the cells after incubation at 40Cwith interferon, but a period of active metabo-lism at 370C was necessary to develop an anti-viral state (38).The nature of the interaction at 40C between

interferon and cells is partially understood.After incubation with interferon at 40C, treat-ment of cells with trypsin in the cold inhibitedthe development of antiviral activity. It ap-peared that interferon was bound at 40C to cellsin superficial sites that were accessible to tryp-sin (8, 38). After a few minutes at 370C, how-ever, a complex series of changes took place.Interferon that had been bound to the cell be-came trypsin resistant, yet could still be re-covered in an active form by extraction of thecells. With further incubation, cell-associatedinterferon tended to elute into the culture fluid(8, 126).The role of the portion of the interferon that

did become cell associated is not entirely clearat present (76). There is no direct evidence thatthis interferon was bound to a specific cellularinterferon receptor; however, interferon treat-ment has been shown to alter the binding ofcholera toxin or thyrotropin (TSH) to plasmamembranes (75) and to decrease the sensitivityof cells to diphtheria toxin (97). All of thesebiologically active substances bind to fairlywell-characterized, specific receptors. The inhi-bition of their binding by interferon meanseither that interferon directly competes withthese substances for their binding sites or thatinterferon treatment brings about a generalalteration in membranes; one manifestation ofthe latter would be a decreased binding of otherbiologically active substances. If, however, theformer is the case, it would appear that distinctbinding sites must exist for interferon.The location, chemical nature, and specificity

of such putative binding sites have been inves-tigated. One study indicated that if such sitesexist, they must be on the external surface ofthe plasma membrane. Interferon has one unu-sual biological property. The cell producing thehumoral factor (interferon) may also be an ef-fector cell. In nature this is not likely to beimportant, because the interferon-producingcell is already virus-infected and thus probablybeyond salvation by interferon treatment. Inone tissue culture study, however, human fi-broblasts were stimulated to produce interferonby the double-stranded ribonucleic acid (RNA)inducer polyriboinosinic acid * polyribocytidylicacid [poly(I * C)]. Inclusion of anti-human inter-

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ANTIVIRAL ACTIVITY OF INTERFERONS 545

feron antibody in the medium prevented devel-opment of an antiviral state, even when theantibody was added after interferon synthesishad already been initiated (134). The resultsseemed to indicate that interferon must beexternalized and interact with the outer surfaceof the plasma membrane in order to be effec-tive. Similar results have been obtained in cellsinduced to make interferon and then treatedwith ouabain (79).The receptors for interferon would appear to

be gangliosides or ganglioside-like structureswith an oligosaccharide moiety at the func-tional binding site. Treatment of mouse cellswith Phaseolus vulgaris phytohemagglutinin(PHA), a plant lectin that binds to carbohy-drates, blocked the development of antiviralactivity after subsequent interferon treatment(9). Also, Sepharose-bound interferon lost itsantiviral activity after preincubation with gan-gliosides, especially GM2 and Gr, (10), and solu-ble interferon was bound to Sepharose-ganglio-side beads (10). Sialyl-lactose reversed the abil-ity of gangliosides to inhibit the antiviral ac-tion of interferon, and PHA inhibited inter-feron binding to gangliosides (12). Further-more, ganglioside-deficient mouse cells wereinsensitive to induction of antiviral activity byinterferon; treatment of these cells with gan-gliosides both increased cell membrane gan-glioside content and, in two of three cell linestested, significantly increased cell sensitivity tointerferon treatment. Ifindeed the receptors forinterferons are gangliosides, these receptorswould resemble those of cholera toxin and sev-eral glycopeptide hormones (98, 99).

It is difficult to comment as yet on the speciesspecificity of the putative binding sites for in-terferon. Many substances that have no knownintracellular function bind to cell surfaces. Onecannot imagine that all such binding involvesspecific receptor sites. There is significant bind-ing of some interferons to cells in which theinterferons tested had no known biological ac-tivity (26, 75). The meaning of this is unclear;however, mouse interferon altered the bindingof cholera toxin or TSH to plasma membranesofKB cells, a human cell line that is completelyunresponsive to both mouse and human inter-ferons (75). Similar conclusions have beenreached from studies in rat embryo fibroblaststreated with rat and human interferons. Hu-man amnion interferon has antiviral activity inthese rat cells, but human leukocyte interferondoes not; however, both human interferonsblock the antiviral activity of rat interferon.Thus, human leukocyte interferon must haveinteracted with the rat cells even though it didnot induce antiviral activity (26). In the case of

human interferons, neutralization experimentsappear to indicate that multiple reactive siteson the interferon molecule can interact withcells of different species to induce antiviral ac-tivity (107). These results taken together sug-gested that the species specificity of interferonsdoes not reside solely at the point oftheir initialinteraction with their binding sites and thatbinding of an interferon is a necessary, but nota sufficient, condition for biological activity; itis an overture that may not be followed by theopera.A number of substances that bind to and

alter the cell membrane inhibit interferon ac-tion. Cytochalasin B (109, 142) and colchicine(26) both are active in this respect, although theeffect of the former is probably due to extrusionof the cell nucleus. Mercaptopyridethylbenzim-idazole, which inhibits membrane transport ofnucleosides, blocks the development of anti-viral activity in interferon-treated cells (46). Asmight be expected, chorionic gonadotropin,TSH, and cholera toxin, which bind to ganglio-side receptors, also inhibit interferon action(11, 75). Antibody to a cell surface componentcoded for by human chromosome 21 inhibitedthe antiviral activity of interferon (112), al-though antibody directed nonspecificallyagainst cell surface antigens of human fibro-blasts had no such inhibitory effect (134). Sincechromosome 21 in human cells appeared to benecessary for the development of antiviral ac-tivity after treatment with human interferon(32, 128, 129), these findings suggested thathuman chromosome 21 may determine an anti-gen important in the interferon receptor. Otherstudies, however, suggested that human chro-mosome 21 did not determine the interactionreceptor but that some .other chromosome did(28, 32). Also, there was a suggestion that chro-mosome 16 carried a function that regulatedthe interferon-induced antiviral state (28).

Interferon treatment appears to bring abouta number of changes in plasma membranes ofuninfected cells. Plasma membrane prepara-tions of L-cells exposed to interferon developeda complex alteration in their ability to bindcholera toxin. At low interferon concentrations,binding of toxin was significantly increased;when the interferon concentration was in-creased, however, binding was inhibited (75).Similar complex effects on binding have beenreported with other systems and are thought tobe due to binding of a substance to both anactive (at low concentrations) and inhibitory(at high concentrations) site (98). Interferontreatment also alters the surface charge on L-cells (73), and in AKR mouse cells treated withinterferon there is a significant change in the

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density of purified plasma membrane prepara-

tions. In untreated cells, 80% of the plasmamembrane banded in sucrose at a density of1.20, and 20% banded at 1.23; in interferon-treated cells, 25% banded at 1.20, and 75%banded at 1.23. This change may be related toalterations in plasma membrane gangliosidecontent after interferon treatment. Also, thereis a marked increased in the number of intra-membranous granular particles that can be ob-served by electron microscopy after freeze etch-ing the membranes of interferon-treated AKRcells as compared with untreated controls (E.Grollman, F. T. Jay, and E. H. Chang, manu-script in preparation). Although the signifi-cance of these alterations in plasma mem-branes after interferon treatment is unclear,they are consistent with a profound change thatappears to be induced by exposure to interferonand may well be related to the development ofthe antiviral state.

Development of Antiviral ActivityVery soon after Isaacs and Lindenmann dis-

covered interferon they recognized the neces-sity for metabolic activity for interferon action(85). This finding has been repeatedly con-firmed and is generally thought to mean thatinterferon itself is not antiviral but that thedevelopment of an antiviral state requires theintracellular production of an antiviral sub-stance. There is one study which does suggestthat interferon may itself be antiviral and thatthe necessary metabolic activity was simply totransport interferon into the cell (122). Al-though this notion is difficult to rule out, thepreviously discussed experiments, which indi-cated that interferon must be externalized to beactive (134), make it rather unlikely, unlessthere is some portion of the interferon moleculethat must be reintroduced. In addition, inter-feron bound to an insoluble, Sepharose matrixwas active in inducing antiviral activity (4);this study did not, however, rule out the possi-bility that interferon that might have come offthe Sepharose matrix was actually responsiblefor the induction.

Interferon resembles, in some respects, glyco-peptide hormones and, therefore, what hasbeen learned about the mechanism of poly-peptide hormone action may be useful in uncov-ering clues to the mechanism of interferon ac-tion. As an example, in transmitting its mes-

sage to the cell, TSH is believed to require asequence of events whose main contributors arein turn a specific receptor (probably a ganglio-side), a significant alteration in the state of themembrane, an effector-responsive adenylatecyclase, and cell metabolism susceptible to reg-

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ulation by increased production of cyclic 3',5'-adenosine monophosphate (cAMP) (99). Thereis some evidence that interferon action involvesthe first two steps listed (75, 133). In addition,there is a report that cAMP analogs such asdibutyl-cAMP increased the level of antiviralactivity induced by a preparation of interferon(45). I am not aware of any reported studies onthe effect of interferon treatment on adenylatecyclase activity. I believe that Chany's model ofan interferon-binding site and an associatedbut distinct activation or amplification site thatis actually responsible for the development ofantiviral activity is probably correct (20, 26, 27)and resembles to a great extent the glycopep-tide hormone models.

Several cell functions are known to be re-quired for the induction of antiviral activity byinterferon. Studies (27, 28, 32, 128, 129) haveshown the relationship of specific chromosomesto the induction of antiviral activity in humanand mouse cells. Human cells trisomic for chro-mosome 21 are more sensitive to human inter-feron than cells that are diploid. The latter inturn are significantly more sensitive than cellsmonosomic for chromosome 21 (28, 128). Thehuman interferon activity gene would appearto lie on the distal portion of the long arm ofchromosome 21, since the translocation of thislocus to another chromosome transfers sensitiv-ity to interferon (35). This site is closely linkedto the locus of the enzyme indophenol oxidase(129).The nature of the gene function required for

antiviral activity is to some extent knownthrough studies with antimetabolites. In 1964,Taylor showed that interferon action was in-hibited in cells treated with actinomycin D(130); however, established antiviral activitywas not decreased by treatment with actinomy-cin D (130). Several other inhibitors of RNAsynthesis and ribonucleoside analogs also in-hibited interferon action and, taken together,these studies indicated that cellular RNA syn-thesis is probably necessary for interferon ac-tion (124). These findings have recently beenconfirmed by studies with enucleated cells. Incells in which nuclei were removed by exposureto cytochalasin B, interferon treatment failedto induce antiviral activity. Once established,however, antiviral activity was not reversed byremoving the nucleus (109, 142).The results of several studies with inhibitors

of protein synthesis or amino acid analogs alsosuggested that interferon action required pro-tein synthesis (124). These experiments werenot as convincing as those with actinomycin D,which suggested a need for RNA synthesis,probably because the activity of inhibitors of

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protein synthesis must be reversed in order totest (by virus growth) for the presence of anti-viral activity (48).These results suggest a by now familiar

model: interferon treatment induces the pro-duction of a cellular messenger RNA (mRNA),which is translated to an antiviral protein. It isthe latter that actually inhibits the growth ofviruses. This theory was first suggested to meby J. A. Sonnabend in 1963, and to date noreport has refuted it. By the same token, therehas not yet been a direct demonstration of anantiviral protein or its mRNA.There are two studies purporting to define a

significant difference in a specific RNA constit-uent between interferon-treated and controlcells. Levy et al. (82, 112a) have investigated asmall increase in the size of mRNA's and trans-fer RNAs (tRNA's) from interferon-treatedmouse cells by polyacrylamide gel electrophore-sis. Although these differences were small,they were reported to be consistent. However,almost all of the RNA species examined frominterferon-treated cells seemed to trail behindthe analogous species from extracts of un-treated cell by about the same distance. Since,under the conditions employed, the distancemigrated by an RNA species is thought to varydirectly with the logarithm of its molecularweight, the finding would imply that interferoninduces very large differences in the molecularweight of large RNA species, but quite smalldifferences in small species. There would seemto be no easy explanation for these findings atpresent, but such studies as these are impor-tant since they may uncover significant differ-ences that may exist between the RNAs ofinterferon-treated and control cells.

LOCUS OF THE INTERFERON-IN-DUCED INHIBITION OF VIRUS GROWTHInterferon inhibits the replication of a sur-

prisingly wide variety of viruses, and inci-dently of some nonviral infectious agents (62).Many of these viruses and infectious agentswould appear to have very little in common. Anumber of virologists have, therefore, investi-gated this problem and employed several virus-cell systems in attempts to find a specific virusfunction which is blocked in interferon-treatedcells. At least four possible sites of action havebeen seriously suggested by a number of labo-ratories. It is unfortunate that most of thesesuggested sites of action involve phases of virusreplication about which we known quite little.Perhaps this is more a comment on the generalstate of the science (art) of virology than oninterferon research; on the other hand, it mayalso be symptomatic of the perversity of the

latter. At any rate, I shall discuss each of thetheories about interferon action currently invogue in the order that they generally appearin the virus replication cycle.

Evidence that Interferon Treatment InhibitsVirus Uncoating

Until recently, there seemed little reason forseriously considering this as a site of action forinterferon. Several studies with infectious RNAfrom poliovirus or western equine encephalitisvirus indicated that interferon inhibited thereplication of these RNA forms (33, 55, 59).Barring hydrolysis of the RNAs by ribonucle-ase (RNase) left from the interferon prepara-tions used to treat the cells, the data clearlyindicated that interferon must act at a sitebeyond the uncoating step. Uncoating is alsonot inhibited in interferon-treated cells infectedwith reovirus (135).

Studies with simian virus 40 (SV40) seemed,however, to contradict the findings with infec-tious RNA (140). In agreement with the find-ings of Oxman et al. (96, 102, 103), the produc-tion of SV40 early mRNA and T antigen wasmarkedly inhibited in interferon-treated mon-key cells, when these cells were infected withintact SV40. If, however, infectious SV40 de-oxyribonucleic acid (DNA) was employed, theresults were directly opposite those obtainedwith infectious RNA. The synthesis of earlySV40 mRNA and T antigen were only slightlyinhibited in interferon-treated cells infectedwith the SV40 DNA (140). This would appear toindicate that in interferon-treated cells, SV40uncoating, or a step soon after it, was inhibited,and that this is the explanation for the appar-ent inhibition of SV40-directed transcriptionunder these conditions. A similar block in un-coating or an event soon thereafter has beendescribed at nonpermissive conditions for theSV40 mutant, tslOl (113).The results in interferon-treated cells in-

fected with SV40 DNA are clear-cut, but havenot as yet been confirmed. The most likelyexplanation for the finding was discussedabove, but there is one other possible explana-tion. In the SV40 systems employed in thisstudy, there is a multiplicity-dependent revers-ibility of the interferon-induced inhibition ofvirus replication (140). This has been reportedin another system (60). To carry out the experi-ments with SV40 DNA, 0.075 or 0.75 jig ofSV40DNA was employed. This amount of infectiousDNA could conceivably be equivalent to a highvirus-cell multiplicity, if any great part of theDNA actually induced infection. The explana-tion for these extraordinary findings may lie,therefore, in the SV40 multiplicity reversal of

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interferon action also reported in the same pub-lication (140). Still, the possibility that inter-feron action involves, at least under some con-

ditions, an inhibition of viral uncoating is im-portant, and until such time as this work isdirectly contradicted, any theory that attemptsto explain interferon action will have to makeprovision for these findings.

Evidence that Interferon Treatment InhibitsTranscription of the Viral Genome

The work in this area of interferon researchis quite controversial, and, like scripture, can

be quoted to support any side of an argument.In this case the disagreement revolves aroundwhether interferon treatment causes an inhibi-tion in the primary transcription of the geneticinformation of viruses. All of the possible an-swers (yes, no, or sometimes) can be found invarious publications.

Part of the confusion undoubtedly is due tothe close relationship between viral RNA syn-thesis and viral protein synthesis (124). If avirus requires a polymerase not present in cellsand has no endogenous RNA polymerase as a

structural element, inhibition of viral proteinsynthesis will result in inhibition of all virus-directed RNA synthesis, because the viral po-lymerase is among the proteins whose synthe-sis would be inhibited. Arboviruses and picor-naviruses, among the RNA viruses commonlyused in interferon research, are lacking struc-tural polymerases.

In the case of those agents containing struc-tural polymerases, analysis ofthe site of inhibi-tion can be quite complicated. Since a polymer-ase is already present, inhibition ofprotein syn-thesis does not cause inhibition of early RNAsynthesis. Primary transcription takes place inthese cells, but secondary transcription, whichdepends on the elaboration ofnew polymerases,will be inhibited. Results obtained employingsuch systems require careful analysis, sincesignificant inhibition of RNA synthesis doesnot necessarily indicate that primary RNA syn-thesis is the site of action.Furthermore, the use of inhibitors of protein

synthesis in these experiments introduces someadditional problems. First, the endogenousviral polymerases may be subject to negativecontrol by viral proteins produced early in theinfection process. When an inhibitor of proteinsynthesis is employed, the level of primaryRNA synthesis in controls may be elevated(13). This could lead to difficulties in interpre-tation of data suggesting inhibition of primarytranscription by interferon. In addition, relatedto the general problem of using inhibitors ofprotein synthesis is a problem relating specifi-

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cally to the use of cycloheximide, which hasbeen the most commonly employed inhibitor ofprotein synthesis; under these conditions, cy-heximide inhibits the elongation of peptides, itprotects and stabilizes mRNA (3, 123). Inter-feron treatment might alter this stabilization,if it tended to restrict the initiation of viralprotein syntehsis; under these conditions, cy-cloheximide might not have protective effectson mRNA in interferon-treated cells. This couldwell lead to large differences in the concentra-tions of intracellular mRNA in cells treatedwith cycloheximide alone as compared withcells treated with both cycloheximide and inter-feron. These differences would not of course beindicative of an interferon effect on primarytranscription.Among RNA viruses, there have been de-

tailed reports on the effect of interferon on theprimary transcription of vesicular stomatitisvirus (VSV), influenza virus, and reovirus. Inthe case of DNA viruses, vaccinia virus andSV40 have been well studied.

Inhibition of primary transcripton of VSVhas been reported in human (88), chick (89),and monkey cells (91). All three studies em-ployed cycloheximide to block secondary tran-scription and actinomycin D to inhibit hostRNA synthesis, and then measured viral RNAsynthesis in the presence or absence of inter-feron. In the experiments performed in humancells, there was a quantitative decrease in viralmRNA but no qualitative change (88). Similarconclusions were drawn from experimentscarried out in chick cells infected with in-fluenza Ao/WSN, but an entirely differentmethod was employed. 32P-labeled virus infec-tion was established, and at different timesafter infection, RNase-resistant RNA wasdetermined before and after annealing. In con-trol cells treated with cycloheximide, up to 20%ofthe infecting RNA became RNase insensitiveafter annealing. In actinomycin D-treated orinterferon-treated cells, almost none of theviral RNA became RNase resistant (7).These results certainly suggested that at

least one effect of interferon was to inhibit pri-mary transcription of RNA viruses with struc-tural polymerases. Contradictory results have,however, been obtained in a study of interferoninhibition of VSV replication in interferon-treated monkey or human cells. With concen-trations of interferon that significantly in-hibited virus replication in these systems, therewas only a 30% decrease in the specific activityof viral RNA synthesis in the presence of cyclo-heximide and interferon in monkey cells ascompared with those treated with cyclohexi-mide alone. In the human cells, the comparable

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figure was 53%. Those viral RNA forms thatwere most sensitive to inhibition by interferonwere forms that required the elaboration of anew polymerase to produce, i.e., were due tosecondary transcription. In addition, a concen-tration of interferon that inhibited viral RNAsynthesis by only 10% inhibited viral proteinsynthesis by 60% (6). The increment of inhibi-tion seen with both cycloheximide and inter-feron over that with cycloheximide alone wasfelt to be due to the above-described protectiveeffect of cycloheximide on mRNA and its possi-ble absence in interferon-treated cells (3, 123).A study of the effect of interferon on early

production of complementary RNA in VSV orinfluenza virus-infected chick or mouse cellsalso reached the conclusion that inhibition ofprimary transcription was probably not thebasis of interferon action. The results of thisstudy are, however, ambiguous to me becausethe authors seemed to ignore what might besignificant differences in hybridization data be-tween cycloheximide- or interferon-treated cellextracts. In fact, in view of a possible protectiveeffect of cycloheximide on mRNA, a cyclohexi-mide control for an interferon-treated cell cul-ture may give very misleading results, and itwas not surprising, therefore, that some differ-ences were indeed noted in this study; thesedifferences were ignored, presumably becausethe authors doubted their significance (111).

In an important study on the mechanism ofinhibition of reovirus replication by interferon,the authors employed a different tack, whichobviated the use of cycloheximide. Among thetemperature-sensitive mutants of reovirus type3, ts447 is blocked in its formation of progenyRNA at nonpermissive temperatures (38.50C);therefore, only primary transcription takesplace at this temperature. A study of thegrowth of ts447 in interferon-treated cells incu-bated at 38.50C indicated that a concentrationof interferon that inhibited virus yields by 80%,inhibited primary transcription by only 12%(135). Under approximately the same condi-tions, however, virus-directed translation wassignificantly inhibited (see below).One additional finding in interferon-treated

cells infected with an RNA virus will be dis-cussed in this section although it does not,strictly speaking, deal with an inhibition oftranscription. Marcus et al. (92) have also re-ported that treatment with chick interferoncaused an elevation of the enzymatic activity ofa membrane-associated RNase, with optimalactivity at an alkaline pH (assays were run atpH 8). If this is a general finding, it couldexplain some ofthe divergent observations thathave been made on interferon action in various

laboratories. It would, for instance, account forwhy viral transcription seems to be inhibited insome interferon-treated cells, whereas in oth-ers, viral translation seems to be the site ofinterferon action. By this theory of Marcus etal. (92), what results one obtains would dependon what was being measured. Intracellularviral mRNA concentrations would be decreasedand virus-directed translation would be in-hibited. In cell-free systems measuring viraltranslation, the increased nuclease in extractsof interferon-treated cells might hydrolyze theadded viral mRNA, and this in turn wouldcause an apparent primary inhibition in trans-lation.

In order for these notions to be considered asa general mechanism of interferon action, how-ever, it would have to be shown that increasedalkaline RNase was induced by homologous in-terferon in cells of species other than chicks andonly chick cells were used in the study of Mar-cus et al. (92); indeed, Maenner and Brandnerhave reported no increase in nuclease activityin poly(I 0)-treated monkey, hamster, quail,or duck cell extracts (86a). In addition, no speci-ficity was shown by this system, in that theRNase activity was tested only with VSVmRNA. Since interferon action appears to showspecificity for virus functions, an RNase that isthought to be responsible for interferon actionshould show a restricted spectrum of activity.

Vaccinia virus was the first of the DNA vi-ruses to be studied intensively with respect tointerferon (50). In many ways vaccinia virusinfection lends itself well to this sort of study,because the virus contains an RNA polymerasethat is activated with removal of the outer coatof the virus. The polymerase, which is locatedin the viral core, then elaborates viral mRNA,which is translated to yield several active pro-teins, including one that inhibits activity of thevirus structural RNA polymerase and anotherthat is responsible for final uncoating of theviral DNA (87).The effects of interferon on this well-studied

system are fairly clear-cut in both mouse (64,65, 94) and chick cells (36, 65, 93). Early vacci-nia mRNA synthesis was increased (64), butfinal uncoating of the viral DNA was inhibited(87). Although the production of viral mRNAdepends on a virus-associated polymerase, theinhibition of the activity of the polymerase andthe uncoating of the viral DNA depend on syn-thesis of new proteins. It would appear in thissystem, then, that the site of interferon-in-duced antiviral action must lie between virus-directed transcription and translation.

In one study, however, inhibition of earlyvaccinia virus mRNA synthesis was reported in

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interferon-treated chick cells (13). This resultmay have been due to the use of cycloheximidein this particular study, but similar experi-ments in both mouse and chick cells with orwithout cycloheximide have repeatedly shownthat transcription of vaccinia virus mRNA isincreased rather than decreased in interferon-treated cells (93).A study performed in frog polyhedral cyto-

plasmic deoxyribovirus (FPCD)-infected fat-head minnow (FHM) cells indicated that theearly mRNA synthesis by FPCD was inhibitedby FHM interferon. The results also showedthat FPCD production was inhibited by FHMinterferon and that FPCD virus-associated po-lymerase activity was not inhibited by an earlyvirus protein; however, there was only a 57%inhibition of early viral mRNA synthesis by 10U of FHM interferon, a concentration that re-duced virus replication by about 90%o, and a75% mRNA inhibition by 100 U under condi-tions in which 20 U completely abolished virusreplication. In addition, cycloheximide was em-ployed in some of these studies (53). These re-sults would appear to be open to the same criti-cisms that can be made of some of the studieswith RNA virus-use of cycloheximide and arelatively small effect on primary RNA synthe-sis as compared with large effects on viral repli-cation.SV40 differs from the other DNA viruses dis-

cussed in that it lacks a structural RNA polym-erase and so at least early in infection mustdepend on a cellular polymerase. Interferontreatment inhibited SV40 T antigen productionin acutely infected, but not in transformed,cells (101, 102). Transformation of mouse 3T3cells by SV40 was also inhibited by interferontreatment (131). These results were consistentwith a primary effect on either virus uncoating(140) or virus-directed transcription (103) ortranslation (52).Oxman and Levin next investigated the ef-

fect of interferon treatment on early SV40-spe-cific RNA synthesis in monkey cells (103). Theresults indicated a marked inhibition of veryearly RNA synthesis under conditions where nolate RNA could be formed, that is, in cellstreated with enough cytosine arabinoside (204g/ml) to inhibit viral DNA synthesis by morethan 99%. A subsequent study showed that theinhibition of early SV40 RNA synthesis in in-terferon-treated cells was probably not the re-sult of inhibition of virus adsorption, penetra-tion, or uncoating; or of increased degradationof either the viral DNA template or viral RNA;or, finally, to an inhibition of translation of avery early mRNA that might have a secondary

inhibitory effect on further early viral RNAsynthesis (96). These results with SV40 weresomewhat unexpected, because the virus mustuse a cell polymerase early in infection.Although these studies are of great interest,

the previously mentioned findings that sug-gested an altered uncoating of SV40 virus ininterferon-treated cells could account for theinhibition ofearly SV40 RNA synthesis (140). Itwill be important to see whether these experi-ments with infectious DNA of SV40 can berepeated.One other study with the SV40 system is of

present interest. SV40 RNA prepared in vitrowith SV40 DNA and a bacterial polymerasewas microinjected into SV40-permissive mon-key cells that had been treated with interferon.Under these conditions, T-antigen productionwas blocked, although it was evident in con-trols microinjected but not treated with inter-feron (52). This result indicated that, in inter-feron-treated and SV40-infected cells, transla-tion of virus genetic information may be in-hibited; however, this result does not necessar-ily mean that inhibition of translation is animportant mechanism of interferon action intbe SV40 system in vivo.Although many of the papers reviewed in

this section suggested that interferon treat-ment caused an inhibition in early virus-di-rected transcription, in the case of most virusgroups studied there is at least one publicationthat indicated no direct effect of interferontreatment on primary transcription. With RNAviruses having virus-associated polymerases,the most meaningful studies of interferon ac-tion at this point would appear to be those thatemploy viral mutants blocked just after pri-mary transcription has been completed. Onlyone study has so far been reported with such asystem (135), and the general conclusionreached was that, although there was someinhibitory effect of interferon treatment onearly reoviruses RNA synthesis, it was notenough to account for the profound inhibition ofvirus replication. The authors felt that in inter-feron-treated cells, transport of subviral parti-cles from phagocytic vacuoles and lysosomes,where they were formed, to locations in thecytoplasm where they transcribe RNA mightbe slowed by interferon; however, they ad-vanced no direct evidence for this hypothesis(135).As for the DNA viruses, the weight of the

evidence in the case of vaccinia virus infectionis that interferon treatment has no inhibitoryeffect on primary viral RNA synthesis. Thejury is still out on SV40, however. I, for one,

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look forward with great anticipation to furtherpublications on the effect of interferon treat-ment on the replication of SV40 DNA.

Evidence that Interferon Treatment InhibitsViral Protein Synthesis

It should be noted again, at the onset of thediscussion of this material, that viral proteinand RNA synthesis are so interdependent thatit is often difficult to distinguish which of thetwo is the primary site of action of an antiviralsubstance such as interferon, since progenyRNA molecules are usually responsible formost of the total virus-directed protein andRNA synthesis that is carried on during infec-tion. Therefore, whether interferon treatmentacts directly to inhibit RNA or protein synthe-sis early in infection, later both transcriptionand translation are profoundly inhibited (124).In order to show which ofthe two is the primarysite of interferon action, it is necessary to studyits effect under conditions in which viral RNAand protein synthesis are clearly dissociated.This has been effected in two ways: either bystudying viral protein synthesis in cell-free sys-tems; or, among RNA viruses, by studying ininfected cells the messenger function of paren-tal (input) RNA of viruses the genome ofwhichis an mRNA (positive-stranded RNA viruses) orof viral mRNA that is made by using the inputRNA as a template in association with a viralpolymerase (negative-stranded RNA viruses).There have also been important studies on oneDNA virus. Experiments on viral mRNA func-tion in cell-free systems derived from inter-feron-treated cells will also be discussed below.

Semliki Forest virus (SFV, an arbovirus) andmengovirus (a picornavirus) have been used todetermine whether positive-stranded RNA vi-rus messenger function is a primary site ofinterferon action. Of the negative-strandedRNA viruses, VSV and reovirus protein syn-thesis have been employed. Among the DNAviruses, there has been extensive study of theeffect of interferon treatment only on the earlyprotein synthesis of vaccinia virus.

Observations in virus-infected cells. In infec-tion with SFV, inhibition of viral protein syn-thesis in interferon-treated cells does not ap-pear to be a result of inhibition of viral RNAsynthesis. Under conditions in which viralRNA synthesis was almost completely in-hibited, early SFV protein synthesis was unaf-fected. If cells were treated with interferon,however, no virus-specific proteins could beidentified (39). In this system, therefore, paren-tal RNA does not seem to be translated; theRNA of parental SFV also does not become

RNase resistant (42, 49). The latter observationwould indicate that the infecting virus fails toenter the double-stranded form and so cannotbecome part of a replication complex as such. InSFV infection the replication complex is amembranous structure associated with theviral polymerase and single- and double-stranded viral RNA (43, 54). One report indi-cated that in interferon-treated cells, parentalRNA of SFV entered into a membranousstructure that appeared to be a replication com-plex. The RNA of this structure remained inthe single-stranded form, presumably becausethe viral polymerase was not produced (51).Some insight into the mechanism of how in-

terferon might inhibit viral protein synthesiswas provided by studies on mengovirus-infectedL-cells. Less radioactivity from input radioac-tive mengovirus was precipitated in a post-mi-tochondrial fraction from interferon-treated L-cells than from untreated control cells. Thisprecipitated fraction was found to contain 50Sand 240S components, both of which had de-creased activity in the extract from interferon-treated cells. The author felt that the 240S com-ponent represented viral polysomes and the 50Scomponent represented a complex between theviral RNA and the 40S ribosomal subunit (81).Although interferon treatment did appear toinhibit the formation of these structures, theirnature was not clearly established, and thelevels ofradioactivity were very low. Therefore,the significance of these findings must remainuncertain.Only one study has been carried out on the

effect of interferon treatment on protein syn-thesis by the negative-stranded RNA virusVSV (141). The results indicated that 3 h afterinfection in interferon-treated rabbit kidneycells, the formation of all structural and non-structural VSV proteins was inhibited. ViralRNA synthesis was not studied, but the inhibi-tion of viral protein synthesis was so profoundthat, if decreased RNA synthesis were the pri-mary factor, only a very marked inhibitioncould possibly account for these results. Thefindings in interferon-treated, VSV-infectedcells (see above) indicated that, even if it doesoccur, inhibition of virus-directed primarytranscription might not be as great as would benecessary to completely account for the ob-served inhibition of virus-directed protein syn-thesis.

In the study of the effect of interferon on thegrowth ofthe reovirus mutant ts446, the resultsof which were discussed above with respect toprimary viral transcription, inhibition of viralprotein synthesis was also studied (135). Under

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conditions in which only primary transcriptiontook place, there was a 20% inhibition in viralRNA synthesis but a 40 to 72% inhibition of thesynthesis of various individual virus-specificpolypeptides. The apparent inhibition of viralRNA synthesis did not seem great enough tothe authors to account for all of the observedinhibition of virus translation.

In interferon-treated cells infected with vac-

cinia virus, as indicated above, all (36, 64, 87,93, 94) but one (13) of several studies indicatedthat early viral mRNA synthesis was not in-hibited. The vaccinia virus mRNA formed ininterferon-treated cells has a normal content ofpolyadenylic acid (70), but does not associatewith ribosomes to form polyribosomes as read-ily as does mRNA ordinarily formed early invaccinia virus infections (19, 64, 94, 95). Thisfailure to form polyribosomes readily is proba-bly the result of an inhibition of the initiationsteps in viral protein synthesis, although someinhibition ofchain elongation was also reported(95). There was a suggestion that the site ofinhibition of initiation of peptide synthesismight be in the formation of a complex betweena vaccinia virus mRNA-protein (ribonucleopro-tein) and the 40S ribosomal subunit (95); how-ever, the findings did not conclusively showthis.SV40 is the only other DNA virus in which

virus-directed protein synthesis in interferon-treated cells has been studied at all. In inter-feron-treated cells there is marked inhibition ofthe synthesis of viral T antigen in lytic infec-tions (102). This could have been due, however,either to the previously discussed inhibition ofvirus uncoating (140) or to inhibition of virus-directed transcription (103). As previouslynoted, there was inhibition ofthe translation ofSV40 mRNA microinjected into interferon-treated cells (52). If this as yet unconfirmedfinding is correct, it suggests that inhibition ofSV40 translation might take place in inter-feron-treated cells; however, significant inhibi-tion of virus uncoating (140) or transcription(103) would preclude there being very muchSV40 mRNA produced in interferon-treatedcells.

Observations in cell-free systems. All of theobservations that have been made on the effectsof interferon in cell-free systems have been car-ried out since 1966. Although late in starting,this has been a rapidly developing field, andmany interesting and potentially important ob-servations have been made; however, the re-sults here have also been somewhat disappoint-ing to me. Although it apparently has beenpossible in cell-free systems to imitate the in-terferon-induced inhibition of virus-directed

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translation, this has not as yet led to firm con-clusions on the basic mechanism of interferon'saction, and, as will be seen from the discussionof the results obtained so far in these studies,there is even some disagreement as to whichsystem best represents what is going on in theinterferon-treated, virus-infected intact cell.Most studies in cell-free systems have been

carried out by investigators convinced at theonset that the interferon-induced antiviralstate was aimed at virus-directed translation.So far, it has been impossible to mimic thepurported inhibitory action of interferon ontranscription, although in the absence of publi-cations on the subject, I am not sure how hardsuch studies have been pursued. The activity ofviral polymerases is markedly decreased in ex-tracts from the cytoplasm of interferon-treatedcells, but this could have been due to a decreasein the production of the enzyme rather than toan inhibtion of its action (125). Neither inter-feron, nor extracts from interferon-treatedcells, virus-infected or uninfected, inhibitedviral polymerase activities (J. A. Sonnabend,personal communication). This is not to saythat such inhibitory activity will not be demon-strated, but so far, there are no reports ofcondi-tions where such inhibitory activity could berepeatedly found.In the study of interferon's effect on virus-

directed protein synthesis, two sorts of systemshave been employed. In the earlier group ofexperiments, viral RNA was incubated withextracts from control or interferon-treated cells,and the interactions were analyzed with respectto the binding ofthe RNA to the cellular compo-nents. Later, when cell-free systems employingcomponents from animal cells became availablefor the production of specific polypeptides, sev-eral laboratories began to study the ability ofviral RNA to stimulate amino acid incorpora-tion by cytoplasmic fractions from interferon-treated and control cells.Marcus and Salb (90), employing viral RNA-

binding studies, postulated that interferon in-duced the production of a new translationalinhibitory protein that could combine with ribo-somes to inhibit their ability to translate viral(but not cellular) mRNA. When they mixedtritiated Sindbis virus RNA with cytoplasmicextracts containing 74S monomeric ribosomes,a 250S polysomal structure was formed at 00C.On incubation at 370C, this 250S polysome wasbroken down, and it was thought that this rep-resented the translation of the mRNA becauseno breakdown occurred in the presence of cyclo-heximide. When ribosomes from interferon-treated cells were used, however, they boundviral RNA to a decreased extent, but more im-

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portantly, the breakdown of the 250S polysomeat 370C was decreased. This was taken to meanthat these ribosomes were unable to translateviral mRNA.Employing similar methods in the mengovi-

rus-L-cell system, Carter and Levy (22) showedthat less viral RNA was bound at 00C to ribo-somes from interferon-treated cells to formpolysomes than to ribosomes from control cells.A later publication using a similar system sug-gested, however, that more, not less, viral RNAwas bound under these conditions (30).

Results from other laboratories were directlycontradictory to the findings of Marcus andSalb. Kerr et al. (69) reported that no discretepolysomes were formed when viral RNA wasincubated with ribosomes at 4°C, and there wasno difference in the attachment of viral RNA toribosomes from interferon-treated or controlcells. When the mixtures were incubated at370C there was breakdown of the complexesthat had been formed at 40C, but this break-down was not correlated with protein synthesis.Indeed, RNase activity was probably responsi-ble for the breakdown of the viral RNA-ribo-some complexes observed at 37°C, since therewas RNase activity in cell sap and ribosomepreparations. Similar observations were madeby R. Lockart, Jr. (personal communication).These results, taken together, were quite de-

pressing. There is no strong evidence that theobservations of Marcus and Salb (90) or Carterand Levy (22) were related to virus-directedprotein synthesis. The observations on the ef-fect of interferon on the system could not berepeated by Kerr et al. (69). No further confirm-atory publications have appeared to strengthenthe binding theories advanced by these au-

thors. It may yet turn out that these notions arethe correct explanations for the antiviral activ-ity ofinterferons, but it will probably not be thedata in these publications that will prove thebinding theories to be correct.The first publication that attempted to em-

ploy viral RNA as a messenger in a cell-freesystem was also by Carter and Levy (23). Theyshowed that, although both polyuridylic acid[poly(U)]- and tobacco mosaic virus (TMV)RNA-directed protein synthesis were not in-hibited in cell-free amino acid-incorporatingsystems derived from interferon-treated cells,mengovirus RNA-directed synthesis was. Theyfelt that the lack of activity of the ribosomeswas due to their alteration as a consequence ofinterferon treatment ofthe cell from which theyhad been derived. The nature of the productsstimulated by mengovirus RNA was not inves-tigated. Moreover, the work was difficult torepeat, partially because the salt concentra-

tions employed in the incubation mixtures wereomitted from the published form of the manu-script and partially because, until relativelyrecently, it has been difficult in other hands tosignificantly stimulate animal cell systemswith TMV RNA to incorporate amino acid intospecific polypeptides (74).

Since 1972, the pace of activity in this area ofinterferon research has quickened. This hasbeen due, in great part, to the development ofanimal cell-free protein-synthesizing systemsthat could translate both viral and cell mRNA'swith fidelity. Some very puzzling results havebeen published using such systems in inter-feron studies, and the contrasting findings ofseveral laboratories have only partially beenreconciled.One ofthe basic disagreements in this field is

whether the antiviral activity of interferon ismanifest in extracts of interferon-treated cellsin the absence ofsome activating step. Workingwith L-cells, a group at Mill Hill, London (44),observed that only a small decrease in virus-directed amino acid incorporation and in thesize of viral peptides formed was present inextracts derived from cells treated with inter-feron. When the interferon-treated cells werealso infected with vaccinia or encephalomyocar-ditis (EMC) virus there was a marked decreasein the ability oftheir extracts to translate EMCRNA, but the stimulatory action of poly(U) onphenylalanine incorporation was not inhibited.The initiation and the elongation of virus-spe-cific peptides were affected in the extracts frominterferon-treated and virus-infected cells, butinitiation seemed inhibited to a greater extent(68). One additional, and somewhat curious,finding in this system was that EMC peptideformation initiated with formylated methionylinitiator tRNA escaped the major interferoninhibition at the time of peptide chain initia-tion (67). This latter finding remains unex-plained but tended to give rise to the notionthat the interferon effect studied was an inhibi-tion of an early step in the initiation of virusprotein synthesis. It might be important to notehere that, in initiation with formylated methio-nyl initiator tRNA, there was a loss of therequirement for one of the (normally required)initiation factors (121a).Current efforts of Kerr's group have been

directed toward investigation of the apparentnecessity for virus infection of cells to activatethe inhibitory effect ofinterferon in the cell-freesystem derived from them. Addition of poly-(I-C) or double-stranded RNAs from reovirusor P. chrysogenum phage to extracts from in-terferon-treated cells that had not been infectedwith viruses resulted in marked inhibition of

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viral RNA-directed translation (66). Indeed, ad-dition of small volumes of a post-ribosomal su-pernatant fraction from interferon-treated cellsto a mixture of extracts of control cells anddouble-stranded RNA also caused an inhibitionin the translation of viral mRNA, whereas cellsap from interferon-treated cells or double-stranded RNA alone did not cause such inhibi-tion when added to extracts from untreatedcells. The observed inhibition in the presenceof interferon cell sap and double-stranded RNAalso required adenosine 5'-triphosphate (ATP),and preincubation of interferon cell sap withboth ATP and double-stranded RNA greatlyincreased its inhibitory capacity. Both thedouble-stranded RNA and the ATP could beremoved after this preincubation step withoutimpairing the inhibitory activity of the treatedcell sap (114). The results suggested that inter-feron treatment caused the formation of aninactive inhibitor, the activation of which wasdue to double-stranded RNA and probably isrelated to a phosphorylation step. The activatedinhibitor then interacted with the protein-synthesizing system to inhibit virus-directedtranslation. I presume that the addition ofdouble-stranded RNAs to the extracts frominterferon-treated cells served the same func-tion as did infection of intact, interferon-treated cells with EMC or vaccinia virus, butthis remains to be proven. These results are ofadded interest because the regulation ofproteinsynthesis in reticulocyte lysates required phos-phorylation of an initiator methionyl-tRNA-binding factor by a protein kinase that is pres-ent in a translational inhibitor from heme-deficient systems (80).

After addition of double-stranded RNA to ex-tracts from interferon-treated mouse cells, frac-tionation indicated that the inhibitory effectwas associated with both the microsomal andcell sap preparations. Addition of microliterquantities of cell sap from interferon-treatedcells to cell-free systems from control cells madethem sensitive to inhibition by double-strandedRNA forms, but the inhibition induced by dou-ble-stranded RNA in interferon cell sap wasnot, in their hands, reversed by the addition oftRNA. The abnormal distribution ofEMC poly-peptides synthesized in systems inhibited byinterferon cell sap and double-stranded RNAresembles that obtained on translation ofEMCRNA in cell-free systems from interferon-treated, virus-infected cells. Finally, althoughthere was an enhanced breakdown of EMCRNA in inhibited systems, it was not clearwhether this was due to an induced nucleaseactivity or to the degradation of mRNA notengaged in translation in inhibited systems

(66a). This uncertainty is somewhat reminis-cent of the above discussion of the purportedinhibition of viral transcription in the presenceof cycloheximide.A group from Yale has suggested that treat-

ment with double-stranded RNA of extractsfrom interferon-treated Ehrlich ascites tumorcells resulted in induction of an endonucleaseactivity that degraded viral RNA (20a). Inthese extracts there was an ATP-dependentphosphorylation of at least two proteins, theapparently critical one having a molecularweight of 67,000 (78). A group from Israel (143)has presented similar findings, and in bothstudies (78, 143) the phosphoprotein was ribo-some-associated but could be removed by a saltwash. After addition of double-stranded RNAthe more rapid degradation of viral mRNA inextracts from interferon-treated cells requiredATP. The degradation was biphasic: double-stranded RNA and ATP were required for thefirst phase (activation), whereas in the secondphase (endonuclease action), neither had to bepresent (119a).

In many respects these findings were similarto those of the group at Mill Hill, with thedifference that the latter group considered thedegradation possibly to be secondary to the in-hibition of protein synthesis and not its cause(66a). In addition, Kerr's group reported (114a)finding at least three major phosphorylatedsubstances in their extracts after addition ofATP and double-stranded RNA, one substancewith a molecular weight of about 60,000, an-other of about 30,000, and the third of low mo-lecular weight. The activated inhibitor itselfwas also of low molecular weight and was sta-ble to 900C for 5 to 15 min. The heated butactive inhibitor had no protein kinase activityand did not function as a kinase activator incell-free systems. It is possible that this inhibi-tor was identical to the low-molecular-weightphosphorylated component.

Lest the reader feel, however, that this as-pect of the mechanism of action of interferon isclose to being solved, it should be pointed outimmediately that there is also a well-nourishedbody of literature which insists that extracts ofuninfected, interferon-treated cells show anti-viral activity with no particular manipulationor activation. In mixed cell-free protein-synthe-sizing systems containing ribosomes fromKrebs ascites cells and cell sap fractions frominterferon-treated chick fibroblasts, EMC RNAwas translated somewhat less efficiently thanin mixed systems containing control cell sap(65a). Also, the same Israel- and Yale-basedgroups that carried out the just-discussed workon a ribosome-associated phosphoprotein acti-

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vated by interferon treatment have publishedseveral important studies on inhibition of viralmRNA translation in extracts from uninfectedcells.

In the Israeli studies, extracts from unin-fected L-cells treated with interferon translatedthe RNA of mengovirus less efficiently thanextracts from control cells (37). The concentra-tion range of interferon used was higher thanwas necessary in the previously discussed stud-ies with cells that were both interferon-treatedand later infected with virus (44). The virusinhibitory effect was associated with a ribo-somal factor that could be washed off the ribo-some by a buffer containing 0.5 M KCl. Some-what similar results were obtained by thegroup at Yale University with a system em-ploying extracts from Ehrlich ascites tumorcells and EMC RNA or reovirus mRNA (57).The inhibitory activity studied by the Yalegroup was not dialyzable and seemed to beribosome associated. During incubation of theextracts at 30TC for 90 min, however, at leastsome of the translational inhibitory activitywas released from ribosomes into the superna-tant and therefore did not sediment at 200,000x g for 150 min. The specificity of this inhibi-tory activity was also investigated. Althoughthe antiviral activity of mouse interferon inintact L-cells was associated with a specific in-hibition of viral, but not of cellular, proteinsynthesis, the translation of both viral andcellular mRNA's was inhibited in incubationmixtures employing extracts from interferon-treated L-cells (56); therefore, the cell-freesystem did not seem to reflect very well whatwas going on in the intact cells. The Israeligroup also found the same lack of discrimina-tion by their interferon-treated, L-cell-derivedsystem (37). The group from Mill Hill had re-ported a similar finding, but they had usedextracts from virus-infected cells in whichcellular protein synthesis was markedly in-hibited; therefore, it was not surprising thattranslation of cell mRNA's would be inhibitedin their systems (44). In general, the inabilityof extracts from interferon-treated cells to dis-tinguish between host and viral mRNA's incell-free systems has been disappointing be-cause there is excellent discrimination in intactcells (note, however, the single exceptionbelow).A further development in the studies on inhi-

bition of translation in extracts from unin-fected, interferon-treated cells indicated thatthe interferon-induced inhibition could be cor-rected by the addition of tRNA from animal,but not bacterial, sources (30, 31, 58). Onlysome species of animal tRNA's seemed very

active in this respect. In extracts from inter-feron-treated cells, kinetic studies showed thattranslation of viral mRNA proceeded normallyin L-cells but stopped after 20 to 30 min. Addi-tion of tRNA allowed translation to resumenormally. The restoration of translation of glo-bin mRNA by tRNA seemed to require differenttRNA's than did restoration for viral mRNA(31). Most of the inhibitory activity was di-rected against elongation of peptide chains, andinhibition of chain initiation seemed secondaryto the effect on elongation. In contrast to thefindings of the Mill Hill group there was adecreased binding of formylated methionyl ini-tiator tRNA to the initiation complex in ex-tracts from interferon-treated cells (31). In ef-fect, therefore, the results suggested that non-functional polysomes were formed in extracts ofinterferon-treated cells and that this was due tointerferon action. The formation of such inac-tive polysomes was, in turn, a result of a defectin some species oftRNA (31, 58). The nature ofthis tRNA was further elucidated by chromato-graphic purification of minor species of leucyl-tRNA (143). Addition of some of these tRNAforms allowed proper translation of mengovirusRNA in extracts from interferon-treated cells,whereas others permitted globin mRNA trans-lation. In spite of these findings, the authorsfelt that the main difference between extractsfrom control cells and extracts from interferon-treated cells was the presence in the latter of aribosome-associated inhibitor of translationthat blocked peptide chain initiation and elon-gation when some tRNA's were present in lim-ited concentrations (143).

Again, the group at Yale had similar find-ings in extracts from Ehrlich ascites cells, ex-cept that viral mRNA translation proceeded ata lower rate for 30 min in extracts from inter-feron-treated cells before halting; but, large,inactive polysomes were formed. This effectwas also reversed with animal tRNA's. Thebasic defect here was traced to an impairmentof amino acid acceptor activity of lysyl-, seryl-,and, especially, of leucyl-tRNA's. This was dueto a faster rate of inactivation of these tRNAforms in extracts from interferon-treated cellsthan from control cells (119).There were, however, some disturbing find-

ings related to this neat explanation for inter-feron action (29, 119). There was no differencein the amounts of tRNA from interferon-treated or control cells required to correct theblock in translation in extracts from interferon-treated cells. In addition, extracts from inter-feron-treated cells inactivated leucine-specifictRNA's from interferon-treated or control cellsat the same rate. Finally, and most impor-

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tantly, filtration of an extract from interferon-treated cells through Sephadex G-25 or dialysiswas necessary for the extract to develop thecapacity to rapidly inactivate leucyl-tRNA. Inextracts that had not been so treated, there wasonly very marginal inhibition of the transla-tion of viral mRNA (119).These results suggested that interferon treat-

ment alone does tend to alter the cytoplasmicextracts in the following manner: after passagethrough Sephadex or dialysis, an activity isproduced that tends to destabilize the chargingcapacity of some species of tRNA. This appearsto be an effect of interferon treatment, but therelationship of this to the antiviral activitydeveloped in interferon-treated cells is very farfrom being clear. It may be that these findingsare related to those previously discussed inthat, in the case of extracts from uninfectedcells, either dialysis or filtration throughSephadex acts as an activation step, just asdoes the addition of double-stranded RNA (66);however, the effects of the activation by dialy-sis or Sephadex filtration (119) seem differentfrom those seen with extracts treated withdouble-stranded RNA (66) or with extractsfrom interferon-treated and virus-infected cells(44). It remains to be determined whether allof these can be somewhat pulled together asbeing basically a single effect of interferontreatment viewed under different conditions.Three additional publications in this area of

research merit discussion at this point. All areof potential interest but are so far unconfirmed.Samuel and Joklik (116) investigated the trans-lation of reovirus and vaccinia virus mRNA's inextracts of uninfected Krebs II ascites cells.Normally, such extracts translated endogenousmRNA, and added poly(U), Krebs cell or L-cellmRNA's, or viral mRNA's quite well. Whenthe cells were treated with interferon in theascites state, their extracts translated viralmRNA's poorly, but they did translate theother mRNA's mentioned as well as extractsfrom control cells. The unusual feature of thissystem was that such treatment with inter-feron actually had to be carried out in intactmice by intraperitoneal injections of high-titermouse interferon preparations.The results of Samuel and Joklik (116) also

suggested that the inhibition of virus mRNAtranslation was due to a ribosome-associatedfactor that could be recovered by treatment ofthe ribosomes with 1.0 M KC1. In addition, a 0.3to 0.6 M KCI wash fraction of the ribosomesfrom interferon-treated cells contained a pep-tide with a molecular weight of 48,000, whichwas not found in ribosomes or washes fromribosomes of untreated cells. The 0.3 to 0.6 M

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KC1 wash fraction also inhibited the transla-tion of viral mRNA's in extracts from untreatedKrebs II ascites cells.

In further work with this system, Samuel(115a) found that formation of reovirus methio-nyl-X initiation dipeptides was only slightlyinhibited in extracts of interferon-treated cells.Also, mouse ascites cell tRNA partially re-versed the inhibition of reovirus mRNA trans-lation, and reovirus 32P-labeled mRNA was sta-ble after a 15-min incubation with extracts frominterferon-treated cells. These results sug-gested that the interferon-induced inhibition ofviral mRNA translation took place at a stepsubsequent to formation of the first peptidebond and involved participation of tRNA.These findings are ofgreat interest, since it is

the only work I am aware of that claims to findthe same specificity of interferon action in cell-free systems derived from uninfected cells as isfound in intact cells. It also suggested that thespecific antiviral activity of interferon treat-ment was mediated by a ribosome-associatedpolypeptide that inhibited virus-directed pro-tein synthesis but had no effect on cellular pro-tein synthesis. Unfortunately, this work hasnot yet been confirmed. Attempts in my labora-tory to repeat the findings of Samuel and Joklikwere unsuccessful (L. Pinkus, unpublished ob-servations). Therefore, although the conclu-sions reached by this work would go a long wayin establishing the biochemical basis for theantiviral action of interferon, final judgmentmust await its confirmation by other groups.Another rather exciting finding by the Yale

group must also await confirmation (120, 120a).This was the report of an inhibition of reovirusmRNA methylation in extracts of interferon-treated Ehrlich ascites cells. Most, but not all,viral (and cellular) mRNA's contain blockedand methylated 5' ends, and this terminalmethylation was shown to be necessary for thetranslation of reovirus mRNA in extracts fromanimal cells. The Yale group demonstratedthat the methylation of unmethylated reovirusmRNA was impaired in extracts from inter-feron-treated cells. This impairment was notdue to cleavage, irreversible inactivation of un-methylated reovirus mRNA, depletion ofmethyl donors, or accumulation of methylationinhibitors in the reaction mixture. The methyl-ation inhibitor was a heat-labile macromole-cule that decreased methylation of 5'-terminalguanylate residues. In a study of the in vitrorate of reovirus mRNA methylation, methyla-tion by core-associated enzymes was inhibitedby extracts from interferon-treated cells (120a).Again, to my knowledge, there has been noconfirmation of this work in this or any other

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system, so that its significance must remain indoubt. Moreover, interferon treatment inhibitsreplication ofEMC virus, which appears not tohave 5'-terminal methyl groups (L. Pinkus, un-published data). However, demonstration of aspecific inhibitory effect on methylation ofviralmRNA's would also go far in establishing yetanother biochemical basis of interferon action.A last contribution ofpotential interest is the

finding that extracts from cells treated withpartially purified mouse interferon caused thedeacylation of aminoacylated TMV RNA or ofEMC RNA. There was no alteration of amino-acylated tRNA, and a similar hydrolyzing ac-tivity was present in crude interferon prepara-tions (118). The chief problem with this work isthat there is as yet no known function in ani-mal cells for amino acids linked to viral RNAsand, therefore, hydrolysis of such structures isofunknown significance. Also, the experimentsin this study did not, to my mind, entirely ruleout the possibility that the activity observedwas due to a contaminating ribonucleolytic ac-

tivity.Evidence that Interferon Inhibits TerminalEvents in the Replication Cycle of Murine

Leukemia VirusesThe last major group of findings to be dis-

cussed is concerned with an apparent inter-feron-induced inhibition of the assembly ofRNA tumor viruses. Studies on this observa-tion have been going on for less time than thoseon transcription or translation; perhaps that iswhy contradictions and paradoxes are some-what less common here.

It has been known for some time that RNAtumor viruses are sensitive to interferon (5) andthat in interferon-treated cells newly infectedwith murine leukemia viruses (MLV), virusyields are significantly inhibited (117). Surpris-ingly, there was also inhibition of virus yieldsin interferon-treated rat and mouse cells chron-ically infected with a murine RNA tumor virus(61, 132a). This was unexpected because inter-feron was thought to be effective only in situa-tions where cells were treated before virus in-fection (124). Replication of endogenous type CRNA tumor viruses was sensitive to interferontreatment (17, 47), whereas high concentra-tions of interferon did not seem to preventinduction of intracisternal type A particlesafter bromodeoxyuridine or dimethyl sulfoxidetreatment (17); however, quantitation of A-particles is quite difficult, and their nature is indispute. Another group found that in theirsystem, mouse mammary tumor virus (B-particle) production was also sensitive to inter-feron treatment (J. Strauchen, N. Young, andR. Friedman, manuscript in preparation).

The work on RNA tumor viruses and inter-feron took an unexpected twist when evidencewas uncovered simultaneously in two laborato-ries that inhibition ofRNA tumor virus produc-tion was not correlated with inhibition of someintracellular steps in virus replication (14, 15,40, 47). In interferon-treated AKR cells in whichthere was marked inhibition of production ofboth endogenous MLV particles and infectiousMLV, the intracellular concentration of viralp30, group-specific (gs) antigen was unaffectedor even increased (40, 47, 108); furthermore,transmission electron micrographs indicatedthat the number of cell-associated virus parti-cles was not depressed in interferon-treatedMO-P cells, which were chronic producers ofKirsten murine sarcoma virus MSV (MLV)(14).More detailed studies followed these initial

observations. S. Z. Shapiro, M. Strand, and A.Billiau (121) found that synthesis of the pro-teins p30, gp 69/71, and p15 was not inhibited ininterferon-treated mouse 3T3 fibroblast cells.Synthesis and cleavage of the precursors ofthese proteins were also unaffected. In inter-feron-treated AKR cells, scanning electronmicrograph (SEM) studies confirmed and ex-tended the finding that there was no change inthe number of cell-associated virus particles.SEM (24) and transmission micrographs (16) infact clearly indicated that in mouse systems thenumber of surface particles was increased. Thismay have been the reason why cell-associatedp30 (gs) antigen was increased in these systemsafter interferon treatment. Since the viralRNA-dependent DNA polymerase also provedto be insensitive to interferon treatment inAKR cells (40), intracellular concentrations ofall of the groups of known MLV structuralproteins seemed unaffected by interferon treat-ment. Therefore, interferon inhibition of MLVproduction probably does not involve inhibitionof virus protein synthesis.One interesting fact to turn up in these stud-

ies was that as long as interferon was presentin the medium ofAKR cells that were infectedwith an endogenous MLV, virus productionwas inhibited. After removal of the interferon,full virus production again resumed and,within 24 h, it was equal to that of untreatedcells (40). The reason for this probably wasthat, although MLV production was greatlyinhibited by the interferon treatment, theprovirus remained integrated into the cellulargenome and upon removal of interferon, therewas no reason why normal production of viruscould not resume. This explanation was similarto that given for why interferon treatment didnot inhibit production of T antigen by an inte-

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grated SV40 genome (101, 105) (see below).These findings differed from those in infectionswith a lytic virus where repeated treatment ofcells with interferon may result in completesuppression of virus replication and apparentlyin disappearance of all traces of the viral ge-nome from cultures (41, 91).A number of cell cultures taken from differ-

ent mouse fibroblast strains chronically in-fected with MLVs were tested for sensitivity tothe virus-inhibitory effect of interferon (using alytic virus infection) and for the ability of inter-feron to inhibit chronic MLV production (40). Inall cell lines sensitive to interferon, MLV pro-duction was inhibited. In interferon-resistantlines there was no effect on MLV production.Pitha et al. (108) also tested the effect of inter-feron on several types ofMLV infection in AKRcells. In the case of exogenous infection, virusproduction was delayed but not suppressed,since virus production began when interferonwas removed. During inhibition by interferonof exogenous or induced infection, the findingswere similar to those previously discussed inthat the interferon block appeared to occur be-fore virus assembly so that, although there wasno inhibition of viral p30 (gs) antigen, therewas a decrease in released virus. They alsoreported, however, a decrease in cell-associatedviral particles, and in this respect their paperdiffered from previous reports on chronicallyinfected cells. Their findings in chronically in-fected cells also differed in one very interestingrespect from those previously discussed in thatthey found that the interferon-induced inhibi-tion of assembly and release seemed to result ina relatively small decrease in virus particlerelease but a much greater inhibition in theproduction of infectious MLV (108). This obser-vation has since been confirmed in both chronicand acute Moloney MLV infection in TB cells(25, 138). It would seem, therefore, that al-though in some systems interferon treatmentresulted in marked inhibition of virus release,in others, particle production was almost nor-mal, but the virus released was quite deficientin infectivity. In view of this conclusion, itwould be interesting to check a report (which isan apparent exception to the above-discussedfinding) that, in an interferon-sensitive cellline, interferon treatment seemed not to inhibitMLV production. Allen et al. (2) found that inJLS-V9R cells, interferon inhibited VSV pro-duction but had little effect on MLV particleproduction. A study of the infectivity of theviral particles produced would have been inter-esting in this system, for here, as in TB cells(25, 138) or in the AKR system of Pitha et al.(108), the interferon-treated cells may have re-

leased almost as many particles as control cells;however, the released particles may have beendeficient in infectivity.One of the most important recent observa-

tions in RNA tumor virology was the discoverythat latent RNA tumor virus infections couldbe activated by treatment of cells with halogen-ated thymidine analogs or by inhibitors of pro-tein synthesis (1, 86). It was natural that inves-tigators would attempt to study the effect ofinterferon treatment on this induction phenom-enon, and it was not difficult to demonstratethat interferon pretreatment (i.e., before induc-tion) inhibited the yield of MLV obtained inKirsten sarcoma virus-transformed BALB/3T3cells (KBALB/3T3) exposed to iododeoxyuridine(IUdR) (18). Interpretation of these results wasdifficult. Only a small percentage of the cells ina culture exposed to IUdR go on to producevirus, and the virus induced could have reinsfected KBALB/3T3 cells; therefore, it was un-clear whether the interferon treatment in-hibited virus induction or only the subsequentinfection of susceptible cells in the induced cul-ture. As noted above, similar findings wereobtained in AKR-2B cells induced by IUdR,except that marked inhibition of virus yieldswas obtained when interferon was present dur-ing and after IUdR treatment. The interferontreatment inhibited the number of cells produc-ing MLV but had no effect on p30 (gs) antigenproduction (108). Again, however, these resultswere complicated by the fact that the effects ofthe interferon treatment rapidly wore off whenthe interferon was removed, and the virus pro-duced could then reinfect susceptible cells inthe culture.One study did, however, suggest that inter-

feron might inhibit an event in the inductionprocess itself (110). Cycloheximide activatedKBALB/3T3 cells to produce only a xenotropicMLV that could infect rat cells but not mousecells (1). In a system using cycloheximide in-duction of KBALB/3T3 mouse cells with ratembryo kidney as detector cells, interferontreatment also decreased the virus yield, so itappeared that some event in the induction proc-ess was inhibited by interferon treatment.The findings ofWu et al. (139) differed in two

important respects from those just described.They found that interferon treatment had noeffect on the induction by IUdR of an N-tropicvirus from KBALB cells, whereas induction ofa xenotropic virus was inhibited in the samesystem. Also, the intracellular concentration ofviral gs antigen was decreased when virus pro-duction was inhibited by interferon treatment.In other studies, the tropism of virus seemed tomake no difference as far as interferon treat-

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ment was concerned, since N, B, NB, or xeno-tropic virus production and the replication ofthe NZB agent were inhibited (40). Also, inseveral other studies, the intracellular concen-trations of p30 (gs) antigen were not decreasedby interferon treatment, and indeed were insome cases actually increased (47, 108). Thereasons for the differences between the resultsof Wu et al. (139) and those of other investiga-tors are not clear. In the case of the p30 (gs)antigen, it may be that the differences are dueto the methods employed for detecting the anti-gen, since Wu et al. (139) used a complementfixation technique, whereas others employed aradioimmunoprecipitation inhibition assay (47,108).As the results obtained with interferon treat-

ment of MLV-infected cells seem at least super-ficially to be inconsistent with the findings inother systems, it would be useful to pin downthe site of this action, if possible, to a specificevent in the virus replication cycle. In order totry to do this, advantage has been taken of theproperties of a temperature-sensitive (ts) mu-tant of Moloney MLV, ts3 (136, 137). At nonper-missive temperatures (390C), this mutant failsto bud normally from the surface of TB cells.When the temperature was lowered, however,ts3 virus particles collected on the cell surfacewere released within 30 min, even in the pres-ence of cycloheximide. The SEM of TB cellsinfected with ts3 at 390C, therefore, strikinglyresembled those from interferon-treated AKRcells infected with MLV. It was of interest toestablish the temporal relationship in thegrowth cycle between the site of the ts3 muta-tion and the interferon-sensitive step.TB cells chronically (25) or acutely (138) in-

fected with the ts3 mutant were employed forthese experiments. In all cases in this sort ofstudy, there was a very rapid release of virusparticles in interferon-treated cells after a tem-perature downshift (25, 138). As previouslyfound by Pitha et al. (108) there was a muchgreater inhibition of viral infectivity than ofvirus particle production; however, the earlyrelease of viral antigen and transcriptase-con-taining viral particles after the temperaturedownshift was about as rapid in interferon-treated as in control cells. The results sug-gested that, in interferon-treated TB cells in-fected with ts3 and held at a nonpermissivetemperature, there was a population of virusparticles that was released from the cell surfaceimmediately upon the temperature downshift.These particles must, therefore, have been in-sensitive to the effect of interferon, and theresults taken together would suggest that theblock in ts3 replication at 390C must occur just

after the interferon-induced block. The resultsindicated that the morphogenesis and release ofMLV is a complex event and that interferontreatment inhibits a comparatively early stagein the process. Morphologically, many more vi-rus particles were in a very early buddingphase in the interferon-treated cells than incontrols (138), a result that also tended to con-firm the notion that interferon inhibited anearly event in the morphogenesis of MLVs. Thedecrease in infectivity of the particles that areactually released from the interferon-treatedcells in some systems (25, 108, 138) is probablyalso related to an interferon effect on this earlystep in morphogenesis.

INTERFERON TREATMENT ISINEFFECTIVE IN SYSTEMS IN WHICHTHE SV40 GENOME IS INTEGRATEDINTO AN INTERFERON-RESISTANT

VIRUS OR HOST GENOMEA special case where interferon is inactive in

a situation where normal inhibitory activitymight be expected requires additional con-sideration. Interferon treatment inhibited lyticinfection with SV40 virus (101). Not only wasinfectious virion formation inhibited but alsoproduction of T-antigen, an early gene product,was decreased (102). In mouse cells, SV40 doesnot undergo a complete replication cycle; never-theless, treatment with interferon inhibitedboth viral T-antigen production and viral-induced cell transformation (131). The stimula-tion of cellular DNA synthesis in BHK-21hamster cells after infection with polyomavirus (which is closely related to SV40) was alsoinhibited by treatment with hamster interferon(34). All of these findings clearly indicated thatSV40 and polyoma virus functions are sensitiveto interferon treatment. Evidence has alreadybeen discussed indicating that the site of inter-feron action in this case may be at any one ofseveral stages in the virus replication cycleincluding the uncoating step, or virus-directedtranscription or translation.

It was, therefore, surprising that in SV40virus-transformed mouse cells the production ofSV40 T-antigen was insensitive to interferontreatment in spite of the fact that such treat-ment made them resistant to VSV infection.That is, the same interferon system that recog-nized and inhibited new lytic infections withSV40 and other viruses apparently failed toinhibit viral fimction when it was exercised byan integrated tumor virus genome (101). Thisinterpretation was strengthened by findings incells infected with an adeno-SV40 hybrid virusthat is infectious and presents an interestinggenetic combination of an interferon-sensitive

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(SV40 virus) with an interferon-insensitive (ad-enovirus) genome. In stimultaneous infection ofcells with both complete viruses the sensitivityof adenovirus or SV40 T-antigen productionwas characteristic of infection with either virusseparately; however, in infection with theSV40-adenovirus hybrid, production of both T-antigens was as resistant as adenovirus T-antigen production in infection with adenovirusalone (105).There is strong independent evidence that

the SV40 genome is covalently linked to theadenovirus genome in the hybrid (104) or tocellular DNA in SV40-transformed cells (115).The mRNA produced by the integrated SV40genome contains host sequences (84), and themRNA of the hybrid contains both adenovirusand SV40 sequences (104). The resistance ofSV40 T-antigen production to interferon treat-ment in the case of integrated genomes mayindicate that the primary sequence of nucleo-tides in the genome does not determine sensi-tivity to interferon. Other sites on the genomesuch as those concerned with initiation or con-trol of genetic expression would then have to bethe loci of interferon action, and the presence ofhost or interferon-resistant virus RNA se-quences would render the virus mRNA resist-ant to interferon action. This inference doesnot, however, take into account the previouslydiscussed finding of Yamamoto et al. (140) thatinterferon inhibited the uncoating of SV40 vi-rus. If this finding is correct, the reason whyintegrated SV40 genomes seem resistant to in-terferon treatment would be quite clear: thevirus uncoating is not a step necessary for ac-tivity of the viral genome.

DISCUSSIONInterferon would appear to bind to glycolipids

or glycolipid-like structures on the plasmamembrane (10, 75, 133). This binding activitymay induce chemical, physical, and morpholog-ical alterations in the plasma membranes, andthese alterations may be directly related to theinduction of antiviral activity in cells in whichsuch activity is produced. Binding alone is notsufficient to bring about antiviral activity,though it induces some alterations in the mem-brane (75). Although nothing specific is knownabout what steps must follow binding in orderto give rise to antiviral activity, there is evi-dence that cAMP (45) may be involved in thisprocess and that cellular RNA (130) and proteinsynthesis (48) are necessary for the develop-ment of antiviral activity; it has been thoughtby many that an antiviral substance must beinduced by interferon treatment and that this isthe active moiety in the inhibition ofviral repli-

cation. These generalities about interferon ac-tion resemble to some extent what is knownabout the biological activities of glycopeptidehormones and bacterial toxins. One peptidecomponent of the hormone or toxin is responsi-ble for binding to specific glycolipid or glyco-lipid-like sites on the plasma membrane, andthis binding effects alterations in the state ofthe membrane. Another portion of the proteinacts through an adenyl cyclase-cAMP mechan-sim and is responsible for carrying out the spe-cific intracellular steps that characterize theaction of the hormone or toxin (99). There arethus some similarities to what is alreadyknown of the induction of interferon action.Perhaps, the establishment of interferon-in-duced antiviral activity will closely resemblethese systems.There has been great interest in the mecha-

nism of action of the putative antiviral sub-stance that is thought to be responsible forinterferon's activity in inhibiting virus replica-tion. The notion that interferon acts to inhibitprimary transcription of viral mRNA is basedfor the most part on findings in cells infectedwith viruses that have a structural polymerase(7, 89) or with SV40 (103). In the case of theformer, use of cycloheximide in many studiesseems to me to have led to the erroneous conclu-sion that primary transcription by viruses isdecreased in interferon-treated cells. This ispossible because cycloheximide treatment inthe absence of interferon might tend to protectmRNA's by forming stable polysomes (3, 123).Recent findings suggest that after interferontreatment, however, initiation of virus proteinsynthesis is inhibited and, if this is so, cyclo-heximide treatment would have less of a ten-dency to stabilize viral mRNA in interferon-treated cells (37, 44). Furthermore, in a study ofa reovirus infection that employed a tempera-ture-sensitive mutant blocked at nonpermis-sive conditions just after primary transcription,interferon treatment had very little effect onthe production of viral mRNA (135). Somewhatsimilar results have been obtained with ananalogous temperature-sensitive mutant ofVSV (P. I. Marcus, personal communication).To me, the results of all of these studies, there-fore, do not conclusively indicate an interferoneffect on primary transcription.However, Oxman et al. (96, 103), on the basis

of carefully conducted studies on SV40-infectedcells, have also reached the conclusion that in-terferon treatment inhibits virus-directed tran-scription early in infection. In spite of the carethat Oxman et al. (96, 103) took to reach thisconclusion, the work of Yamamoto et al. (140)would seem to indicate that the effect of inter-

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feron may be on a step even earlier than pri-mary transcription of SV40.

In summary, I feel work published to thepresent time does not argue compellingly thatinterferon has a significant inhibitory effect onviral transcription. In addition, studies withtemperature-sensitive mutants ofVSV and reo-

virus (135) present data to suggest that there isno significant effect on viral transcription.Related to possible inhibition of translation

in interferon-treated cells are reports of in-duced ribonucleolytic activity in cells after in-terferon treatment (92) or in extracts from suchcells to which double-stranded RNA was added(119a). Although such a putative mechanism ofaction of interferon is of great possible interest,several observations suggest caution in accept-ing this as necessarily the explanation for in-terferon action. Repeated observations in vac-cinia virus-infected interferon-treated cells in-dicated that viral mRNA production was in-creased over that found in appropriate controls(64, 94). This would be unlikely if interferoninduced an RNase activity that was responsiblefor inhibiting virus replication. The RNase ac-

tivity induced by addition of double-strandedRNA to cell-free systems from interferon-treated cells may not be related to antiviralactivity in these systems, because the kinaseactivity induced in such extracts resulted in thephosphorylation of several proteins clearly notrelated to the antiviral state and even of his-tones added to the system (114a). It is thereforepossible that the RNase activity induced insuch extracts was related to the kinase activa-tion but was not functional in interferon-treated cells and, indeed, could be detected onlyin extracts in which virus-directed translationhad already been decreased as a consequence ofan inhibition of viral mRNA-ribosome associa-tion induced by interferon treatment (66a).

Studies in intact cells clearly point to a mech-anism that involves inhibition of virus-directedtranslation under conditions where viral tran-scription did not take place (39). Several studiesin cell-free systems involving translation ofvirus genetic information suggest that inter-feron treatment results in the production ofan inactive precursor of the antiviral sub-stance (78, 114, 143); some aspect of infectionwith virus (possibly production of a double-stranded RNA) may be the most important acti-vator in vivo (66). The role of virus infection inthese systems may be mimicked by incubatinguninfected, interferon-treated cells in abuffered salt medium to reduce their level ofprotein synthesis (68). Cell-free systems frominterferon-treated cells incubated in this way

have a reduced capacity to translate viral

mRNA. Thus, virus-induced inhibition of hostprotein synthesis might be responsible for theactivation of the interferon-induced antiviralfactor. In cell-free systems, addition of double-stranded RNA or passage through Sephadexmay also activate the system (120). This activa-tion step may involve a phosphorylation processrequiring ATP (78, 114, 143). Assuming all ofthis is correct, what virus function does theantiviral substance block? The best bet to mymind, at present, is a step in the initiation ofprotein synthesis. Results with formylated ini-tiator tRNA point to a function involving anearly event in the initiation ofprotein synthesisas the site directly affected in interferon-treated cells (67). Also, the nature ofthe mRNAmay be very important to interferon action,because SV40 mRNA with host sequences frominterferon-insensitive adenovirus is translatednormally in interferon-treated cells (101, 105).Inhibition of virus-directed translation at theinitiation site might allow for the remarkablespecificity shown by interferon..There is, however, the unusual inhibitory

effect of interferon treatment on the replicationof RNA tumor viruses to consider. In thesesystems, virus protein and RNA synthesis donot seem to be decreased; however, virus as-sembly is disrupted, and this results in theproduction of very little extracellular virus (vi-rus remains attached to the cell surface) or ofvirus with a greatly decreased infectivity (17,47, 108). The lack of effect on these virus biosyn-thetic functions is probably related to the inte-gration of the provirus of RNA tumor virusesinto the cellular genome. As in the case of someSV40-transformed cells, the viral mRNA pro-duced under these circumstances may includehost sequences that act to prevent the inter-feron-induced mechanism from recognizing thegenome as viral (101).The decrease in yields of infectious RNA tu-

mor viruses could, on the other hand, be due tointerferon-induced alterations in the plasmamembrane, as could the reported inhibition ofavery early step in SV40 replication which mayinvolve virus uncoating (140). Such alterationsmay preclude normal assembly of murine leu-kemia viruses or uncoating and transcription ofSV40. In the case of other viruses that assembleon the cell surface and bud from the plasmamembrane (such as arboviruses or myxovi-ruses) the viral genome is not integrated, andthe primary effect of interferon would be toinhibit the translation of virus genetic informa-tion. It might be, however, that, under somecircumstances in lytic infections, virus with de-creased infectivity could be produced in inter-feron-treated cells.

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If interferon has a single mechanism of ac-tion, it might be summarized as follows. Inter-ferons bind to a specific receptor on the cellsurface. In cells with the proper effector appa-ratus (sensitive cells) this binding causeschanges in the cell membrane which, possiblythrough a cAMP mechanism, result in the pro-duction of an inactive precursor of an antiviralsubstance. After viral infection and formationof a double-stranded viral RNA, this precursoris activated in a step involving phosphorylationto produce an antiviral substance that selec-tively inhibits a step in the initiation oftransla-tion of viral mRNA. Although this scheme isspeculative, the speculation is based on the re-sults of many excellent studies. If, however,Ockham's razor does not apply here, interferontreatment may inhibit several or all ofthe stepsin virus replication discussed in this review. Analteration induced in the plasma membrane byinterferon treatment might help to account forthe inhibition of MLV production and the un-coating of SV40, as well as for some of theputative non-antiviral actions of interferons.These include inhibition of cell replication (72,77, 106, 132), enhancement of phagocytosis (62),regulation of the immune response (63), in-creased specific cytotoxicity of sensitized lym-phocytes (83), and increased susceptibility tothe toxicity of poly(I C) (127).There seem to be at least two "fail-safe" steps

in the mechansim proposed. The correct inter-feron must be bound to the cell surface in orderto produce the inactive precursor of the anti-viral substance. Then, virus infection must fol-low to activate the precursor. One reasonableexplanation for this intricate series of stepsmight be that interferon treatment affects thecell in ways other than simply to produce anantiviral activity. These would include theabove-mentioned reports of interferon inhibit-ing cell replication and mediating the immuneresponse. Ifthese reports are correct, it is likelythat the effects of interferon treatment must becarefully regulated by the cell, and such "fail-safe" control mechanisms might be quite impor-tant.

Lastly, one has to wonder why such a compli-cated series of biological processes as the inter-feron mechanism evolved to control virus infec-tions. Interferon is an extracellular proteinwhose induction and production are carefullyregulated. When interferon is bound to theproper cell, further actions are induced andthese, also with careful regulation, result in anintracellular antiviral activity. This seems anextraordinarily complex mechanism for an ap-parently simple end, the inhibition of virusgrowth. Perhaps, what have been considered

here as "other" actions of interferon, the controlofcell growth and expression are, in reality, themain point of it all. On the other hand, ifinduction of antiviral activity is indeed themain biological role of interferon, nature wouldseem to have found it necessary to work inexceedingly complex ways. The only solace fora scientist working on interferon is that otherbiological mechanisms about which we knowsome details, such as the complement and bloodclotting systems, or the inflammatory responseseem, if anything, a great deal more complexthan interferon action.

LITERATURE CITED1. Aaronson, S. H., and C. Y. Dunn. 1974. Endog-

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2. Allen, P. T., H. Schellekens, L. J. L. D. VanGriensven, and A. Billiau. 1976. Differentialsensitivity of Rauscher murine leukemia vi-rus (MuLV-R) to interferons in two inter-feron-responsive cell lines. J. Gen. Virol.31:429-435.

3. Allende, C. C., J. E. Allende, and R. A. Firtel.1974. The degradation of ribonucleic acidsinjected into Xenopus laevis oocytes cell. Cell2:189-196.

4. Ankel, H., C. Chany, B. Galliot, M. J. Cheva-lier, and M. Robert. 1973. Antiviral effect ofinterferon covalently bound to sepharose.Proc. Natl. Acad. Sci. U.S.A. 70:2360-2363.

5. Bader, J. P. 1962. Production of interferon bychick embryo cells exposed to Rous sarcomavirus. Virology 16:436-443.

6. Baxt, B., J. A. Sonnabend, and R. Bablanian.1977. Effects of interferon on vesicular sto-matitis virus transcription and translation.J. Gen. Virol. 35:325-334.

7. Bean, W. J., Jr., and R. W. Simpson. 1973.Primary transcription of the influenza virusgenome in permissive cells. Virology 56:646-651.

8. Berman, B., and J. Vilcek. 1974. Cellular bind-ing characteristics of human interferon. Vi-rology 57:378-386.

9. Besancon, F., and H. Ankel. 1974. Inhibition ofinterferon action by plant lectins. Nature(London) 250:784-786.

10. Besancon, F., and H. Ankel. 1974. Binding ofinterferon to gangliosides. Nature (London)252:478-480.

11. Besancon, F., and H. Ankel. 1976. Inhibitionde l'action de l'interf6ron par des hormonesglycoproteiques. C. R. Acad. Sci. Paris283:1807-1810.

12. Besancon, F., H. Ankel, and S. Basu. 1976.Specificity and reversibility of interferonganglioside interaction. Nature (London)259:576-578.

13. Bialy, H. S., and C. Colby. 1972. Inhibition ofearly vaccinia virus ribonucleic acid synthe-sis in interferon-treated chicken embryo fi-

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