Antiviral Activity of Interferons

25 Pages·2002·4.62 MB·English

BAcTzmuoLoGIcAL Ruvizws, Sept. 1977, p. 543-567 Vol. 41, No. 3
Copyright0 1977 AmericanSocietyforMicrobiology PrintedinU.S.A.
Antiviral Activity of Interferons
ROBERTM, FRIEDMAN
LaboratoryofExperimentalPathology,NationalInstituteofArthritis,Metabolism,andDigestiveDiseases,
Bethesda, Maryland20014
INTRODUCTION.............................................................. 543
ESTABLISHMENT OFTHE ANTIVIRAL STATE ........... 544
....................
Interferon Binding................... 544
.............................
Development ofAntiviral Activity 546
LOCUSOFTHE INTERFERON-IN.D.U..C.E..D..I.N..H.I.B..I.T.I.O..N..O.F..V..I.R..U.S.GROWTH.. 547
EvidencethatInterferonTreatment InhibitsVirusUncoating ....... 547
...........
Evidencethat Interferon Treatment InhibitsTranscription oftheViral Genome. 547
EvidencethatInterferonTreatment Inhibits Viral ProteinSynthesis ..... 550
......
Observations invirus-infected cells 551
...........................
Observations incell-free systems ....... 552
....................
Evidencethat InterferonTreatment Inhibits Terminal Events inthe Replication
CycleofMurine LeukemiaViruses ........ ................. 557
INTERFERON TREATMENT IS INEFFECTIVE IN SYSTEMS IN WHICH THE
SV40 GENOME IS INTEGRATED INTO AN INTERFERON-RESISTANT
VIRUS ORA HOSTGENOME........................... 559
DISCUSSION ............................. 560
LITERATURE CITED........................... 562
Principlesshouldnotbeunnecessarilymultiplied. has there been any great optimism that this
-William ofOckham, about 1320 canbe accomplished (71).
I beseech you, in the bowels ofChrist, think it Interferon assays have added to this diffi-
possibleyou may bemistaken. culty because they have been exclusively bio-
-Oliver Cromwell, 1650
logical and arebased ontheability ofaprepa-
rationtoinhibittheproductionofavirusorofa
INTRODUCTION viral product in infected cells. They are time-
consuming and relatively inaccurate. Cells
Interferons are proteins, the production of must be treated with interferon for several
whichcanbeinducedinanimalcellsbyavari- hours before virus infection until antiviral ac-
ety of stimulating substances; interferons in- tivity develops. Because ofthe inherent inac-
hibit a wide range of viruses by inducing an curacies of the biological assays, a two- to
intracellular antiviral state, yetmostinterfer- threefold inhibition is considered just signifi-
onsareanimalspeciesspecificintheirrangeof cant;therefore, alevelofuncertaintyispresent
antiviral activity. Interferon wasdiscovered in that is usually considered intolerable in abio-
1957 (85),buttothepresenttimetherehasbeen chemical orbiophysical system.
no entirely statisfactory explanation ofits po- One other problem has been the multiple
tent antiviral effect. Partofthereasonforthis substances that bear the name interferon in
lies in the impressive biological activity ofin- any one species. There are several proteins of
terferons. Arecentestimateofthespecificanti- differing molecular weights thathave an anti-
viralactivityofhumaninterferonwasatleast2 viral effect and are produced in differing cell
x 108 reference units per mgofprotein, and it systems. Humanbuffy coatinterferoncontains
maybe 10to 100timesmorepotent (71). Witha three antiviral proteins, two with molecular
molecular weight of about 25,000, this would weights of about 20,000 and one ofmolecular
meanthat 1 activeunitpermlispresentin (at weight 15,000. Interferon produced in human
most) a 10-12 M interferon solution. Cholera diploid fibroblasts antigenically resembles one
and diphtheria toxins are marginally active at of the proteins with a molecular weight of
10-9 M; therefore, on a molar basis interferon 20,000 but isdistinct from the other buffy coat
exceedsthespecificactivityofsomeofthemost interferons. Finally,humanTlymphocytespro-
potentbiologicallyactive substances. Asacon- duce an "immune" interferon in response to
sequence of this, interferon preparations con- antigens and mitogens; this is adistinct inter-
tainingverylargeamountsofantiviralactivity feron species. All of these act to produce an
turn out to have extremely small amounts of intracellular antiviral state, but it is unclear
interferon. It is, therefore, verydifficult to pu- whether theyhave the same mechanism ofac-
rify interferons completely, and only recently tion (107).
543
544 FRIEDMAN BACTERIOL. REV.
Finally, results of studies on the antiviral mediately infected at370C with arapidlyrepli-
activity ofinterferons have often been confus- catingvirus. If,however,thecellswereallowed
ingandcontradictory. Insomecasesitisuncer- to incubate at 370C for even 1 or 2 h, antiviral
tain whether the reported effect wasdueto an activity was evident. There is, therefore, an
interferon, becausetheconcentrationofimpur- alteration in the cells after incubation at 40C
itiesininterferonpreparationsfarexceededthe with interferon, but a period ofactive metabo-
concentrationofinterferon. Also, withdistress- lism at 370C was necessary to develop an anti-
ing frequency, studies on the mechanism of viral state (38).
actionofinterferonwhichappearedtoheralda The natureofthe interaction at40Cbetween
real breakthrough in our understanding have interferon and cells is partially understood.
seemedtolackthatmostfundamentalrequire- After incubation with interferon at 40C, treat-
ment ofscience, reproducibility. ment ofcells with trypsin inthe cold inhibited
Thisreviewwill concern itselfwiththe anti- the development of antiviral activity. It ap-
viral activity ofinterferons. There isvery con- pearedthatinterferonwasboundat40Ctocells
vincing evidence that interferon alsohasother insuperficial sitesthatwereaccessible totryp-
effects on cells, such as inhibition ofcell and sin (8, 38). After a few minutes at 370C, how-
tumor growth (72, 77, 106, 132), alteration in ever, a complex series of changes took place.
levelsofcellularenzymes (100), andregulation Interferon that had been bound to the cell be-
ofthe immune system (63). Although publica- came trypsin resistant, yet could still be re-
tions onsomeoftheseobservationsareappear- covered in an active form by extraction ofthe
ing with increasing frequency, there are still cells. With further incubation, cell-associated
too few reports towarrant a detailed review of interferontendedtoeluteintotheculturefluid
these atthistime. (8, 126).
The role ofthe portion ofthe interferon that
ESTABLISHMENT OF THE ANTIVIRAL did become cell associated is not entirely clear
STATE atpresent (76). Thereisnodirectevidencethat
this interferon was bound to a specific cellular
When cells are treated with an appropriate interferon receptor; however, interferon treat-
interferon, theydevelopanantiviral state, and
ment has been shown to alter the binding of
a wide variety ofviruses grow poorly in such cholera toxin or thyrotropin (TSH) to plasma
cells. In the absence of virus infection, how-
membranes (75) andtodecreasethesensitivity
ever, it is usually difficult to show whether or of cells to diphtheria toxin (97). All of these
notacellhasbeentreatedwithinterferon. One biologically active substances bind to fairly
can arbitrarily look at two aspects ofthe anti- well-characterized, specific receptors. The inhi-
viral effect ofinterferon cells: first, how inter- bition of their binding by interferon means
feron treatment alters the uninfected cell to either that interferon directly competes with
produceanantiviralstate; andsecond, thefate these substances for theirbinding sites or that
ofviruses in an interferon-treated cell. In this interferon treatment brings about a general
section I will discuss what is known about the alteration in membranes; one manifestation of
inductionoftheantiviral state. Untilrecently, thelatterwouldbeadecreasedbindingofother
most studies on this phenomenon have para- biologically active substances. If, however, the
doxically had to involve virus infection at one formeristhecase, itwouldappearthatdistinct
point or another in order to test the degree of binding sites must exist for interferon.
antiviral activity developed. Thelocation, chemicalnature,andspecificity
ofsuchputativebinding siteshavebeeninves-
Interferon Binding
tigated. One study indicated that ifsuch sites
In spite of the very low concentrations of exist, they must be on the external surface of
interferon that are required to induce an anti- theplasmamembrane. Interferonhasoneunu-
viral state, thereis strongevidencethatonly a sualbiologicalproperty. Thecellproducingthe
small portion of the interferon in a solution humoral factor (interferon) may also be an ef-
interacts directly with cells to induce antiviral fector cell. In nature this is not likely to be
activity. Nodetectableinterferonwasremoved important, because the interferon-producing
fromapreparationthatwasrepeatedlyusedto cell isalreadyvirus-infected andthusprobably
induce antiviral activity (21). There is, how- beyond salvation by interferon treatment. In
ever, someevidencethatinterferonisboundto one tissue culture study, however, human fi-
thesurfaceofcellsinwhichanantiviralstateis broblastswerestimulatedtoproduceinterferon
later induced. When cells were treated with by the double-stranded ribonucleic acid (RNA)
interferon at 40C, no antiviral activity was inducer polyriboinosinic acid*polyribocytidylic
present ifthe cells were washed and then im- acid [poly(I*C)]. Inclusionofanti-humaninter-
VOL. 41, 1977 ANTIVIRAL ACTIVITY OF INTERFERONS 545
feron antibodyinthemediumpreventeddevel- humaninterferons, neutralizationexperiments
opment of an antiviral state, even when the appear to indicate that multiple reactive sites
antibody was added after interferon synthesis on the interferon molecule can interact with
had already been initiated (134). The results cells ofdifferent species to induce antiviral ac-
seemed to indicate that interferon must be tivity (107). These results taken together sug-
externalizedandinteractwiththeoutersurface gestedthatthespeciesspecificityofinterferons
ofthe plasma membrane in order to be effec- doesnotresidesolelyatthepointoftheirinitial
tive. Similarresultshavebeenobtainedincells interaction with their binding sites and that
induced to make interferon and then treated bindingofaninterferonis anecessary, butnot
with ouabain (79). a sufficient, condition for biological activity; it
The receptors for interferon would appearto is an overture that may notbefollowedby the
be gangliosides or ganglioside-like structures opera.
with an oligosaccharide moiety at the func- A number of substances that bind to and
tional binding site. Treatment of mouse cells alter the cell membrane inhibit interferon ac-
with Phaseolus vulgaris phytohemagglutinin tion. Cytochalasin B (109, 142) and colchicine
(PHA), a plant lectin that binds to carbohy- (26)bothareactiveinthisrespect,althoughthe
drates, blocked the development of antiviral effectoftheformerisprobablyduetoextrusion
activity after subsequent interferon treatment ofthecellnucleus. Mercaptopyridethylbenzim-
(9). Also, Sepharose-bound interferon lost its idazole, which inhibits membrane transport of
antiviralactivityafterpreincubationwithgan- nucleosides, blocks the development of anti-
gliosides, especiallyGM2andGr, (10), andsolu- viralactivityininterferon-treatedcells(46). As
bleinterferonwasboundtoSepharose-ganglio- might be expected, chorionic gonadotropin,
sidebeads (10). Sialyl-lactosereversedtheabil- TSH, andcholeratoxin, whichbindtoganglio-
ity ofgangliosides to inhibit the antiviral ac- side receptors, also inhibit interferon action
tion of interferon, and PHA inhibited inter- (11, 75). Antibody to a cell surface component
feron binding to gangliosides (12). Further- coded for by human chromosome 21 inhibited
more, ganglioside-deficient mouse cells were the antiviral activity of interferon (112), al-
insensitive to induction ofantiviral activityby though antibody directed nonspecifically
interferon; treatment of these cells with gan- against cell surface antigens of human fibro-
gliosides both increased cell membrane gan- blastshadnosuchinhibitoryeffect(134). Since
glioside content and, in two ofthree cell lines chromosome 21 in human cells appeared to be
tested, significantlyincreasedcellsensitivityto necessary for the development ofantiviral ac-
interferontreatment. Ifindeedthereceptorsfor tivity after treatment with human interferon
interferons are gangliosides, these receptors (32, 128, 129), these findings suggested that
wouldresemble those ofcholera toxinandsev- humanchromosome21maydetermineananti-
eral glycopeptide hormones (98, 99). genimportantintheinterferonreceptor. Other
Itisdifficulttocommentasyetonthespecies studies, however, suggested that human chro-
specificity ofthe putative binding sites for in- mosome 21 did not determine the interaction
terferon. Manysubstancesthathavenoknown receptor but that some .other chromosome did
intracellularfunctionbindtocellsurfaces. One (28, 32). Also,therewasasuggestionthatchro-
cannot imagine that all such binding involves mosome 16 carried a function that regulated
specificreceptorsites. Thereissignificantbind- the interferon-induced antiviral state (28).
ing of some interferons to cells in which the Interferon treatment appears to bring about
interferons tested had no knownbiological ac- a number ofchanges in plasma membranes of
tivity (26, 75). The meaningofthis is unclear; uninfected cells. Plasma membrane prepara-
however, mouse interferon alteredthe binding tions ofL-cells exposedto interferon developed
ofcholera toxin orTSH to plasma membranes a complex alteration in their ability to bind
ofKBcells,ahumancelllinethatiscompletely choleratoxin. Atlowinterferonconcentrations,
unresponsive to both mouse and human inter- binding of toxin was significantly increased;
ferons (75). Similar conclusions have been when the interferon concentration was in-
reached from studies in rat embryo fibroblasts creased, however, binding was inhibited (75).
treated with rat and human interferons. Hu- Similar complex effects on binding have been
manamnioninterferonhasantiviralactivityin reportedwithothersystemsandarethoughtto
theseratcells,buthumanleukocyteinterferon be due to binding of a substance to both an
does not; however, both human interferons active (at low concentrations) and inhibitory
block the antiviral activity of rat interferon. (at high concentrations) site (98). Interferon
Thus, human leukocyte interferon must have treatment also alters the surface charge on L-
interacted withtheratcellseventhough itdid cells (73), andinAKRmousecellstreatedwith
notinduce antiviral activity (26). Inthecaseof interferon there is a significant change in the
546 FRIEDMAN BACTERIOL. REV.
density ofpurified plasma membrane prepara- ulation by increased production ofcyclic 3',5'-
tions. In untreated cells, 80% of the plasma adenosine monophosphate (cAMP) (99). There
membrane banded in sucrose at a density of issomeevidencethatinterferonactioninvolves
1.20, and 20% banded at 1.23; in interferon- the first two steps listed (75, 133). In addition,
treated cells, 25% banded at 1.20, and 75% there is a report that cAMP analogs such as
banded at 1.23. This change may be related to dibutyl-cAMP increased the level of antiviral
alterations in plasma membrane ganglioside activity induced by a preparation ofinterferon
content after interferon treatment. Also, there (45). I amnot aware ofany reported studies on
is a marked increased in the number ofintra- the effect ofinterferon treatment on adenylate
membranousgranularparticlesthatcanbeob- cyclaseactivity. IbelievethatChany'smodelof
servedbyelectron microscopyafterfreezeetch- an interferon-binding site and an associated
ing the membranes ofinterferon-treated AKR butdistinctactivationoramplificationsitethat
cells as compared with untreated controls (E. is actually responsible for the development of
Grollman, F. T. Jay, and E. H. Chang, manu- antiviralactivityisprobablycorrect (20, 26, 27)
script in preparation). Although the signifi- and resembles to a great extent the glycopep-
cance of these alterations in plasma mem- tide hormone models.
branes after interferon treatment is unclear, Several cell functions are known to be re-
theyareconsistentwithaprofoundchangethat quiredforthe induction ofantiviral activityby
appearstobeinducedbyexposureto interferon interferon. Studies (27, 28, 32, 128, 129) have
and may well be related to the development of showntherelationshipofspecificchromosomes
the antiviral state. to the induction ofantiviral activity in human
andmousecells. Humancellstrisomicforchro-
Development ofAntiviral Activity
mosome 21 are more sensitive tohuman inter-
Very soon after Isaacs and Lindenmann dis- feron than cells that are diploid. The latter in
covered interferon they recognized the neces- turn are significantly more sensitivethancells
sity for metabolic activity for interferon action monosomic for chromosome 21 (28, 128). The
(85). This finding has been repeatedly con- human interferon activity gene would appear
firmed and is generally thought to mean that to lie on the distal portion ofthe long arm of
interferon itself is not antiviral but that the chromosome 21, since the translocation ofthis
development ofan antiviral state requires the locustoanotherchromosometransferssensitiv-
intracellular production of an antiviral sub- ityto interferon (35). This site iscloselylinked
stance. There is one study which does suggest to the locus ofthe enzyme indophenol oxidase
that interferonmayitselfbe antiviral andthat (129).
the necessary metabolic activity was simply to The nature ofthe gene function required for
transport interferon into the cell (122). Al- antiviral activity is to some extent known
though this notion is difficult to rule out, the through studies with antimetabolites. In 1964,
previously discussed experiments, which indi- Taylor showed that interferon action was in-
catedthatinterferonmustbeexternalizedtobe hibited in cells treated with actinomycin D
active (134), make it rather unlikely, unless (130); however, established antiviral activity
thereissomeportionoftheinterferonmolecule wasnotdecreasedbytreatmentwithactinomy-
that must be reintroduced. In addition, inter- cin D (130). Several other inhibitors of RNA
feron bound to an insoluble, Sepharose matrix synthesis and ribonucleoside analogs also in-
was active in inducing antiviral activity (4); hibited interferon action and, taken together,
this study did not, however, rule outthe possi- these studies indicated that cellular RNA syn-
bility that interferon that mighthave come off thesis is probably necessary for interferon ac-
the Sepharose matrix was actually responsible tion (124). These findings have recently been
forthe induction. confirmed by studies with enucleated cells. In
Interferonresembles, insomerespects, glyco- cellsinwhichnucleiwereremovedbyexposure
peptide hormones and, therefore, what has to cytochalasin B, interferon treatment failed
been learned about the mechanism of poly- to induce antiviral activity. Once established,
peptidehormoneactionmaybeusefulinuncov- however, antiviralactivitywasnotreversedby
ering clues to the mechanism ofinterferon ac- removing the nucleus (109, 142).
tion. As an example, in transmitting its mes- The resultsofseveral studieswith inhibitors
sage to the cell, TSH is believed to require a ofprotein synthesis or amino acid analogs also
sequenceofeventswhosemaincontributorsare suggested that interferon action required pro-
in turn a specific receptor (probably a ganglio- tein synthesis (124). These experiments were
side), asignificant alterationinthestateofthe notasconvincingasthosewith actinomycin D,
membrane, an effector-responsive adenylate which suggested a need for RNA synthesis,
cyclase, andcellmetabolism susceptible toreg- probably because the activity of inhibitors of
VOL. 41, 1977 ANTIVIRAL ACTIVITY OF INTERFERONS 547
protein synthesis must be reversed in order to latter. At any rate, I shall discuss each ofthe
test (by virus growth) for the presence ofanti- theories about interferon action currently in
viral activity (48). vogue in the order that they generally appear
These results suggest a by now familiar in the virusreplication cycle.
model: interferon treatment induces the pro-
duction ofa cellular messenger RNA (mRNA), Evidence that Interferon Treatment Inhibits
whichistranslatedtoanantiviralprotein. Itis Virus Uncoating
the latter that actually inhibits the growth of Untilrecently, there seemedlittlereasonfor
viruses. This theory was first suggested to me seriously considering this as asiteofactionfor
by J. A. Sonnabend in 1963, and to date no interferon. SeveralstudieswithinfectiousRNA
report hasrefuted it. Bythe sametoken, there from poliovirus or western equine encephalitis
has not yet been a direct demonstration ofan virus indicated that interferon inhibited the
antiviral protein or its mRNA. replication of these RNA forms (33, 55, 59).
There are two studies purportingto define a Barring hydrolysis ofthe RNAs by ribonucle-
significant difference inaspecific RNAconstit- ase (RNase) left from the interferon prepara-
uent between interferon-treated and control tions used to treat the cells, the data clearly
cells. Levyet al. (82, 112a) haveinvestigated a indicated that interferon must act at a site
smallincreaseinthesizeofmRNA'sandtrans- beyond the uncoating step. Uncoating is also
fer RNAs (tRNA's) from interferon-treated notinhibitedininterferon-treatedcellsinfected
mousecellsbypolyacrylamide gelelectrophore- with reovirus (135).
sis. Although these differences were small, Studies with simianvirus40 (SV40) seemed,
they were reported to be consistent. However, however, to contradict the findings with infec-
almost all ofthe RNA species examined from tious RNA (140). In agreement with the find-
interferon-treated cells seemed to trail behind ings ofOxman etal. (96, 102, 103), theproduc-
the analogous species from extracts of un- tion of SV40 early mRNA and T antigen was
treated cellby aboutthe same distance. Since, markedly inhibited in interferon-treated mon-
under the conditions employed, the distance key cells, when these cells were infected with
migratedbyanRNAspecies isthoughttovary intact SV40. If, however, infectious SV40 de-
directly with the logarithm of its molecular oxyribonucleic acid (DNA) was employed, the
weight, thefindingwouldimplythatinterferon results were directly opposite those obtained
inducesvery largedifferences inthe molecular with infectious RNA. The synthesis of early
weight of large RNA species, but quite small SV40 mRNA and T antigen were only slightly
differences insmall species. There would seem inhibited in interferon-treated cells infected
to be no easy explanation for these findings at withtheSV40DNA(140).Thiswouldappearto
present, but such studies as these are impor- indicate that in interferon-treated cells, SV40
tant since they may uncover significant differ- uncoating, orastepsoonafterit,wasinhibited,
ences that may exist between the RNAs of and that this is the explanation forthe appar-
interferon-treated and control cells. ent inhibition of SV40-directed transcription
under these conditions. A similarblock in un-
LOCUS OF THE INTERFERON-IN-
coating or an event soon thereafter has been
DUCED INHIBITION OF VIRUS GROWTH
described at nonpermissive conditions for the
Interferon inhibits the replication of a sur- SV40mutant, tslOl (113).
prisingly wide variety of viruses, and inci- The results in interferon-treated cells in-
dently ofsome nonviral infectious agents (62). fected with SV40 DNA are clear-cut, buthave
Many of these viruses and infectious agents not as yet been confirmed. The most likely
would appeartohave very little incommon. A explanation for the finding was discussed
number ofvirologists have, therefore, investi- above, butthere is one otherpossible explana-
gatedthisproblemandemployedseveralvirus- tion. In the SV40 systems employed in this
cell systems inattemptstofindaspecific virus study, thereisamultiplicity-dependent revers-
function which is blocked in interferon-treated ibility of the interferon-induced inhibition of
cells. Atleastfourpossible sites ofactionhave virus replication (140). This has been reported
been seriously suggested by a number oflabo- inanothersystem (60). Tocarryouttheexperi-
ratories. It is unfortunate that most of these mentswithSV40DNA, 0.075or0.75 jigofSV40
suggestedsitesofactioninvolvephasesofvirus DNA was employed. This amount ofinfectious
replication aboutwhich we known quite little. DNAcouldconceivablybeequivalenttoahigh
Perhapsthisismoreacommentonthegeneral virus-cell multiplicity, ifany great part ofthe
state of the science (art) of virology than on DNA actually induced infection. The explana-
interferon research; on the other hand, it may tion for these extraordinary findings may lie,
also be symptomatic of the perversity of the therefore, in the SV40 multiplicity reversal of
548 FRIEDMAN BACTERIOL. REV.
interferonactionalsoreportedinthe pub- cally to the use of cycloheximide, which has
same
lication (140). Still, the possibility that inter- been themostcommonly employed inhibitor of
feron action involves, at least under some con- protein synthesis; under these conditions, cy-
ditions, an inhibition ofviral uncoating is im- heximide inhibits the elongationofpeptides, it
portant, and until such time as this work is protects and stabilizes mRNA (3, 123). Inter-
directly contradicted, anytheorythatattempts feron treatment might alter this stabilization,
to explain interferon action will have to make if it tended to restrict the initiation of viral
provision for these findings. protein syntehsis; under these conditions, cy-
cloheximide might not have protective effects
Evidence that Interferon Treatment Inhibits
onmRNAininterferon-treatedcells. Thiscould
Transcription ofthe Viral Genome well lead to large differences in the concentra-
The work in this area ofinterferon research tions of intracellular mRNA in cells treated
is quite controversial, and, like scripture, can with cycloheximide alone as compared with
be quoted to support any side ofan argument. cellstreatedwithbothcycloheximideandinter-
In this case the disagreement revolves around feron. These differences would notofcoursebe
whetherinterferontreatmentcauses aninhibi- indicative of an interferon effect on primary
tion intheprimarytranscription ofthegenetic transcription.
information ofviruses. All ofthe possible an- Among RNA viruses, there have been de-
swers (yes, no, or sometimes) can be found in tailed reports on the effect ofinterferon onthe
various publications. primary transcription of vesicular stomatitis
Part ofthe confusion undoubtedly is due to virus (VSV), influenza virus, and reovirus. In
the close relationship between viral RNA syn- the case of DNA viruses, vaccinia virus and
thesis and viral protein synthesis (124). If a SV40 have been well studied.
virusrequiresapolymerasenotpresentincells Inhibition of primary transcripton of VSV
and has no endogenous RNA polymerase as a has been reported in human (88), chick (89),
structural element, inhibition ofviral protein and monkey cells (91). All three studies em-
synthesis will result in inhibition ofall virus- ployed cycloheximide to block secondary tran-
directed RNA synthesis, because the viral po- scription and actinomycin D to inhibit host
lymerase is among the proteins whose synthe- RNA synthesis, andthen measured viral RNA
sis would be inhibited. Arboviruses and picor- synthesis in the presence or absence of inter-
naviruses, among the RNA viruses commonly feron. Inthe experiments performed in human
used in interferon research, are lacking struc- cells, therewasaquantitativedecreaseinviral
tural polymerases. mRNAbut no qualitative change (88). Similar
In the case ofthose agents containing struc- conclusions were drawn from experiments
turalpolymerases, analysisofthesiteofinhibi- carried out in chick cells infected with in-
tioncanbequitecomplicated. Sinceapolymer- fluenza Ao/WSN, but an entirely different
aseisalreadypresent,inhibitionofproteinsyn- method was employed. 32P-labeled virus infec-
thesis does not cause inhibition ofearly RNA tion was established, and at different times
synthesis. Primarytranscriptiontakesplacein after infection, RNase-resistant RNA was
these cells, but secondary transcription, which determined before and after annealing. In con-
dependsontheelaborationofnewpolymerases, trolcellstreatedwithcycloheximide, upto20%
will be inhibited. Results obtained employing oftheinfectingRNAbecameRNaseinsensitive
such systems require careful analysis, since after annealing. In actinomycin D-treated or
significant inhibition of RNA synthesis does interferon-treated cells, almost none of the
notnecessarilyindicatethatprimaryRNAsyn- viral RNA became RNase resistant (7).
thesis is the site ofaction. These results certainly suggested that at
Furthermore, the use ofinhibitors ofprotein leastone effect ofinterferon wasto inhibitpri-
synthesisintheseexperimentsintroducessome mary transcription ofRNA viruses with struc-
additional problems. First, the endogenous tural polymerases. Contradictory resultshave,
viral polymerases may be subject to negative however, beenobtainedinastudyofinterferon
control by viral proteins produced early in the inhibition of VSV replication in interferon-
infection process. When an inhibitor ofprotein treated monkey or human cells. With concen-
synthesis is employed, the level of primary trations of interferon that significantly in-
RNA synthesis in controls may be elevated hibitedvirusreplicationinthesesystems,there
(13). This could lead to difficulties in interpre- wasonlya30%decreaseinthespecificactivity
tation ofdata suggesting inhibition ofprimary ofviralRNAsynthesisinthepresenceofcyclo-
transcriptionbyinterferon. Inaddition, related heximide and interferon in monkey cells as
to the general problem of using inhibitors of compared with those treated with cyclohexi-
protein synthesis is a problem relating specifi- midealone. Inthehumancells,thecomparable
VOL. 41, 1977 ANTIVIRAL ACTIVITY OF INTERFERONS 549
figure was 53%. Those viral RNA forms that laboratories. Itwould, forinstance, accountfor
weremost sensitive to inhibitionby interferon whyviraltranscriptionseemstobeinhibitedin
were forms that required the elaboration ofa some interferon-treated cells, whereas in oth-
new polymerase to produce, i.e., were due to ers, viral translation seems to be the site of
secondary transcription. In addition, aconcen- interferon action. By this theory ofMarcus et
tration ofinterferon that inhibited viral RNA al. (92), whatresultsoneobtainswoulddepend
synthesis by only 10% inhibited viral protein on what was being measured. Intracellular
synthesis by 60% (6). The increment ofinhibi- viralmRNAconcentrationswouldbedecreased
tion seen with both cycloheximide and inter- and virus-directed translation would be in-
feron over that with cycloheximide alone was hibited. In cell-free systems measuring viral
felt to be dueto the above-described protective translation, the increased nuclease in extracts
effectofcycloheximide onmRNAanditspossi- ofinterferon-treated cells might hydrolyze the
ble absence in interferon-treated cells (3, 123). added viral mRNA, and this in turn would
A study of the effect of interferon on early cause anapparentprimaryinhibitionintrans-
production ofcomplementary RNA in VSV or lation.
influenza virus-infected chick or mouse cells Inorderforthese notionstobeconsidered as
also reached the conclusion that inhibition of ageneralmechanismofinterferonaction, how-
primary transcription was probably not the ever, itwouldhave tobe shownthatincreased
basis of interferon action. The results of this alkalineRNasewasinducedbyhomologousin-
study are, however, ambiguous to me because terferonincellsofspeciesotherthanchicksand
the authors seemed to ignore what might be only chickcellswere used inthe studyofMar-
significantdifferencesinhybridizationdatabe- cus et al. (92); indeed, Maenner and Brandner
tween cycloheximide- or interferon-treated cell have reported no increase in nuclease activity
extracts. Infact, inviewofapossibleprotective in poly(I 0)-treated monkey, hamster, quail,
effect ofcycloheximide on mRNA, a cyclohexi- orduckcellextracts(86a).Inaddition,nospeci-
mide control for an interferon-treated cell cul- ficity was shown by this system, in that the
ture may give very misleading results, and it RNase activity was tested only with VSV
wasnotsurprising, therefore, thatsomediffer- mRNA. Sinceinterferonactionappearstoshow
ences were indeed noted in this study; these specificityforvirusfunctions, anRNasethatis
differences were ignored, presumably because thought to be responsible for interferon action
the authors doubtedtheir significance (111). shouldshowarestrictedspectrumofactivity.
In an important study on the mechanism of Vaccinia virus was the first ofthe DNA vi-
inhibitionofreovirusreplicationbyinterferon, ruses to be studied intensively with respect to
the authors employed a different tack, which interferon (50). In many ways vaccinia virus
obviated the use ofcycloheximide. Among the infection lends itselfwell to this sort ofstudy,
temperature-sensitive mutantsofreovirustype becausetheviruscontains an RNApolymerase
3, ts447 is blocked in its formation ofprogeny thatisactivatedwithremovaloftheoutercoat
RNA at nonpermissive temperatures (38.50C); ofthe virus. The polymerase, which is located
therefore, only primary transcription takes inthe viral core, then elaborates viral mRNA,
place at this temperature. A study of the which is translated to yield several active pro-
growthofts447 ininterferon-treated cellsincu- teins, includingonethatinhibitsactivityofthe
bated at 38.50C indicated that a concentration virus structural RNA polymerase and another
ofinterferonthatinhibitedvirusyieldsby80%, that is responsible for final uncoating of the
inhibited primary transcription by only 12% viral DNA (87).
(135). Under approximately the same condi- The effects ofinterferon on this well-studied
tions, however, virus-directed translation was system are fairly clear-cut in both mouse (64,
significantly inhibited (see below). 65, 94) and chickcells (36, 65, 93). Early vacci-
One additional finding in interferon-treated nia mRNA synthesis was increased (64), but
cells infected with an RNA virus will be dis- finaluncoating oftheviral DNAwas inhibited
cussed in this section although it does not, (87). Although the production of viral mRNA
strictly speaking, deal with an inhibition of depends on a virus-associated polymerase, the
transcription. Marcus et al. (92) have also re- inhibitionoftheactivityofthepolymerase and
ported that treatment with chick interferon theuncoatingoftheviral DNAdependon syn-
causedanelevationoftheenzymaticactivityof thesis ofnew proteins. It would appear in this
a membrane-associated RNase, with optimal system, then, that the site of interferon-in-
activity atan alkaline pH (assays were run at duced antiviral action must lie between virus-
pH 8). If this is a general finding, it could directed transcription and translation.
explainsomeofthedivergentobservationsthat In one study, however, inhibition of early
havebeenmadeoninterferonactioninvarious vacciniavirusmRNAsynthesiswasreportedin
550 FRIEDMAN BACTERIOL. REV.
interferon-treated chick cells (13). This result inhibitory effect on further early viral RNA
mayhave beenduetotheuseofcycloheximide synthesis (96). These results with SV40 were
in this particular study, but similar experi- somewhat unexpected, because the virus must
ments in both mouse and chick cells with or use a cell polymerase early in infection.
without cycloheximide have repeatedly shown Although these studies are ofgreat interest,
that transcription of vaccinia virus mRNA is the previously mentioned findings that sug-
increased rather than decreased in interferon- gested an altered uncoating of SV40 virus in
treated cells (93). interferon-treated cells could account for the
A study performed in frog polyhedral cyto- inhibitionofearlySV40RNAsynthesis(140). It
plasmic deoxyribovirus (FPCD)-infected fat- will be important to see whether these experi-
head minnow (FHM) cells indicated that the ments with infectious DNA of SV40 can be
early mRNA synthesis byFPCD was inhibited repeated.
by FHM interferon. The results also showed One other study with the SV40 system is of
that FPCD production was inhibited by FHM present interest. SV40 RNA prepared in vitro
interferon and that FPCD virus-associated po- with SV40 DNA and a bacterial polymerase
lymeraseactivitywasnotinhibitedbyanearly was microinjected into SV40-permissive mon-
virus protein; however, there was only a 57% keycellsthathadbeentreatedwithinterferon.
inhibition ofearlyviral mRNAsynthesisby 10 Under these conditions, T-antigen production
U ofFHM interferon, a concentration that re- was blocked, although it was evident in con-
duced virus replication by about 90%o, and a trols microinjected but not treated with inter-
75% mRNA inhibition by 100 U under condi- feron (52). This result indicated that, in inter-
tions in which 20 U completely abolishedvirus feron-treated and SV40-infected cells, transla-
replication. Inaddition, cycloheximidewasem- tion of virus genetic information may be in-
ployed in some ofthese studies (53). These re- hibited; however, thisresultdoesnotnecessar-
sultswouldappeartobeopentothesamecriti- ily mean that inhibition of translation is an
cisms that can be made ofsome ofthe studies important mechanism of interferon action in
with RNA virus-use of cycloheximide and a tbe SV40 system in vivo.
relativelysmalleffectonprimaryRNAsynthe- Although many of the papers reviewed in
sisascomparedwithlargeeffectsonviralrepli- this section suggested that interferon treat-
cation. ment caused an inhibition in early virus-di-
SV40differsfromtheotherDNAvirusesdis- rected transcription, in the case ofmost virus
cussedinthatitlacksastructuralRNApolym- groups studied there is at leastonepublication
erase and so at least early in infection must that indicated no direct effect of interferon
depend on a cellular polymerase. Interferon treatmentonprimarytranscription. WithRNA
treatmentinhibitedSV40Tantigenproduction viruses having virus-associated polymerases,
in acutely infected, but not in transformed, the most meaningful studies of interferon ac-
cells (101, 102). Transformation of mouse 3T3 tionatthispointwouldappeartobethosethat
cells by SV40 was also inhibited by interferon employ viral mutants blocked just after pri-
treatment (131). These results were consistent mary transcription has been completed. Only
withaprimaryeffectoneithervirusuncoating one study has so farbeenreportedwith such a
(140) or virus-directed transcription (103) or system (135), and the general conclusion
translation (52). reached was that, although there was some
Oxman and Levin next investigated the ef- inhibitory effect of interferon treatment on
fect ofinterferon treatment onearly SV40-spe- early reoviruses RNA synthesis, it was not
cific RNA synthesis in monkey cells (103). The enoughtoaccountfortheprofoundinhibitionof
results indicated a marked inhibition of very virusreplication. Theauthorsfeltthatininter-
earlyRNAsynthesisunderconditionswhereno feron-treated cells, transport ofsubviral parti-
late RNA could be formed, that is, in cells cles from phagocytic vacuoles and lysosomes,
treated with enough cytosine arabinoside (20 where they were formed, to locations in the
4g/ml) to inhibit viral DNA synthesisbymore cytoplasm where they transcribe RNA might
than 99%. Asubsequent studyshowedthatthe be slowed by interferon; however, they ad-
inhibition ofearly SV40 RNA synthesis in in- vanced no direct evidence for this hypothesis
terferon-treated cells was probably not the re- (135).
sult ofinhibition ofvirus adsorption, penetra- As for the DNA viruses, the weight of the
tion, or uncoating; or ofincreased degradation evidence in the case ofvaccinia virus infection
ofeithertheviral DNAtemplateorviralRNA; is that interferon treatment has no inhibitory
or, finally, to an inhibition oftranslation ofa effect on primary viral RNA synthesis. The
veryearlymRNAthatmighthaveasecondary jury is still out on SV40, however. I, for one,
VOL. 41, 1977 ANTIVIRAL ACTIVITY OF INTERFERONS 551
lookforwardwithgreat anticipationtofurther RNaseresistant (42, 49). Thelatterobservation
publications on the effect of interferon treat- would indicate that the infecting virus fails to
ment on the replication ofSV40 DNA. enter the double-stranded form and so cannot
becomepartofareplicationcomplexassuch.In
Evidence that Interferon Treatment Inhibits
SFV infection the replication complex is a
Viral Protein Synthesis
membranous structure associated with the
It should be noted again, at the onset ofthe viral polymerase and single- and double-
discussion ofthis material, that viral protein stranded viral RNA (43, 54). One report indi-
andRNA synthesis are so interdependentthat cated that in interferon-treated cells, parental
it is often difficult to distinguish which ofthe RNA of SFV entered into a membranous
twoistheprimarysiteofactionofanantiviral structurethatappearedtobeareplicationcom-
substance such as interferon, since progeny plex. The RNA ofthis structure remained in
RNA molecules are usually responsible for the single-stranded form, presumably because
most of the total virus-directed protein and the viral polymerase was not produced (51).
RNA synthesis that is carried onduringinfec- Some insight intothe mechanism ofhow in-
tion. Therefore, whether interferon treatment terferon might inhibit viral protein synthesis
acts directly to inhibit RNA orprotein synthe- wasprovidedbystudiesonmengovirus-infected
sis early in infection, later both transcription L-cells. Less radioactivity from input radioac-
and translation areprofoundly inhibited (124). tive mengovirus was precipitated in apost-mi-
Inordertoshowwhichofthetwoistheprimary tochondrial fraction from interferon-treated L-
siteofinterferonaction, itisnecessarytostudy cells than from untreated control cells. This
its effect under conditions in which viral RNA precipitated fraction was found to contain 50S
and protein synthesis are clearly dissociated. and 240S components, both of which had de-
This has been effected in two ways: either by creased activity in the extract from interferon-
studyingviralproteinsynthesisincell-freesys- treatedcells. Theauthorfeltthatthe240Scom-
tems; or, among RNA viruses, by studying in ponentrepresentedviralpolysomesandthe50S
infected cells the messenger function ofparen- component represented acomplexbetweenthe
tal (input)RNAofvirusesthegenomeofwhich viral RNA andthe 40Sribosomal subunit (81).
isanmRNA (positive-stranded RNAviruses) or Although interferon treatment did appear to
ofviralmRNAthatismadebyusingtheinput inhibit the formation ofthese structures, their
RNA as a template in association with a viral nature was not clearly established, and the
polymerase (negative-stranded RNA viruses). levelsofradioactivitywereverylow.Therefore,
There have alsobeen important studiesonone the significance ofthese findings must remain
DNAvirus. Experiments onviralmRNAfunc- uncertain.
tion in cell-free systems derived from inter- Only one study has been carried out on the
feron-treated cellswillalsobediscussedbelow. effect of interferon treatment on protein syn-
SemlikiForestvirus (SFV,anarbovirus)and thesis by the negative-stranded RNA virus
mengovirus (apicornavirus) havebeenusedto VSV (141). The results indicatedthat3 hafter
determine whether positive-stranded RNA vi- infection in interferon-treated rabbit kidney
rus messenger function is a primary site of cells, the formation ofall structural and non-
interferon action. Of the negative-stranded structural VSV proteins was inhibited. Viral
RNA viruses, VSV and reovirus protein syn- RNAsynthesiswasnotstudied, buttheinhibi-
thesis have been employed. Among the DNA tion ofviral protein synthesis was so profound
viruses, there has been extensive study ofthe that, ifdecreased RNA synthesis werethe pri-
effectofinterferontreatmentonlyonthe early mary factor, only a very marked inhibition
protein synthesis ofvaccinia virus. could possibly account for these results. The
Observationsinvirus-infectedcells. Ininfec- findings in interferon-treated, VSV-infected
tion with SFV, inhibition ofviral protein syn- cells (see above) indicated that, even ifit does
thesis in interferon-treated cells does not ap- occur, inhibition of virus-directed primary
pear to be a result ofinhibition ofviral RNA transcription mightnotbeasgreataswouldbe
synthesis. Under conditions in which viral necessary to completely account for the ob-
RNA synthesis was almost completely in- served inhibition ofvirus-directed protein syn-
hibited, early SFV protein synthesiswasunaf- thesis.
fected. If cells were treated with interferon, Inthe study ofthe effectofinterferon onthe
however, no virus-specific proteins could be growthofthereovirusmutantts446,theresults
identified (39). Inthissystem,therefore,paren- ofwhich were discussed above with respect to
tal RNA does not seem to be translated; the primary viral transcription, inhibition ofviral
RNA of parental SFV also does not become proteinsynthesiswasalsostudied (135). Under
552 FRIEDMAN BACTERIOL. REV.
conditions inwhichonly primary transcription translation, thishasnot asyetledto firmcon-
took place, there was a 20% inhibition in viral clusionsonthebasicmechanismofinterferon's
RNAsynthesisbuta40to72%inhibitionofthe action, and, aswillbeseenfromthediscussion
synthesis of various individual virus-specific ofthe results obtained so far in these studies,
polypeptides. The apparent inhibition ofviral there is even some disagreement as to which
RNA synthesis did not seem great enough to system bestrepresents what is goingon in the
the authors to account for all ofthe observed interferon-treated, virus-infected intact cell.
inhibition ofvirus translation. Most studies in cell-free systems have been
In interferon-treated cells infected with vac- carried out by investigators convinced at the
cinia virus, as indicated above, all (36, 64, 87, onset that the interferon-induced antiviral
93, 94) but one (13) ofseveral studies indicated state was aimed at virus-directed translation.
that early viral mRNA synthesis was not in- So far, it has been impossible to mimic the
hibited. The vaccinia virus mRNA formed in purported inhibitory action of interferon on
interferon-treated cellshasanormalcontentof transcription, althoughintheabsenceofpubli-
polyadenylic acid (70), but does not associate cations on the subject, I am not sure how hard
with ribosomes to form polyribosomes as read- suchstudieshavebeenpursued. Theactivityof
ily as does mRNA ordinarily formed early in viral polymerases ismarkedly decreased in ex-
vaccinia virus infections (19, 64, 94, 95). This tracts from the cytoplasm ofinterferon-treated
failure to form polyribosomes readily is proba- cells,butthiscouldhavebeenduetoadecrease
bly the result ofan inhibition ofthe initiation inthe production ofthe enzyme ratherthan to
steps in viral protein synthesis, althoughsome an inhibtion ofits action (125). Neither inter-
inhibitionofchainelongationwasalsoreported feron, nor extracts from interferon-treated
(95). There was a suggestion that the site of cells, virus-infected or uninfected, inhibited
inhibition of initiation of peptide synthesis viral polymerase activities (J. A. Sonnabend,
mightbeintheformationofacomplexbetween personal communication). This is not to say
avacciniavirusmRNA-protein (ribonucleopro- thatsuchinhibitoryactivitywillnotbedemon-
tein) and the 40S ribosomal subunit (95); how- strated,butsofar,therearenoreportsofcondi-
ever, the findings did not conclusively show tions where such inhibitory activity could be
this. repeatedly found.
SV40 is the only other DNA virus in which In the study of interferon's effect on virus-
virus-directed protein synthesis in interferon- directed proteinsynthesis, twosortsofsystems
treated cells has been studied at all. In inter- have been employed. In the earlier group of
feron-treatedcellsthereismarkedinhibitionof experiments, viral RNA was incubated with
the synthesis ofviral T antigen in lytic infec- extractsfromcontrolorinterferon-treatedcells,
tions (102). Thiscouldhavebeendue, however, andtheinteractionswereanalyzedwithrespect
either to the previously discussed inhibition of tothebindingoftheRNAtothecellularcompo-
virus uncoating (140) or to inhibition ofvirus- nents. Later, whencell-freesystemsemploying
directed transcription (103). As previously componentsfromanimalcellsbecameavailable
noted, therewasinhibition ofthetranslationof forthe production ofspecific polypeptides, sev-
SV40 mRNA microinjected into interferon- eral laboratories began to study the ability of
treated cells (52). If this as yet unconfirmed viral RNA to stimulate amino acid incorpora-
findingiscorrect, it suggests that inhibition of tion by cytoplasmic fractions from interferon-
SV40 translation might take place in inter- treated and control cells.
feron-treated cells; however, significant inhibi- Marcus andSalb (90), employingviral RNA-
tion of virus uncoating (140) or transcription binding studies, postulated that interferon in-
(103) would preclude there being very much duced the production of a new translational
SV40 mRNA produced in interferon-treated inhibitoryproteinthatcouldcombinewithribo-
cells. somes to inhibit their ability to translate viral
Observations in cell-free systems. All ofthe (but not cellular) mRNA. When they mixed
observationsthathavebeenmadeontheeffects tritiated Sindbis virus RNA with cytoplasmic
ofinterferonincell-freesystemshavebeencar- extracts containing 74S monomeric ribosomes,
ried out since 1966. Although late in starting, a 250S polysomal structure was formed at 00C.
this has been a rapidly developing field, and On incubation at 370C, this 250Spolysome was
manyinterestingandpotentiallyimportantob- broken down, and itwasthought thatthisrep-
servations have been made; however, the re- resented the translation ofthe mRNAbecause
sultsherehavealsobeensomewhatdisappoint- nobreakdownoccurredinthepresenceofcyclo-
ing to me. Although it apparently has been heximide. When ribosomes from interferon-
possible in cell-free systems to imitate the in- treated cells were used, however, they bound
terferon-induced inhibition of virus-directed viral RNA to a decreased extent, but more im-