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Interferons.

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pyrrole pigments 1857. Rapid progress may be expected
in the investigation of new members of this class of
compounds by the establishment of optimum degradation conditions for each type of pigment, operation on
the micro scale, and combination of sensitive separation methods with modern analytical aids.
M y own investigations on the phycobilins were mainly
carried out in collaboration with Dr. P. 0’ Carra, Department of Biochemistry, University College, Galway
(Eire). I am grateful to Professor C . OhEocha for
his cooperation. Thanks are due to the Deutsche Forschungsgemeinschaft for generous support.
[85] A brief review of t h e most important works is given
in 1401.
Received: September 18, 1969
[A 762 IE]
German version: Angew. Chem. 82, 527 (1970)
Translated by Express Translation Service, London
Interferons
By Y. K. S. Murthy and Hans-Peter Anders[*J
Interferons are proteins which bring about a nonspecific and nonimmunological defence
reaction against virus infections in vertebrates. Interferon formation is induced in vivo
and in vitro by viruses and other agents, e.g. endotoxins, nucleic acids, synthetic anionic
copolymers, and phytohemagglutinins. All attempts to produce pure interferons have so
far been unsuccessful. Interferons can only exert an antiviral action when the cellular
RNA and protein synthesis is intact.
1. Introduction
protected against a virulent pantropic variant of the
same virus. (For reviews of viral interference see c5-71.)
It has long been known that the formation of antibodies is not the only defence a n organism has to
protect itself against a virus infection. One of these
nonimmunological defences is “viral interference”,
i.e. the adverse effect that various viruses can produce
on the multiplication of another virus when the two
are present in the same host.
In 1937 Findlay and MacCallum L11 showed that treatment with Rift Valley fever virus protected monkeys
against subsequent challenge with the immunologically
unrelated yellow fever virus. Shortly after infection of
rabbit skin with vaccinia virus, 0rskov and Andersen 121
observed the appearance of a “local” antibody,
although no antibodies could yet be demonstrated in
the serum. In 1962 the authors discussed this observation as a possible interferon effect. Lenette and
Koprowskic31 were able to demonstrate a slight antiviral effect in infected chick- and mouse-embryo
cultures even after the viruses had been removed.
Matumoto et al.141 showed that mice that had been
infected with a neurotropic Rift Valley fever virus were
In 1957 fsaacs and Lindenmann 181 succeeded in clearly
differentiating the principle of viral interference from
that of specific immunity. These authors treated the
chorioallantoic membrane of chick embryos with
inactivated influenza virus incapable of multiplication.
After three hours at 37°C pieces of membrane with
adsorbed virus were added to a buffer solution and
incubated for 24 hours. After removal of the tissue a
substance having an antiviral action was found in the
buffer solution, which the authors called “interferon”
Subsequently, it has been found that interferons or
similar substances are formed in the cells of many
vertebrates in response to viral infection.
The effect of these interferons also extends to Bedsonia
organisms, bacteria, protozoa, and some oncogenic
viruses, e .g. leukemia viruses @,101. Today it is
generally accepted that interferons play an essential
role in the formation of a host’s nonspecific resistance
to superinfection with a second virus. It is difficult to
decide whether the effects ofviral interference described
before the discovery of interferons were due to interferon action or to other causes, but it is certain that
[*I Dr. Y . K. S. Murthy and Dr. H.-P. Anders
Schering AG, Hauptlaboratorium
1 Berlin 65, Mullerstrasse 170-1 72 (Germany)
151 W. Henle, J. Immunology 64, 203 (1950).
111 G. M. Findfay and F. 0 . MacCallum, J. Pathol. Bacteriol.
44, 405 (1937).
[6] R . W. Schlesinger in T. M . Rivers and F. C. Horsfall: Viral
and Rickettsia1 Infections of Man. Pitman Publ., London 1959,
p. 145ff.
[2] J . Orskov and E. K . Andersen, Acta pathol. microbiol.
scand., Suppl. 37, 621 (1938).
[7] R . Wagner, Bacteriol. Reviews 24, 151 (1960).
[ 3 ] E . H . Lenette and H . Koprowski, 3. exp. Medicine 83, 195
(1946).
[4] M . Matumoto, I . Nishi, and Y . Saburi, C . R. Seances SOC.
Biol. Filiales 153, 1645 (1959).
480
[8] A . Isaacs and J . Lindenmann, Proc. Roy. SOC.(London),
Ser. B 147, 258 (1957).
[9] T. C . Merigan, Symposium on Interferon, Lyons 1969.
[IO] E. F. Wheefoek, Symposium on Interferon, Lyons 1969.
Angew. Chem. infernat. Edit. Vol. 9 (1970) / No. 7
there are other kinds of viral interference in addition
to the interferon system r11-141. (For the current
position in interferon research see also r15-201.)
2. Biological Properties
One of the most interesting properties of interferons
is the nonspecificity of their antiviral action. Interferon
makes the host cell resistant to both R N A and D N A
viruses, including some oncogenic viruses. In addition,
its range of action also extends to bacteria, Bedsonia
organisms, and protozoa. There are, however, considerable differences in the sensitivity of virus strains
to interferon. Ruiz-Gomez and Isaacs [211 were able to
show that 30 times the amount of interferon was
necessary in chick embryo fibroblast cultures t o inhibit Newcastle disease virus (NDV) [**] to the same
degree as O’nyong-nyong virus. Other authors have
also described differences in the sensitivity of viruses
to interferons 122-247. Such differences apparently also
depend on the type of the host cell. Two strains of
vesicular stomatitis virus (VSV), which are inhibited
to different degrees in L cells (a stable mouse fibroblast
cell line), show the same sensitivity in chick embryo
cells [251.
It is very difficult to summarize the sensitivity of
viruses to interferons, partly because of the contradictory results. The arbor viruses are among the most
sensitive, whereas“ Newcastle disease, herpes, adeno,
and Aujeszky’s disease viruses appear to be relatively
resistant. VSV has a moderate sensitivity. Most of the
findings indicate that interferon sensitivity and the
ability to induce interferon formation go in parallel.
An interesting suggestion is that the virulence of
viruses is closely connected with their interferon
sensitivity and their ability to induce interferon
~.__.
-
[Ill A. S . Huang and R. R. Wagner, Virology 30, 173
(1966).
[12] P . I. Marcus and D . H . Carver, J. Virol. (Baltimore) I , 334
(1967).
1131 E. Zebovitz and A . Brown, J. Virol. (Baltimore) 2, 1283
(1968).
[14] M . A. Brat/ and H. Rubin, Virology 35, 395 (1968).
[15] N . B. Finter: Interferons. North Holland, Amsterdam
1966.
[16] G . E. W . Wolstenholme and M . O’Connor, Ciba Foundation, Symposium Interferon, J. A. Churchill Ltd., London 1968.
[17] J. Vilcek: Interferon, Springer, Heidelberg 1969.
1181 M . S. Finkelstein and T . C. Merigan, Calif. Med. 109, 24
(1968).
[19] M . R . Hilleman, Clin. Pharmacol. Therapeutics 9, 517
(1968).
120) R . 2. Lockart, Progr. med. Virology 9, 451 (1967).
[21] J. Ruiz-Gomez and A . Isaacs, Virology 19, 8 (1963).
[ **I Abbreviations: Newcastle disease virus
= NDV
= VSV
Vesicular stomatitis virus
= SFV
Semliki Forest virus
Translation inhibitory protein = T I P
[221 M . H o and J . F. Enders, Virology 9, 446 (1959).
[23] L . A. Glasgow and K. Habel, J. exp. Medicine 115, 503
(1962).
[241 J . Vilcek, Acta Virol. 6, 144 (1962).
1251 R . R . Wagner, A. H . Levy, R . M . Snyder, G . A. Ratcliff,
and D . F. Hyatt, 3. Immunol. 91, 112 (1963).
Angew. Chem. internat. Edit. / Vol. 9 (1970)
No. 7
formation [26,271. The interferon system is less effective
against virulent than against nonvirulent strains.
Another important biological characteristic of interferons is shown by the observation that the inhibitory
effect is much clearer if the cells used in the test come
from homologous animal species. This “species
specificity” was first observed by Tyrell[281 with interferon from calf and chick fibroblasts. Similar findings
were made when chick and rabbit cellsQ91 or chick
and duck cells were used[7.301. In all these cases the
interferon had a weaker effect in heterologous tissue.
Frequently no activity at all can be determined in such
tissues. This “species specificity” of the antiviral
effect - whether absolute or partial - is regarded as
an important characteristic of the interferon system.
Recently, however, the general validity of this “species
specificity” has been questioned. Bucknall1311 found
that the interferon of Macaca monkeys exerted an
antiviral activity in the cells of two other genera (Cercopirhecus and Erythrocebus). Thus, this interferon
not only did not show any “species specificity” but
also no genus specificity. Merigan 1321 observed a
cross-reaction between human and rabbit interferons
in cell cultures. Bucknall proposed that in future the
“species specificity” of the antiviral effect should n o
longer be regarded as a typical interferon property.
The present account of important studies on interferon would be incomplete without any mention of
the antigenicity of this substance. As interferons are
proteins (see Section 4), attempts have been made to
immunize animals with them, but the first experiments
were unsuccessful [33-351.
Paucker et al. 136,371 were the first to obtain an interferon antiserum. For this purpose guinea pigs and
rabbits were injected intraperitoneally with highly
purified interferon samples obtained from NDVtreated L cells. The treatment was repeated several
times. An antiserum that inactivated up to 99 % of the
interferon from L cells was thus obtained. Antibodies
to mouse and chick interferon only neutralized the
activity of the homologous preparations. The two
interferons thus differed in their antigenic properties.
Paucker considered it probable that all the earlier
attempts to obtain an antiserum failed because too
little interferon was given; interferon is obviously a
substance with an extremely high biological activity,
i. e. only relatively few interferon molecules are present
[26] J. F. Enders, Trans. Stud. Coll. Physicians Philadelphia
28, 68 (1960).
[27] R . F. Sellers, G . N . Mowat, J . H . Bennet, and D . A . Barr,
Arch. ges. Virusforsch. 23, 12 (1968).
[28] D . A. J . Tyrell, Nature (London) 184, 452 (1959).
[29] A. Isaacs and M . A . Westwood, Lancet 2, 324 (1959).
[30] M . Ho and J . F. Enders, Virology 9, 446 (1959).
[31] R . A . Bucknall, Nature (London) 216, 1022 (1967).
[32] T. C . Merigan, Interferon Scientific Memoranda, Memo
No. 146/1 (1969).
[33] D . C. Burke and A. Isaacs, Acta Virol. 4, 215 (1960).
[34] J.Lindenmann, Z. Hyg. Infektionskrankh. 146, 287 (1960).
[35] A . Isaacs, Advances Virus Res. 10, 1 (1963).
[361 K. Paucker and K. Cantell, Virology 18, 145 (1962).
[37] K . Paucker, J . Immunol. 94, 371 (1965).
48 1
even in the highly active purified preparations (see
Section 4).
An interesting and so far little-studied property of the
interferon system is the occurrence of the refractory
phase. Cantell and Paucker 1381 established that the
production of interferon ceased 24 hours after the
treatment of L cells with inactivated NDV. N o renewal
of interferon production could be demonstrated after
further treatment with NDV. Ho and Konoc391 and
Youngner and Stinebring 1401 made similar observations
in experiments carried out in vivo. Rabbits were
treated with Sindbis virus or endotoxin from E. coli,
a very potent interferon inducer. When the treatment
was repeated 24 hours later, no renewal of interferon
production could be detected. In the serum of tolerant
animals, Ho et al.1411 found a humoral factor which
inactivated the interferon-inducing capacity of endotoxin.
3. Methods of Determining Interferons
All methods used to determine interferons are based
on the fact that interferons inhibit virus multiplication. Of extreme importance in judging the results
are the experimental conditions, such as the type of
the host cell, the time of preincubation with interferon,
the type and amount of the test virus, and the experimental temperature.
3.1. Reduction of Virus Hemagglutinin Titers
Many viruses, including influenza virus, can form
hemagglutinins. The amount of hemagglutinin that
can be measured is proportional to the quantity of
the virus. Chorioallantoic membranes from chick
embryos are incubated in an interferon-containing
medium and are then infected with influenza virus.
The decrease in the hemagglutinin titer parallels that
in the interferon titer 181.
3.2. Decrease in Hemadsorption
(Quantitative Hemadsorption Method)
The phenomenon of hemadsorption is based on the
fact that many virus-infected cells can adsorb erythrocytes. The cells are incubated with the interferon
preparation and infected, and erythrocytes are then
added. After removing the unadsorbed erythrocytes,
the adsorbed erythrocytes are lyzed; the hemoglobin
released is measured spectrophotometrically. The
method is simple and gives reliable results 1421.
[38] K . Cantell and K . Paucker, Virology 21, 11 (1963).
L391 M . Ho and Y . Kono, J. d i n . Invest. 44, 1059 (1965).
[40JJ . S. Youngner and W. R. Stinebring, Nature (London)
208, 456 (1965).
[411 M . Ho, K . Kono, and M . K . Breinig, Proc. SOC.exp. Biol.
Med. 119, 1227 (1965).
[42]N . B. Finter, Virology 24, 589 (1964).
3.3. The Plaque Inhibition Method
The cells are incubated with the interferon sample and
then infected with a definite amount of a plaqueforming virus. After adsorption of the virus the cells
are covered with a layer of agar. The interferon titer
is given as the reciprocal of the greatest dilution of the
interferon sample that reduces the plaque count by
5 0 % compared with controlsr431. At the same time
this shows that the interferon neither prevents virus
adsorption nor inactivates extracellular viruses.
3.4. Indicator Test
Normal cells excrete acids into the incubation medium.
Phenol red, a constituent of most cell culture media,
thus changes to a yellow color. Infected cells do not
excrete enough acid to produce a change in color,
while interferon-treated cells behave in the same way
as non-infected ones, i. e. the medium becomes yellow.
Puucker [371 used this effect to develop a very reliable
and sensitive method for the determination of interferons.
3.5. Reduction of the Cytopathic Effect
In certain tissues the multiplication of viruses leads to
microscopically detectable cytopathic effects. The inhibition of these effects by pretreatment of the tissue
with interferon preparations is also used for interferon determination.
3.6. Prevention of Incorporation of
"4CI-Thymidine into Virus DNA
Bod0 [441 has described a biochemical method for the
determination of interferons in which the decrease in
the incorporation of [14C]-thymidine into the acidinsoluble DNA fraction is taken as a measure of the
amount of interferon (the cellular DNA was broken
down with DNase in the presence of Triton X-100).
4. The Chemical a n d Physicochemical Properties
of Interferons
Zsaacs and Lindenmann 181 describe interferons as
substances that are not dialyzable, not sedimented a t
100000 x g, partly inactivated by trypsin, precipitated
with a saturated ammonium sulfate solution, and not
undergoing any loss of activity with DNase. They
conclude from this that interferons or a t least their
essential components must consist of protein. Other
research workers have confirmed this hypothesis 1451.
Today, it is generally accepted that the operative part
1431 R . R . Wagner, Virology 13, 323 (1961).
[44] C.Bodo, Interferon Scientific Memoranda, Memo No. 78
(1 968).
[45]K . H . Funtes, see ref. [151, p. 119.
Angew. Chem. internat. Edit. J Vol. 9 (1970)
1 No. 7
of an interferon consists of protein with a small carbohydrate component. It is considered certain that it
does not contain any R N A or DNA.
The well-known methods of protein chemistry have
been used in experiments to isolate interferons. It
must be recalled that the only way of demonstrating
an interferon is by biological tests - a fact that makes
isolation very difficult. Two of the best-known methods
of purification are briefly described in the following
paragraphs.
Larnpson et a/. [461 purified interferon in the following way
from hen’s eggs infected with influenza A virus:
1. The virus and inactive protein were precipitated with
0.15 N HC104.
2. The interferon was precipitated at p H 6 with zinc acetate.
After centrifuging, the sediment was dissolved in 0.2 N HCI,
dialyzed against 0.9 % NaCl solution, and re-precipitated
with zinc acetate.
3. The precipitate was dissolved in 0.01 M phosphate buffer
(pH 6 ) , chromatographed o n a carboxymethylcellulose
column, and eluted using a 0.01 M phosphate buffer (pH 8)
with 0.1 M NaCI.
4. The biologically active fractions were again treated with
zinc acetate.
5. This was followed by another chromatographic separation
o n carboxymethylcellulose, and
6. precipitation with zinc acetate.
7. The operation was finished by zone electrophoresis at
p H 8.9 on Pevikon.
The specific activity of the interferon purified in this way
was 236000 units/mg protein 1471.
A product with a still higher specific activity (1.6~106
units/mg protein) was obtained by Fantes ei a/. 148,491 using
the following procedure:
1. Adsorption of interferon at p H 5 o n Doucil (a synthetic
sodium aluminum silicate) and elution at p H 7.5 with
potassium thiocyanate.
2. Precipitation of the inactive protein at p H 2.
3. Precipitation of further inert protein with 5 volumes of
methyl alcohol.
4. Precipitation o f interferon by neutralization of the solution.
5. Precipitation of interferon in 0.01 M phosphate buffer
(PH 7.5) and filtration through DEAE cellulose.
6. Chromatography of the filtrate a t p H 5.9 on a carboxymethyl-Sephadex column. The interferon-containing fraction
was eluted with a n increasing p H gradient.
20000-fold concentration was obtained with this method of
purification. The overall recovety of the activity was 7 %.
The use of polyacrylamide gel electrophoresis resulted
in a substantial advance in the field of interferon
purification. On the one hand this showed that all the
highly purified products still contain many impurities,
and on the other hand that interferon itself is not
homogeneousr45,5o,511. In polyacrylamide gel electrophoresis the biological activity of a highly purified
[46] G. P. Lampson, A . A. Tytell, M . M . Nemes, and M . R.
Hilleman, Proc. SOC.exp. Biol. Med. 112, 468 (1963).
[47] Interferon units are generally given as the reciprocals of
the greatest dilution that protects cells against a virus attack
under defined conditions.
[48] K . H . Fantes, Nature (London) 207, 1298 (1965).
[49] K. H . Fantes, J. gen. Virol. I , 257 (1967).
[50] T . C. Merigan, C. A . Winget, and C. B. Dixon, J. molecular Biol. 13, 679 (1965).
[51] K . H . Fantes, 11. Int. Sympos. med. appl. Virol., Ft.
Lauderdale.
Angew. Chem. internat. Edit.
J Vol. 9 (1970) 1 No. 7
product is distributed over a broad zone. The biologically active constituents of the interferon preparation
of Fantes er al.
differed both in molecular weight
and in isoelectric point. Other authors too have
concluded that there may be no “interferon” as such;
instead, there are probably several substances with
interferon-like properties which appear simultaneously i n the same animal in response to the same
stimulus153,541. Thus, it seems t o be certain that
different types of cell from the same organism form
different interferons 155,561. Their molecular weight
was between 13000 and 160000L17J.
There is as yet no explanation for these facts. So far
it is not clear whether these substances are preliminary
stages of interferon formation, interferon produced b y
different cells, interferon with different carrier proteins,
or something quite different.
Since the o n l y possible conclusion is that none of the
biologically active substances so far isolated and
investigated was a pure interferon, it seems advisable
to treat with reservation some of the values and properties so far determined e . g . the amino-acid composition.
On the other hand, certain characteristics can be
accepted as generally valid. In all cases the interferons
were neutral or weakly basic proteins or proteincontaining compounds. Their stability over a wide pHrange i s particularly worth mentioning. Lampson ef
al. [571 observed that highly purified chick interferon
was stable at 23 “C for over an hour in the p H range
1-10. These results have been confirmed several
times. Today the stability at p H 2 is regarded as an
important characteristic of all interferons. Most interferons are relatively heat resistant. Lampson et al.
found that purified chick interferon remained fully
active for one hour at 66°C. Some inactivation only
occurred after heating at 76°C for one hour. The
biological activity of interferon is destroyed by
proteolytic enzymes such as trypsin, chymotrypsin,
and pepsin, but not by DNase, RNase, lipase, peptidase, and a-amylase.
It has already been mentioned that the general current
opinion is that interferon o r at least its active part
consists of protein. A Japanese research team 158,591
has described an interferon-like compound, which
was isolated from rabbit skin infected with vaccinia
virus. This compound was reported to be a proteinfree polysaccharide (molecular weight lOOOO), which
[52] K . H . Fantes in G. Rita: The Interferons. Academic Press,
New York 1968, p. 213.
[53] R . R. Wagner and T . S. Smith, see ref. [16], p. 95.
1541 Y. H . Ke, M . Ho, and T . C. Merigan, Nature (London)
211, 541 (1966).
I551 E. Falcott, F. Fournier, and C. Chany, Ann. Inst. Pasteur
111, 241 (1966).
[56] M . Ho and Y . H . Ke, Interferon Scientific Memoranda,
218/1 (1969).
1571 G. P. Lampson, A . A. Tytell, M . M . Nemes, and M . R.
Hilleman, Proc. SOC. exp. Biol. Med. 121, 441 (1965).
[ 5 8 ] Y. Nagano, Y. Kojima, T . Haneishi, and M . Shirasaka,
Jap. J. exp. Med. 36, 535 (1966).
[59] Y. Nagano, Y. Kojima, and R. S. Kanashiro, Jap. J. exp.
Med. 36, 477 (1966).
48 3
was only active in vivo and had no “species specificity”. Whether this substance is the active part of
interferon, which is combined with a species-specific
protein in the animal body and thus forms a “normal”
interferon molecule, or whether it is an interferoninducing compound, has not so far been elucidated.
5. M o d e of Action of Interferon
Isaacs et al.[60,611 observed an increased rate of
glycolysis in interferon-treated cells. In addition, it
was found that there was a decline in the direct
oxidation of glucose via the pentose phosphate cycle
and that less inorganic phosphate was incorporated.
As the same metabolic changes are produced by
chemical uncoupling of oxidative phosphorylation,
Zsaacs et al. came to the conclusion that interferon
likewise acts as an agent of uncoupling oxidative
phosphorylation and that this uncoupling occurs not
in the mitochondria but in the cell nucleus. However,
Lampson et al.[6*1 were able to show later that the
described effects failed to appear when the experiments were carried out with highly purified interferon
preparations. It now appears certain that the uncoupling of oxidative phosphorylation and other
effects on the uninfected cells were produced by
impurities [631.
The following processes must occur before a virus
can multiply within a host cell: Adsorption of the
virus onto the cell surface; penetration of the cell;
and liberation of the viral nucleic acids by removal
of the protein coat.
Interferon has no effect on the adsorption of viruses[64.651. From the fact that the multiplication of
infectious RNA isolated from viruses is inhibited by
interferon, it was concluded that interferon has n o
effect on the penetration of intact virus or the liberation of viral nucleic acids [66,673.
This conclusion does not seem to be absolutely correct, at least for certain D N A viruses, as the recent
findings of Magee et al. 1681 indicate that interferon
probably inhibits the uncoating process in vaccinia
virus. Since it is known that the uncoating enzymes
of some D N A viruses are virus proteinsr691, this
effect can be attributed to an inhibition of virusspecific protein synthesis. It has also been possible to
[60] A. Isaacs, Virology 10, 144 (1960).
[61J A . Isaacs, H. G. Klemperer, and G. Hitchcock, Virology
13, 191 (1961).
[62] G. P. Lampson, A. A . Tytell, M . M . Nemes, and M . R.
Hilkman, Proc. SOC.exp. Biol. Med. 112, 468 (1963).
[63] S . Baron, T. C . Merigan, and L. M . McKerlie, Proc. SOC.
exp. Biol. Med. 121, 50 (1966).
[64] A . Isaacs and D . C . Burke, Brit. med. Bull. 15, 185 (1959).
1651 P . de Somer, A . Prinzie, P . Denys, and E. Schonne, Virology 16, 63 (1962).
1661 M . Ho, Proc. SOC.exp. Biol. Med. 107, 639 (1961).
1671 S. E. Grossberg and J . J . Holland, J. Immunol. 88, 708
(1962).
[68] W. E. Magee, S. Levine, 0 . V. Mille, and R . D . Hamilton,
Virology 35, 505 (1968).
1691 B. Woodson, Bacteriol. Reviews 32, 127 (1968).
484
exdude a direct inactivating action of interferon on
the complete virus particle[70,71l as well as on viral
RNAL721. From this it can be concluded that the
action of interferon must be based on an inhibition of
the synthesis of virus-specific proteins or viral nucleic
acids.
R N A viruses multiply in the following way:
1. The “early” cistrons of the parental RNA molecule (plus
strand) encode directly as m-RNA for the synthesis of
“early” enzymes such as the RNA-dependent polymerase.
2. Through the RNA-dependent R N A polymerase is formed
the “replicative” form of the virus, i.e. a second R N A strand
(minus strand) is synthesized on the parental plus strand
which then acts again as a matrix for the synthesis of plus
strands.
3. Translation of the “late” cistrons and thus synthesis of
the viral coat protein.
4. Combination of viral RNA (plus strand) with coat protein
to form the intact virus.
The synthesis of viral RNA is inhibited in interferontreated cells[73-751. This means that either the synthesis or the activity of RNA-dependent RNA polymerase is interrupted by interferon. Sonnabend, Martin, and Mkcsf751 have shown that interferon has no
effect on the activity of the RNA-polymerase of Semliki Forest virus (SFV) in a cell-free system.
Miner, Ray, and Sirnon[761 studied the effect exerted
by a homogenate of interferon-treated L cells on
Mengo virus RNA polymerase, and established that
the activity of the polymerase was not decreased. The
same authors, however, found that pretreatment of L
cells with interferon clearly decreased their content of
virus-specific RNA polymerase after a Mengo virus
infection. Sonnabend et al. also demonstrated a
decrease in the extractable amount of SFV RNA polymerase when they pretreated L cells with interferon.
This means it is not the action but the synthesis of
viral R N A polymerase that is inhibited.
Marcus and Salbr771 were able to show that although
Sindbis virus RNA and the ribosomes of interferontreated chick embryo cells form a polysome complex,
no amino acids are incorporated. The action of interferon on the multiplication of R N A viruses thus
obviously consists in an inhibition of the translation
of virus-specific m-RNA, and consequently in inhibition of the synthesis df virus proteins.
The multiplication of DNA viruses take place according to
the following series of events:
1. “Early” transcription of the viral DNA molecule with the
formation of “early” viral m-RNA.
2. Translation of the “eariy” m-RNA into “early” proteins.
[70] M. Ho and J . F. Enders, Proc. nat. Acad. Sci. USA 45,
385 (1959).
1711 R. R. Wagner, Virology 13, 323 (1961).
[72] V. Mayer, F. Sokol, and J. Vilcek, Acta Virol. 5, 264
(1961).
[73] C . Cocito, E . de Maeyer, and P. de Somer, Life Sci. 1, 753
(1962).
[74] J. Taylor, Virology 25, 340 (1965).
1751 J . A . Sonnabend, E . M . Martin, and E. MPcs, Nature (London) 213, 365 (1967).
1761 N . Miner, W. J . Ray, and E . H . Simon, Biochem. biophysic. Res. Commun. 24, 264 (1966).
[77] P . I . Marcus and .
I
.
M . Salb, Virology 30, 502 (1966).
Angew. Chem. internat. Edit. J Vol. 9 (19701 1 NO. 7
interferon
4
%
1
fm-RNA
or TIP
TIP-
I
r i b o s o m e pool
producinj
polysome
0
0
0
Figure 1 .
’ibosomes
- TIP
normal
no p r o t e i n
synthesis
on v i r a l
polys ome
complex
Mode of action of interferon according to Marcus and Salb [771 and Hilleman [191.
1 . Interferon penetrates the uninfected cell and induces the synthesis of rn-RNA for TIP. 2. With the help of this rn-RNA the cell synthesizes TIP
3. TIP interacts with the ribosomes, hut does not effect the cellular protein synthesis.
4. A virus penetrates the cell.
5. Although the viral RNA comes in contact with the TIP ribosome complex, no viral protein is synthesized.
3. Synthesis of viral DNA.
4. Formation of “late” m-RNA.
5. Translation of the “late” m-RNA into the viral coat
proteins.
6 . Combination of viral DNA and coat protein to form
intact virus (maturation).
In several systems it has been possible to show that the
synthesis of viral D N A is inhibited in interferontreated cells 178,791.
Joklik and Merigan[sol showed that the synthesis of
viral m-RNA is not affected by interferon in the L
cells/vaccinia virus system. The virus infection produced a disintegration of the host’s polyribosomes,
which was followed by a formation of virus-specific
polyribosomes with viral m-RNA. I n the presence of
interferon the formation of these polyribosomes fails
to occur, with the result that no virus-specific proteins
-and thus no D N A polymerase - are synthesized.
[78] S . N . Ghosh and G. E. Gifford,Virology 27, 186 (1965).
[79] S . Levine, W. E. Magee, R . D . Hamilton, and 0 . V . Miller,
Virology 32, 3 3 (1967).
[SO] W. K . Joklik and T . C. Merigan,
USA 56, 558 (1966).
Angew. Chem. internat. Edit.
Proc. nat. Acad. Sci.
VoI. 9 (1970)J No. 7
Looking for common factors in the inhibitory action
of interferon on the multiplication of RNA and D N A
viruses, it can be established that in both cases it is
the “early” translation of virus-specific message, i. e.
the formation of “early” virus-specific proteins, that is
inhibited. Whether the inhibition is based on the
prevention of reading of the m-RNA in the polyribosome, or whether the formation of the polyribosomes is suppressed, has not been finally resolved.
It is even possible that both effects can occur. The
problem of the site of action of interferon seems to be
solved, but the mode of action at the molecular level
remains an open question.
TayIor 1811 established that actinomycin C, which is
known to inhibit cellular DNA-dependent RNA
synthesis, prevents the antiviral action of interferon.
A similar effect is produced by many inhibitors of
protein biosynthesis such as puromycin [*2,831 and p [81] J . Taylor, Biochem. biophysic. Res. Commun. 14, 447
(1964).
[82] R . M . Friedman and J . A. Sonnabend, J . Immunol. 95, 696
(1965).
f83J S . Levine, Virology 24, 587 (1964).
485
fluorophenylalanine [841. The cellular RNA and protein
synthesis must therefore be intact for the development
of the antiviral action of interferon. It was concluded
that interferon induces the formation of a second
protein, which is the substance with the true antiviral
effect r‘antiviral protein”, “translation inhibitory
protein (TIP)”] [77,851.
Using the model proposed by Jacob and Monod to
explain these processes, the development of the antiviral effect of interferon can be supposed to proceed
largely in accord with Figure 1. There has been no
lack of attempts to provide direct experimental proof
of the existence of TIP. Using a double labeling
method, Vilcek et al. 186,871 were able to demonstrate
that the ribosomal protein fraction of interferontreated cells showed a different distribution of activity
in polyacrylamide electrophoresis from that of normal
cells. The authors found one completely new band
and an increased amino-acid incorporation in two
other protein bands. The identification of one or more
of these proteins as TIP has still not yet been accomplished.
Marcus and SaZb(881 allowed trypsin to react under
mild conditions with the ribosomes of interferontreated cells. At 0 ° C the ribosomes could form a
polysome complex with Sindbis virus RNA, which
rapidly disintegrated again and took up amino acids
at 37°C. Marcus and Salb regarded this as an indication of normal translation, since the corresponding
complex with interferon-treated nontrypsinized ribosomes was not broken down and did not take up any
amino acids.
Surprisingly, Dianzani, Buckler, and Baron 1891 found
that cycloheximide - another inhibitor of protein
biosynthesis - did not neutralize the antiviral action
of interferon. The authors considered that it was
possible that the m-RNA for TIP is formed in the
presence of the inhibitor and remains relatively stable,
and that the period after removal of the cycloheximide
and before addition of the virus is sufficient for
enough TIP to be formed with this m-RNA.
Summing up, it may be said that there is yet no
definite proof of the existence of one or more antiviral
proteins whose synthesis is induced by interferon, but
that the majority of the experimental findings supports
this hypothesis. It should also be added that, if this
theory is confirmed, the formation of m-RNA and
the synthesis of TIP would be the only effects of
interferon on normal, i. e . uninfected cells.
1841 R . M . Friedeman and J . A . Sonnabend, Nature (London)
203, 366 (1964).
[ 8 5 ] J. Taylor, Virology 25, 340 (1965).
[86] M . H . Ng, J . Vilcek, and T . G . Rossman, Bacteriol. Proc.
1969, 147.
I871 J . Vilcek, M . H . Ng, and T . G . Rossman in G . Rita: The
Interferons. Academic Press, New York 1968, p. 185.
I881 P. I. Marcus and J . M . Salb in G . Rita: The Interferons.
Academic Press. New York 1968, p. 111.
I891 F. Dianzani, C . E. Buckler, and S . Baron, Proc. Soc. exp.
Biol. Med. 130, 519 (1969).
486
6. Interferon Inducers
As already mentioned, both intact and inactivated
viruses, i . e . viruses not capable of multiplying, can
induce the formation of interferon. This ability to
induce interferon formation is apparently a feature of
all viruses, no matter to what class they belong. It has
been known for some years that other agents besides
viruses can stimulate interferon formation. Such
agents include bacteria, rickettsiae, phytohemagglutinins, Toxoplasma, Mycoplasma, protozoa, cycloheximide, as well as some anionic copolymers and
nucleic acids.
6.1. Bacteria and Bacterial Endotoxins
Even before the discovery of interferon it was recognized that bacteria have an inhibitory effect on the
growth of some viruses. In 1938 Arrnstrongc901 observed that certain myxovirus infections of mice are
prevented by bacteria. Horsfall and McCarty [91J
established that the multiplication of viruses in the
lungs of mice infected with pneumonia virus was
decreased 10-100 fold if the animals were also treated
with a nonhemolytic Streptococcus MG’ strain.
Youngner and Stinebring[9*] were the first to show
that bacteria can act as interferon-inducers. These
authors infected hens with a virulent strain of Brucella
abortus and found an antiviral activity in the birds’
serum. The substance responsible for this showed the
same physicochemical and biological properties as
interferon.They also found an interferon-like substance
after treating mice with purified E. coli endotoxin [931.
Thus bacterial multiplication is not a prerequisite for
the induction of interferon. Similar results were
obtained by Ho[941 with an endotoxin which he
administered to rabbits. Hopps et al. 1951 were also able
to isolate an interferon-like substance from chick
embryo cells inoculated with Rickettsia tsutsugamuchi.
There are indications that endotoxin treatment does
not induce the synthesis of interferon but only the
release of preformed molecules. Endotoxin-induced
interferon appears earlier in the serum of treated
rabbits than virus-induced interferon, and, in addition,
its formation is not inhibited by actinomycin D 1961.
6.2. Phytohemagglutinin
Phytohemagglutinin is a substance contained in an
extract of Phaseolus vulgaris. Wheelock 1971 established
that this plant product has an antiviral effect in human
1901 C . Armstrong, Public Health Rep. 53, 2004 (1938).
[911 F. L. Horsdall and M. McCarty, J. exp. Medicine 85,623
(1947).
1921 J. S. Youngner and W. R . Stinebring, Science 144, 1022
(1964).
[93] W. R . Srinebring and J. S . Youngner, Nature (London)
204, 712 (1964).
1941 M . ffo, Science (Washington) 145, 1472 (1964).
1951 ff. E. Hopps, S . Khono, M . Khono, and J . E . Smadel, Bacteriol. Proc. 1964, 115.
[96] Y . Khono and M . ffo, Virology 25, 162 (1965).
1971 E. F. Wheelock, Science (Washington) 149, 310 (1965).
Angew. Chem. internat. Edit.
Vol. 9 (1970) No. 7
leukocytes; the cytopathic effect of Sindbis virus was
also decreased. Like interferon, the antiviral substance
can be neither dialyzed nor sedimented.
6.3. Synthetic Anionic Copolymers
In 1966 Regelson [981 found that copolymers of maleic
anhydride and divinyl ether (molecular weight
17000-450000) develop an antiviral activity after
intraperitoneal administration to mice, and this
appears in the serum 24 hours after the injection.
Merigan[99] was able to show that this antiviral
substance had all the known properties of interferon.
The same agent also inhibited virus-induced leukemia
in mice, and in clinical trials it inhibited neoplasm
formation in man [1001. An interferon-inducing effect
could also be demonstrated with copolymers of maleic
anhydride and vinyl methyl ether or vinyl acetate 11011.
The corresponding styrene compounds were not such
good inducers and were more toxic.
Some of these copolymers have been tested clinically.
A maleic anhydride/divinyl ether copolymer with a
molecular weight 17000 was tested in the USA as an
antitumor drug. A definite disadvantage of this compound is that it is not broken down in the body but
is stored in the reticulo-endothelial system 11021. So
far there has been n o success in inducing interferon
formation in vitro. The exact mode of action of these
polymers is not known. It has been argued that the
antiviral action is based not only on the formation of
interferon but also on a direct interaction with the cell
due to the polyanionic structure of the molecule.
6.4. Nucleic Acids
In 1961 Zsaacs~1031put forward the theory that the
formation of interferon is a reaction of the cell to a
“foreign” nucleic acid, in the same way that antibodies are formed by recognizing differences between
foreign and the body’s own proteins.
Rotem et al. 11041 established that mouse liver RNA
inhibits the growth of pox viruses in chicken cell
cultures, exactly in the same way as chicken R N A
inhibits it in mouse cell cultures. A much smaller
inhibition occurred with the homologous RNA.
Zsaacs 11051 continued these investigations and established that nucleic acids did not induce interferon
formation in the homologous cells. The R N A could
do this, however, after treatment with nitrous acid.
Zsaacs concluded that the R N A was so changed by
[98] W. Regelson, Proc. Int. Sympos. on Atherosclerosis and
Reticuloendothelial System, Como/Italy, September 1966.
[99] T. C. Merigan, Nature (London) 214, 416 (1967).
[loo] T . C . Merigan and W.Regelson, Clin. Res. IS, 309 (1967).
[ l o l l T . C. Merigan, Nature (London) 214, 416 (1967).
[lo21 T. C . Merigan, see ref. 1161, p. 50.
[lo31 A . Isaacs, Nature (London) 192, 1247 (1961).
[lo41 Z . Rotem, R . A . Cox, and A . Isaacs, Nature (London)
197, 564 (1963).
[lo51 A. Isaacs, R . A . C o x , and Z . Rotem, Lancet 2,113 (1963).
Angew. Ckem. internat. Edit. j Vol. 9 (1970)1 No. 7
deamination that it was taken for a foreign nucleic
acid by the cells. Although some later findings supported this concept [106-1081, Zsaacs’ theory did not
find general recognition. In 1965 Zsaacs [lo91 reported
that he himself had also obtained “different results”
in further studies.
The theory that the foreign nucleic acids induced interferon formation received further experimental support
from Hilleman’s group. For some years this team
had sought a potent interferon inducer that would be
suitable for clinical use. Hilleman reported attempts
to fractionate viruses into their constituent parts and
to examine the individual fractions for their capacity
to induce interferon formation [1101. These experiments
were unsuccessful.
Braun and Nakano reported [111,1121 that they had
succeeded in stimulating antibody synthesis with some
synthetic oligonucleotides. This prompted Hilleman’s
group to test the interferon-inducing capacity of
synthetic polynucleotides. These investigations were
crowned with success: a few pg of a double-stranded
complex of polyriboinosinic acid and polyribocytidylic
acid (poly-1:C) induced interferon formation in
mice [1131. This interferon protected the animals
against a lethal dose of Columbia SK virus. A
double-stranded complex of polyriboadenylic acid
and polyribouridylic acid (poly-A:U) and a mixture
of polyinosinic acid and cytidylcytidine (I + CpC)
were also active, but to a lesser degree. N o activity,
however,was shown by single-stranded polynucleotides,
and by some oligonucleotides, dinucleotides, nucleotides, and nucleosides. Interferon formation could
also be demonstrated in vitro with poly-1:C.
These findings led to a search for other kinds of
double-stranded RNA. Hilleman and his associates
succeeded in isolating such a double-stranded RNA
from helenine (an antiviral substance produced by
Penicillium funiculosum) 11141. RyteI [1151 was able t o
show that helenine can induce interferon formation
in vivo and in vitro. The double-stranded R N A from
helenine was also an excellent interferon inducer [1161.
Subsequently it was established that the antiviral
principle of statolon, an extract of Penicillium staloniferum is likewise a double-stranded ribonucleic
[lo61 H . Kohlhage and D . Falke, Arch. ges. Virusforsch. 14,404
(1964).
11071 K. E. Jensen, A . L. Neal, R. I . Owens, and J . Warren,
Nature (London) 200, 433 (1963).
[lo81 K . Takano, J . Warren, K . E. Jensen, and A . L . Neol, J.
Bacteriol. 90, 1542 (1965).
[lo91 A. Isaacs, Aust. J. exp. Biol. med. Sci. 43, 405 (1965).
[110] M . R . Hilleman, J. cellular Physiol. 71,43 (1968).
[ill] W. Braun and M . Nakano, Proc. exp. Biol. Med. 119,
701 (1965).
11121 W . Braun and M . Nakano, Science (Washington) 157,
819 (1967).
[113] A. K . Field, A . A . Tytell, G. P . Lampson, and M . R. Hilleman, Proc. nat. Acad. Sci. USA 58, 1004 (1967).
[114] R . E . Shope, J. exp. Medicine 123, 213 (1966).
[115] M . W . Rytel, R . E. Shope, and E. D . Kilbourne, J. exp.
Medicine 123, 577 (1966).
[116] G . P . Lampson, A. A . Tytell, A . K . Field, M . M . Nemes,
and M . R . Hilleman, Proc. nat. Acad. Sci. USA 58, 782 (1967).
487
acid [1171. Another source of naturally occurring RNA
is the replicative form of RNA viruses. The replicative
form of the MS2bacteriophage was isolated and shown
to be an interferon inducer even in very small
amounts [1181. A similar result was obtained with
purified reovirus RNA [1191. Reovirus is one of the few
viruses whose nucleic acid core consists of doublestranded RNA.
It is interesting to note that the ability of poly-I:C to
induce interferon formation can be increased about
100-fold by the addition of DEAE-dextran 11201. This
raises the question why double-stranded ribonucleic
acids are some of the most powerful interferon
inducers. The possibility of these nucleic acids conveying genetic information can be ruled out, since
double-stranded homopolynucleotides such as polyI:C cannot do this.
Two explanations have been suggested. Hilleman
believes that the appearance of the double-stranded
replicative form, one of the first steps in the multiplication of R N A viruses, is normally the starting signal
for interferon synthesis. When double-stranded R N A
is supplied, the cell reacts as if virus multiplication
were occurring, and interferon synthesis begins. The
weak point in this theory is that RNA viruses incapable of multiplication and also D N A viruses are
good interferon inducers, although, according to
present knowledge, there is no double-stranded RNA
in either of these cases.
The surprising finding reported by CoZby and Duesberg11211 is of special interest in this connection. These
authors observed that chick embryo cells infected
with vaccinia virus (a D N A virus) contained 10 times
as much double-stranded RNA as did noninfected
cells. According to the concepts generally accepted
today concerning D N A virus multiplication, there is
as yet no evidence of the presence of double-stranded
R N A after vaccinia virus infection. Likewise, there is
no explanation for the existence of double-stranded
RNA in noninfected cells. However, it is also worth
mentioning that Montagnier[1221 was able to demonstrate double-stranded RNA in normal rat liver
cells.
The second explanation for the good inducer properties of double-stranded R N A is the principle of
“foreign” nucleic acid put forward by Zsaacs. A wellknown characteristic of double-stranded RNA is its
relative stability in the presence of RNase; this gives
it a greater chance of reaching the cell than all the
other types of nucleic acids, which are very quickly
broken down by nucleases.
[117] L. F. EIIis and W. J . Kieinschmidt, Nature (London) 215,
215 (1967).
[118] A. A. Tytell, G . P . Lampson, A . K . Field, and M . R . Hilleman, Proc. nat. Acad. Sci. USA 58, 1719 (1967).
[1191 A. A. TyteN, G . P. Lampson, A. K. Field, and M . R . Hiileman, Proc. nat. Acad. Sci. USA 58, 1719 (1967).
I1201 F. Dianzani, P . Cantagalli, S . Goagnoni, and G . Rita,
Proc. SOC.exp. Biol. Med. 128, 808 (1967).
[121] C. Colby and P. H . Duesberg, Nature (London) 222, 940
(1 969).
I1221 L . Munfagnier, C. R . hebd. Seances Acad. Sci., Ser. D
1968,1417.
488
7. Future Prospects
There has been no lack of ideas and experiments on
the use of the interferon system for the control of
virus diseases. Its broad antiviral spectrum of activity,
its lack of toxicity, and its low antigenicity apparently
make interferon especially suitable for this purpose.
The virus diseases, for which only a limited number of
serologically different types of viral agents are responsible and which in addition lead to prolonged immunity, are today largely kept in check by vaccination.
This is the case with measles, pox virus diseases,
yellow fever, poliomyelitis, etc. In addition to this,
virus chemotherapy has been introduced with the use
of iododeoxyuridine, adamantamine, and methylisatinP-thiosemicarbazone in the treatment of herpes, influenza, and pox virus infections, respectively.
The hope that interferon could heIp in the effective
control of virus diseases was recently strengthened
when Hilleman reported in 1967 that some doublestranded ribonucleic acids were extremely potent interferon inducers. Park and Baron11231 as well as Pollik08[1241 were also able to establish that interferon is
suitable for both prophylactic and therapeutic treatment. The above authors observed that rabbits recovered from herpetic conjunctivitis much quicker
and better after treatment with poly-T:C than the
control animals. This therapeutic effect was observed
both with local and systemic administration. Guerra
et aZ.11251 described the poly-1:C treatment of 22
patients suffering from herpetic keratitis. A good
therapeutic effect was obtained in 17 cases.
The great difficulties which stand in the way of clinical
use of interferon inducers are, in the first place, the
already mentioned refractory phase and, secondly, the
considerable toxicity of most of the inducers known
at present.
The clinical application of interferon itself, i. e. exogenous interferon, is problematic, since it is very difficult
to prepare sufficient quantities of human interferon.
Thirteen years have passed since the discovery of
interferon by Isaacs and Lindenmann, and much new
information has been acquired during this time. The
process of interferon induction on the molecular
level, and the mode of action of interferon are still
obscure in many respects. Another task for the future
is the further purification of interferon, so that its
chemical composition can be investigated. Recently,
Vilcek [I261 discussed an interesting possibility: “Further studies may perhaps lead to the discovery of noninterferon inducers of TIP. Or would such agents be
called interferons ?’
Received: August 25, 1969
[A 761 IEI
German version. Angew. Chem. 82, 489 (1970)
Translated by Express Translation Service, London
[123] J . H. Park and S . Baron, Science (Washington) 162, 811
(1968).
[124] R. Pollikof, P . Cannavale, P . Dixon, and A . Dipupgo,
Bacteriol. Proc. 1969, 150.
[125] R . Guerra, R . Frezzotti, R . Bonanni, F. Dianrani, and G .
Rita, 11. Conf. Antiviral Substances New York, June 1969.
[126] J . Vilcek, see ref. [17], p. 111.
Angew. Chem. internat. Edit.
1 Vol. 9 11970) / No. 7
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