Interferon

Interferon* A Review and Analysis of Recent Observations ROBERT R. WAGNER,

M.D.

Baltimore, Maryland ivrERFERoNis a generic

term for nonviral cellular proteins that inhibit multiplication of animal viruses. It was discovered by Isaacs and Lindenmann [ 71during the course of studies on the well known but theretofore mysterious phenomenon of viral interference. Many investigators had previously noted that dual infection with unrelated viruses, or different strains of the same virus, was often better tolerated by animals or cell cultures than was infection with either virus alone [2]. It had been apparent from the studies of Henle [3] and others that inactivated as well as infectious influenza viruses interfere with superinfecting influenza viruses at an intracellular site. The ingeniously simple experiments of Isaacs and Lindenmann [I] provided an explanation for this phenomenon. They exposed suspended fragments of chick chorioallantoic membrane to interfering influenza virus that had been inactivated by heat or ultraviolet light. These infected allantoic cells released into the medium a substance, called interferon, which could be transferred to uninfected allantoic cells, thus rendering them resistant to fully infectious influenza virus. Since this time virtually every class of animal virus has been found capable of inducing cells to form interferons which inhibit multiplication of the same or totally unrelated viruses (reviewed by Ho 141). Notable exceptions may be the adenoviruses as well as the simian myxovirus SV5 which appears to lack the capacity to cause interference in monkey kidney cell cultures [5]. Also worthy of note is the fact that certain types of viral interference do not appear to be mediated by interferon. Under rather exacting conditions myxoviruses [6] and enteroviruses [7J are capable of altering the cytoplasmic receptor sites for attachment and penetration of superinfecting viruses;

the excluded virus will, of course, fail to multiply. In addition, there is evidence that some viruses, which can induce interferon formation, may sometimes cause intracellular interference which is not demonstrably mediated by interferon [8,9]. These exceptions notwithstanding, it seems evident from the mass of data accumulated since 1957 that interferon induction is by far the most common form of viral interference in animal cells. Parenthetically, the phenomenon of viral interference is of some practical significance in diagnostic virology and for vaccination with live attenuated viruses. For those agents that do not readily cause cytopathology in tissue cultures, such as the arboviruses of the Russian tick-borne encephalitis group [70], their capacity to interfere with a cytopathogenic superinfecting virus may provide convenient methods of isolation and serologic identification. This is particularly true for virologic and immunologic studies of the fastidious rubella virus which can be most readily identified at present by interference assay [ 7 7 1, Viral interference also occurs under field conditions, the best known example of which is the failure of vaccine strains of attenuated poliovirus to multiply and induce immunity in populations heavily infested with other enteroviruses [ 721. The apparent universality of interferon production by cells infected with certain animal viruses is understandably of considerable interest from at least two related standpoints. At the level of cellular biology the synthesis and action of interferon provide potential tools for studying regulation of nucleic acid metabolism and particularly the competitive antagonism between viral and cellular nucleic acids. Of equal or greater interest, perhaps, is the real possibility

I

* From the Department of Microbiology, The Johns Hopkins University School of Medicine, Baltimore, Maryland. Experimental studies in the author’s laboratory were supported by grants from the U. S. Public Health Service and the National Science Foundation. 726

AMERICAN

JOURNAL

OF

MEDICINE

Interferon-that interferon represents a primary host mechanism of cellular defense against viral infection. Much of the available information has been reviewed and analyzed in considerable detail [ [3. I-11. Therefore, this brief review will deal onl!. with selected data, principally as they bear on the theoretic significance of the interferon response as a determinant of virus virulence and, con\.crsely, of tissue immunity to viral infection. PKODIJCTION

AND IDENTIFICATION

Interferons are usually prepared by infecting cell cultures or animals with infectious or inactivated viruses. The factors that control interferon formation will be discussed subsequently in greater detail. Suffice it to say that the evidence now seems conclusive that interferon synthesis is a cellular function shared, in all probability, by all vertebrate cells. The amounts of interferon produced vary considerably, depending on the nature of the infecting virus, the cell type and environmental conditions. It should be borne in mind, however, that viruses merely act as inducing agents. In fact, viruses do not appear to be essential for interferon synthesis. Nonviral nucleic acids [ 151, rickettsiae ] Ici]? or statolon (a complex polyanionic polysaccharide obtained from a penicillium mold) [ 171 stimulate cell cultures to form small amounts of interferon-like substances. Intravenous injection of brucellae [78], enteric bacteria or purified bacterial lipopolysaccharide endotoxins [ 19,201 also results in rapid appearance in animal serum of viral inhibitors with most of the properties of interferon. Whether these substances are identical with virus-induced interferons remains to be proved [ 191. i\lthough most investigators have failed to detect interferon activity in uninfected cell cultures or animal tissues, the question whether trace amounts can be formed without external stimulation has not been resolved. The most thoroughly characwhich should serve as a terized interferon, reference standard for all other interferons, is produced by allantoic cells of chick embryos infected with influenza A virus [27]. To prepare this material, allantoic fluids are harvested 48 to 72 hours after infection, centrifuged to remove most of the virus and treated with acid to inactivate residual virus. Neutralized supernatant fluid constitutes a crude preparation of interferon, almost invariably of high titer. The presence of interferon is determined, by definition, by the capacity to inhibit multiplica-

Il/cl~~n~~ tion of a test virus. The original assay method [I] of measuring the degree to which interferon reduces the yield of influenza hemagglutinin in surviving chorioallantoic membrane fragments is limited to myxoviruses and occasionally gives inconsistent results. Other widely used procedures are inhibition of virus cytopathology or reduction of virus yield in infected monolayer cultures [.??I. The most accurate and the most readily standardized technic is the plaqueinhibition method [_?/I. By this means, interferon activity can be quantitated by determining the dilution at which virus plaque formation in replicate monolayer cultures is reduced by 50 per cent of controls. Detailed analysis of this procedure [2.?] reveals excellent reproducibilit) of results over the range of 20 to 80 per cent reduction of vaccinia virus plaques. The choice of a test virus can only be determined empirically on the basis of its sensitivity to interferon. The most useful test virus has been vesicular stomatitis because of its sensitivity to interferon and its ability to plaque on most vertebrate cells [?J]. Interferons are not virus specific. The same preparation can inhibit multiplication of a wide variety of viruses unrelated to the virus used to induce its formation [d>131. However, interferons exhibit strict specificity of action for cells of the same animal species in which they are prepared. For example, purified interferons of mouse, chick or human origin show no capacity to inhibit viral multiplication in cells of hrterologous species [31. Earlier reports of partial cross reactivity- for cells of different animal species are probably attributable to impurities in crude preparations [Z]. The effectiveness of monkey interferon against viral infection in human cells may be an exception. The identification of an antiviral substance as an interferon is fraught with pitfalls implicit. in reliance on bioassay procedures. Some inhibitors that have been loosely called interferons will undoubtedly be reclassified when purification procedures and, hopefully, physicochemical analysis become standardized. At the present time certain biologic criteria must be met before an inhibitor of viral multiplication can be accepted as an interferon [1,?>13]. Two essential conditions are evidence that the putative interferon does not inactivate viruses directlv but acts on host cells of the same species in which the interferon was prepared. One of the most useful properties is the capacity of all interferons to withstand prolonged exposure to an acid en-

Interferonvironment at pH 2. Most unpurified interferons are also relatively heat stable and retain activity for months on storage at 4”~. or in the frozen state. However, mouse interferon, for example, is considerably more heat labile than chick interferon [2.5]. Interferons are nondialyzable and are not readily sedimented by centrifugation at 100,000 g. Another useful criterion is loss of biologic activity on incubation with proteolytic such as trypsin and chymotrypsin, enzymes, compared with resistance to nucleases and lipases. The most difficult problem, perhaps, is to distinguish the antiviral action of interferon from that of residual interfering virus so often present in crude preparations. However, interferon is readily differentiated from viral protein by demonstrating that antiviral antibody does not influence its action [13]. The expectation that interferon is itself antigenic was not confirmed in early experiments, undoubtedly because the antigenic mass in available preparations was too small to invoke an antibody response on injection into a heterologous host. Failure to induce antibody in a homologous host would not be surprising, of course, owing to possible immunologic tolerance to interferon as a normally occurring cellular protein, Paucker and Cantell [26] finally succeeded in demonstrating anti-interferon antibody in the serum of guinea pigs given injections of mouse interferon. Moreover, Paucker [27] has recently shown that antibody produced by immunization with mouse interferon inhibits the biologic action only of mouse interferon and not interferons produced by cells of other animal species. These experiments appear to provide additional proof that interferons are specific cellular products, similar in biologic properties, but different in primary or tertiary protein structure. PHYSICOCHEMICAL

PROPERTIES

Until very recently, the major stumbling block for accurate characterization of the nature and action of interferons has been the unavailability of purified preparations. It should be noted, however, that some previous educated guesses based on indirect evidence [1,28] have been largely substantiated by later studies. Lampson et al. [29] reported the first reliable purification procedure, which consisted essentially of acid precipitation to remove extraneous proteins, concentration and partial purification of interferon activity by Zn++ precipitation,

Wagner chromatographic separation on carboxymethylcellulose, and electrophoresis in a pevikon supporting medium. These largely empirical procedures resulted in a 4,500-fold purification of interferon with respect to initial protein concentration of allantoic fluid from chick embryos infected with influenza A virus. In their final product 0.0042 pg. of protein was equivalent to 1 unit of interferon. The molecular weight of interferon was estimated to be in the range of 20,000 to 34,000 based on sedimentation of biologic activity. The protein nature of chick embryo interferon was confirmed, and limited analysis of amino acids revealed a content of 7.3 per cent arginine and 11.1 per cent lysine. Trace amounts of carbohydrate were also found but the purified preparation was devoid of nucleic acids and lipids. As expected from the lysinearginine content, interferon exhibited an isoelectric point of a slightly basic protein. The estimate of molecular weight was confirmed by Kreuz and Levy [30] who studied an identical, but unpurified, interferon by indirect methods. These latter investigators reported the sedimentation coefficient of interferon to be approximately 2.2 to 2.3 Svedberg units as determined by velocity sedimentation in cesium chloride. Independent confirmation of molecular size determination was also demonstrated by Sephadex gel filtration of interferon along with a reference protein, human serum albumin [30]. Most significantly, Kreuz and Levy showed that chick interferon is a homogeneous molecular species with respect to its effective buoyant density, gel diffusion and molecular weight calculated from these independent variables. Similar results were obtained in previous studies of several interferons [2.5,37,32]. Merigan [25] purified chick and mouse interferons approximately 6,000-fold and found that, despite their biologic heterogeneity, both interferons exhibited indistinguishable properties of electrophoretic mobility, elution from carboxymethylsephadex, molecular weight and sedimentation in sucrose density gradients [37]. The only physical properties found thus far to distinguish mouse interferon from chick interferon are heat lability and slight differences in molecular size as determined by Sephadex sieve chromatography [25]. MECHANISMS

Certain interferon

OF ANTIVIRAL

ACTION

deductions as to the mechanism of action could be made from early

AMERICAN

JOURNAL

OF

MEDICINE

Interferon-

??.‘)

observations 1IJ?]. It was deemed unlikely that interferon could act directly on infecting virus because the same preparation was found to inhibit multiplication of many unrelated viruses, including DNA and RNA viruses [ 7
perhaps as little as one t~~olcculc of interferon is sufficient to render one ccl1 resistant to viral

VOL.

38,

MAY

1965

infection. Numerous attempts have been made to detertnine whether interferons alter the Inetabolic functions of uninfected cells as a possible basis for their resistance-promoting activity. The thesis has been advanced, for example, that interferon enhances aerobic glycolysis and acts as an uncoupler of oxidative phosphorylation [ 1,?]. Both of these presumed effects wrr-e found to be due to impurities in crude preparations as demonstrated by failure to reproduce thr obser1X/. \Vell vations with purified interferon documented evidence has also been presented to show that interferon-containing fluid slows the division of L cells in suspended cultures [JO]. However, no effect on mitosis was noted in chick embryo fibroblasts exposed to potent chick interferon [33]. It seems apparent that intcrferon-treated cells retain, or regain [.Jo], their capacity to divide and, in so doing, lost their resistance to viral infection [l,?l. In estimating the duration of interferon action, Wagner [.?/.I found that cells treated with interferon became resistant to viral infection 4 to 5 times more rapidly than they regained susceptibility in interferon-free medium. This is a substantial rate of reversion to susceptibility and suggests that interferon, at least in tnodcrate doses, does not produce profound or pertnanent alteration of uninfected cells. Several investigators have also reported that crude or semipurified preparations of interferon inhibit [J7,12] or enhance [4/l the rate of RNA synthesis in normal uninfected cells. Close scrutiny of these data reveals that the changes are slight, certainly when compared to the effect of interferon on viral RNA synthesis, and probably not significant. Cocito et al. [4.3] could not repeat the observations and point out the dangers of drawing conclusions from results obtained with relatively impure preparations of interferon. These negative or questionable data notwithstanding, there is considerable indirect evidence that the host cell plays a significant role in mediating the antiviral action of interferon. It has been known for some time that continuous lines of malignant cells are generally less susceptible to interferon action than are primary non-neoplastic cell cultures [44]. One interesting exception was reported by Cantell and Paucker [4.5] who found that one of two lines of HeLa (,human epidermoid carcinoma) cells was com-

InterferonTABLE I DIFFERENCES IN MOUSE

SUSCEPTIBILITY

FIBROBLASTS

OF TWO

(L

INTERFERONS

OF TWO

CELLS)

TO THE

PREPARED

CLONES

OF

ANTIVIRAL

IN CULTURES

~-929

ACTION

OF EACH

L

CELLCLONE* I

Interferons Prepared in

1 I

xL cells.. yL cells.

.

/

Interferon XL Cells 16 10

Titers

’

on

yL Cells 192 128

* Monolayer flask cultures of XL and yL cell clones were each infected with Newcastle disease virus at an input ratio of approximately 10 virus particles per cell. After incubation at 37 ‘c. for 24 hours the cell-free media were harvested, centrifuged thrice at 100,000 g for 1 hour, then dialyzed against hydrochloric acid at pH 2 for 24 hours, and redialyzed to neutrality against Earle’s salt solution at pH 7.4. The interferon titers of these fluids were determined by exposing either XL or yL cells in drained plate cultures to 0.2 ml. of serial twofold dilutions of each preparation for 2 hours, following which duplicate plates at each dilution were challenged with 100 plaque-forming units of encephalomyocarditis virus and overlayered with agar. The interferon titers are expressed as the reciprocal of the dilution at which the plaque count was reduced to 50 per cent or less of controls. The results represent the means of two experiments.

parable to primary human amnion cells in their production of and sensitivity to interferon. Nevertheless, the generalization holds in most instances [ 731. Rotem et al. [46] extended these observations by examining changes in the interferon sensitivity of hamster cells transformed with carcinogenic hydrocarbons. They found that clones of transformed (neoplastic) cells were far less susceptible to interferon than were normal hamster cells. Wagner [47] cloned two lines of mouse fibroblast L cells and found that they were equally efficient in producing interferon in response to infection with Newcastle disease virus. However, as shown in Table I, interferon prepared in either cell line was far more effective in suppressing plaque formation by encephalomyocarditis virus in one cell line than in the other. These studies suggest that sensitivity to interferon action is a genetically determined function of individual cells. Comparable results have recently been published by Lockhart [47b], who also showed that different lines of L cells vary in their capacity to produce interferon. Some ingeniously designed experiments by Taylor [48] provide important clues for reconciling these variations in cellular responses and for

Wagner explaining the mechanism of interferon action. She took advantage of the fact that the antibiotic actinomycin D inhibits transcription of messenger RNA on a cellular DNA template without affecting viral RNA synthesis in cells infected with Semliki Forest virus. In the absence of interferon, actinomycin-treated and infected cells incorporated tritium-labeled adenosine into viral RNA and the virus multiplied normally. When cells were first exposed to partially purified interferon for 5 hours, then treated overnight with actinomycin (1 Fg.) and infected with virus, the synthesis of viral RNA and the yield of progeny virus were markedly reduced. However, if the cells were treated with actinomycin before they were exposed to interferon, viral RNA synthesis and viral multiplication were not inhibited by interferon. These data are interpreted as evidence that interferon acts by inducing cellular synthesis of a new messenger RNA which, in turn, presumably codes for the synthesis of a new cellular protein. This interferon-induced protein appears to be the true active component in the inhibition of viral RNA synthesis. Lockhart [49] confirmed Taylor’s results and also showed that actinomycin can reverse the antiviral action of interferon for a period of 2 to 3 hours. Additional weight has been lent to these hypotheses by reports that selective inhibitors of protein synthesis, p-fluorophenylalanine [50] and puromycin [49,57], also block interferon action. The interferon-induced protein that inhibits viral RNA synthesis has not yet been identified, but the circumstantial evidence for its existence seems excellent. Moreover, these studies provide potential explanations for the species specificity of interferons and the variability in response of cells of the same species. BIOSYNTHESIS

OF INTERFERON

The process by which the host produces interferon is crucial to an understanding of its presumed role in tissue resistance and recovery from viral infection. Accumulated data provide overwhelming evidence that the capacity to synthesize interferon is a predetermined latent property of uninfected cells [ 741. The alternative explanation, that the genetic information for interferon synthesis is encoded in viral nucleic acid, is untenable. The earliest studies [7] showed that viral infectivity is not essential for interferon production. Even more conclusive is the fact that completely unrelated viruses can induce AMERICAN

JOURNAL

OF

MEDICINE

Interferon-IVagner the formation in identical cells of interferons that are indistinguishable on the basis of biologic and physical properties [25]. By the same token, a single virus induces the formation of distinguishable interferons in cells of different animal species. Lastly, interferon-like substances are produced in cell cultures or animals by nonviral nucleic acids and bacterial products. The major unanswered questions, therefore, are how cells make interferon and what role viruses play in inducing them to do so. Two outstanding possibilities must be considered: (1) that interferons exist in all cells as biologically inactive precursors which are activated by viruses or other agents; or (2) that interferons are synthesized de nouo in response to induction by these agents. Owing to technical difficulties, no serious attempts have yet been made to generate interferon in cell-free systems or to study incorporation of labeled amino acids into biologically active material. The problem has been approached experimentally by studying the effect of selective antagonists of cellular nucleic acid and protein synthesis. The antagonist with the greatest value has been actinomycin D because of its capacity to block cellular RNA and, consequently, protein synthesis without inhibiting RNA viruses. Two other major requirements for such studies are accurate methods for assaying interferon and availability of cellvirus systems in which rapid synthesis permits kinetic analysis. Heller [~5_3]has found that Chikungunya virus, a group A arbovirus, induces rapid formation of interferon at a linear rate after a lag period of only some hours. Cells pretreated with actinomycin produce little or no interferon. Wagner [5.3] observed that actinomycin had a similar suppressive effect on interferon formation in chick cells infected with myxoviruses or L cells infected with Eastern equine encephalitis virus. In an extension of these studies 1541, it was noted that actinomycin blocks interferon synthesis in chick or L cells only when it is added to cultures during the first 4 hours after infection with Chikungunya or Newcastle disease viruses, respectively. Almost identical observations have been made by Ho [55] and by Levy et al. [56a] using other virus cell systems. These studies support the contention that interferon production is not under genetic control of viral nucleic acids. The conclusion also seems valid [54] that interferon does not exist in the cell as a preformed precursor but requires formation of a ” 0 I. . 38,

MAY

1965

new messenger RNA from information encoded in cellular DNA. The failure of actinomycin to interrupt interferon synthesis late in viral infection suggests that the bulk of the messenger RNA is formed early and, once formed, is relatively stable. Further evidence that the cell synthesizes interferon de nova has been obtained by demonstrating that puromycin j3%~,5fjb] and p-fluorophenylalanine [566] block interferon production. Unlike actinomycin, these inhibitors of protein synthesis are effective throughout the period of active interferon synthesis. Other cell poisons have also been studied but their effect on interferon production is more difficult to interpret. Inhibition of cellular DXA synthesis by the halogenated pyrimidine, S-fluoro-2’-deoxyuridine, was not found to influence interferon synthesis, whereas the antibiotic mitomycin C does to a very slight extent [56a,.56b]. These findings suggest that cellular DNA synthesis is not essential for interferon production, although Holmes et al. [.56c] report variable degrees of inhibition of interferon synthesis by L cells exposed to large doses of S-iodo2 ‘-deoxyuridine. However, exposure of cells to ultraviolet light prior to viral infection markedly reduces interferon synthesis without diminishing (or in fact enhancing) viral multiplication [47,57]. Another interesting observation is that cortisone suppresses interferon synthesis [.58] as do a group of carcinogenic hydrocarbons, prominent among them methylcholanthrene, whereas several noncarcinogenic hydrocarbons have no effect [.59]. The androgen A’, 17crmethyltestosterone is also said to inhibit interferon production despite its propensity to increase cellular protein synthesis [60 j. Studies such as these may eventually shed some light on the previously observed capacity of steroids and carcinogens to enhance viral multiplication and to activate latent viral infections. Owing to their obscure mechanism of action, the use of these substances unfortunately sheds little light on the nature of interferon biosynthesis. Among the major unsolved problems is the role played by infecting viruses in inducing interferon synthesis. All that can be said at the present time is that some viruses are better inducers than others. The fact that different viruses can stimulate interferon production in the same cells suggests that the cellular response is not due to recognition of any specific molecular configuration of viral nucleic acid or protein. In this respect interferon induction is un-

Interferonlike synthesis of inducible enzymes stimulated by specific substrates or their analogues acting as derepressors [67]. The nonspecificity of interferon induction is highlighted by the aforementioned effects of nonviral nucleic acids and bacterial polysaccharides. Nevertheless, the analogy of interferon synthesis to that of inducible enzymes [74,54] cannot be dismissed lightly 1551, particularly in view of the requirements for new messenger RNA [54,566]. In this regard it is interesting to note that Kleinschmidt et al. [77] speculate on the possibility that the anionic polysaccharide, present in the penicillium product statolon, may induce interferon synthesis by combining with a normal histone repressor. RELATIONSHIP

OF ENDOGENOUS TO VIRUS

INTERFERON

VIRULENCE

The virulence of a virus, which can be defined only in terms of susceptibility of a specified host, is correlated with the rate and degree of viral multiplication in that host. Soon after its discovery, interferon was suspected of being one possible determinant of virus virulence [I, 131. Although certain virulent viruses can induce interferon production [62], less virulent viruses are more likely to do so and with greater efficiency [73]. The thesis has been advanced in several earlier reviews [ 14,631 that interferon produced endogenously during the course of a viral infection may feed back into the system and inhibit further multiplication of the same virus that induced its formation. For example, multiplication of influenza virus in chick allantoic membrane ceases at the time that interferon can be detected in the same tissue [62]. Also, Newcastle disease virus induces abundant interferon production in cells of animal species that do not support its multiplication, whereas little or no interferon is produced in susceptible chick embryo cells [64] unless the virus is inactivated if environmental conditions, [55]. Moreover, such as temperature [64], are altered, multiplication of infecting virus may be inhibited coincident with increased production of interferon. A particularly important point is emphasized by Gresser and Enders [65] who studied virus virulence and interferon production in mixed populations of human amnion cells. They found that virus-resistant stable amnion cells appeared to produce enough interferon to protect virus-susceptible primary amnion cells in the same culture. These data raise the important question whether tissue

Wugner resistance to infection can be determined by relatively few cells with the specialized function of producing interferon. It should be noted that sensitivity to the action of interferon may also influence virus virulence. The infectivity of Newcastle disease virus, for example, is unaffected by a dose of interferon a thousand times greater than that required to inhibit Eastern equine encephalitis virus 1281. Virus mutants with altered sensitivity to interferon have also been isolated. A relatively avirulent small plaque variant of vesicular stomatitis virus was found to be 4 to 8 times more susceptible to interferon than the parent virulent variant [.Z]. Similar correlations between virulence and susceptibility to interferon action have been made with foot-and-mouth disease [66] and Semliki Forest viruses [67]. Much less impressive are reports of differences in the abilities of virulent and avirulent mutants of the same virus to induce interferon production. Additional evidence for a relationship between viral multiplication and interferon production has been obtained by the use of actinomycin. The rationale behind these experiments is the capacity of actinomycin to inhibit interferon synthesis without directly affecting an inducing RNA virus [.54]. If endogenous interferon prevents multiplication of the infecting virus, exposure of cells to actinomycin should result in enhancement of virus yield. This prediction proved to be correct under limited conditions of abortive viral infection. Heller [52] showed that inhibition by actinomycin of interferon production in chick embryo cells results in a higher yield and longer persistence of Chikungunya virus. Similarly, the extremely limited growth of Eastern equine encephalitis virus in mouse L cells is enhanced some lo- to 300-fold, depending on multiplicity, by prior exposure to actinomytin [53]. However, caution must be exercised in interpreting these data because actinomycin undoubtedly alters other cell functions in addition to those that determine RNA and interferon synthesis. In an attempt to design a more critical experiment, we have recently compared the dose of actinomycin required to cut off interferon synthesis with the dose required to enhance virus yield. The dose-response curves shown in Figure 1 demonstrate that the same amount of actinomycin that inhibits interferon synthesis by L cells infected with Newcastle disease virus also increases the yield of Eastern equine encephalitis virus in identical cultures. AMERICAN

JoURNAL

OF

MEDICINE

Thus far. we have been discussing virulence in terms of the effect that the cell, through its capacity to synthesize interferon, has on the virus. Another way of looking at the problem is is to ask what effect the virus has on the cell. As previously mentioned, chick embryo cells often fail to produce interferon in response to infection with virulent myxoviruses unless the infecting viruses are first inactivated by ultraviolet light [ 7$5]. Furthermore, fully infectious influenza virus can prevent interferon induction by radiated virus of the same type, a phenomenon that Lindenmann [6S] has called inverse interference. Similar observations have been made with parainfluenza 3 virus [9]. Isaacs [64] also found that virulent myxoviruses inhibit interferon synthesis in chick embryo cells doubly infected with avirulent Chikungunya virus. Hermodsson [69] has extended these findings considerably in a well designed series of experiments. He found that Newcastle disease virus multiplies poorly in calf kidney cells and produces abundant amounts of interferon, whereas parainfluenza 3 virus multiplies well, produces no interferon and destroys the cells. If cultures chronically infected with Newcastle disease virus are superinfected with parainfluenza 3 virus, interferon synthesis ceases and the Newcastle disease virus begins to multiply. Similar effects were noted with dual infection by avirulent and virulent strains of parainfluenza 3 virus [9]. We have investigated this phenomenon in somewhat greater detail by testing the effect of hrghly virulent vesicular stomatitis virus on the interferon synthesizing capacity of mouse L cells and Krebs-2 carcinoma cells [47]. Interferon synthesis begins in suspended cultures of either cell type by 3 or 4 hours after infection with Newcastle disease virus. If these infected cells are superinfected with vesicular stomatitis virus within 3 or 4 hours, no interferon is produced. When superinfection with vesicular stomatitis virus is delayed until 6 hours, interferon synthesis continues at a normal rate. From these experiments it is concluded that avirulent Newcastle disease virus switches on the interferonsynthesizing machinery of the cell and virulent vesicular stomatitis virus switches it off. Moreover, the effective period during which vesicular stomatitis virus inhibits interferon synthesis corresponds almost exactly to that of actinomycin. Huang [70] in our laboratory has found that actinomycin and vesicular stomatitis virus VOL.

38,

MAY

1965

8I

i

4-

<;]____;__ i

2

0

.Ol

.033

ACTINOMYCIN

.I DOSE,

.3

“w w

1.0

pg./ml.

FIG. 1. Inhibition of interferon synthesis and corresponding enhancement of viral multiplication as functions of dose of actinomycin D. Interferon production was determined by infecting a series of monolayer cultures of L cells with Newcastle disease virus at a multiplicity of approximately 5. Centrifuged and acidified fuids from duplicate cultures were assayed for interferon activity 24 hours after infection by plaque inhibition of encephalomyocarditis virus. The effect of actinomycin on viral multiplication was determined by infecting identical L cell cultures with Eastern equine encephalitis (EEE) virus at a multiplicity of 0.1. The final yield of virus was measured by plating dilutions of fluids from 24 hour cultures on chick embryo cells and counting the number of plaque-forming units (PFU) 48 hours later. Actinomycin at the indicated doses was added to the culture media at the end of virus adsorption.

both inhibit RNA synthesis of Krebs-2 cells to a comparable degree and at a comparable rate. Earlier studies with enteroviruses suggest that virulence may be associated with the capacity of a virus to turn off RNA synthesis in the cell that it infects [71,72]. As a corollary to this thesis, the possibility should be entertained that inhibition of cellular RNA synthesis by a virulent virus destroys the capacity of that cell to produce interferon. Aurelian and Roizman [73] have recently provided some evidence in support of this hypothesis from studies with a continuous line of dog kidney (DK) cells infected with virulent and avirulent mutants of herpes simplex virus. One mutant, dk+, multiplies in DK cells, inhibits cellular RNA synthesis and fails to induce interferon formation. The other mutant, dk-. does

734

Interferon-

not multiply in DK cells, does not inhibit cellular RNA synthesis for at least 6 hours except when infected at very high multiplicity, and induces interferon production. INTERFERON

AND

VIRAL

DISEASES

The factors that determine the outcome of viral infections in animals (including man) are not well understood. Part of the difficulty stems from the multiplicity of genetic and environmental forces [74] that are difficult to control experimentally or, needless to say, clinically. Even the role of specific circulating antibody is not easy to assess, particularly in acute infections on first exposure to a virus. It seems fair to state that antibody responses to many acute viral infections are often too little and too late to influence the outcome [63,74]. Moreover, viral infections in immunologically incompetent hosts are not necessarily more severe. The spread of infection may be self-limiting, depending once again on virus virulence, despite the fact that the supply of susceptible cells is not readily exhausted. For some years it has seemed likely that intrinsic host resistance is partially determined by local tissue factors, prominent among them being the interferon mechanism [ 73,63,74]. Unfortunately, much of the data from earlier studies on interferon in animals are difficult to interpret and some are contradictory. One problem had been the technical difficulty of detecting interferon in infected mammalian tissues [28]. However, Baron et al. [75] have recently demonstrated respectable amounts of interferon in tissues of mice infected with influenza and other viruses. A number of reports have also suggested that interferon appears in the blood [76], cerebrospinal fluid [77] and pharyngeal washings [78] of man during infection with various viruses. However, only in chick embryos [62] and mice [75] has any close temporal relationship been found between onset of interferon synthesis and recovery from viral infection. Other studies have attempted to implicate interferon as a primary factor in increased resistance to viral infection that occurs with increasing age of the host. Baron and Isaacs [79] presented data which suggest that marked susceptibility of very young chick embryos to influenza virus is related to deficiency in production of interferon and insusceptibility to its action. Heineberg et al. [80] reported that suckling mice produce little interferon and die of infection with Coxsackie B virus, whereas adult

Wugner mice survive, ostensibly because greater amounts of interferon are produced in their tissues. Death of adult mice infected with Coxsackie B virus and stressed by exposure to an environmental temperature of 4’~. has also been attributed to failure to produce interferon [87]. However, contrary data can also be cited. Vainio et al. [82] found that more, rather than less, interferon was produced in the brains of genetically susceptible mice infected with West Nile virus than was produced in resistant mice. Suckling mice also die from infection with Sindbis virus, despite the fact that their brains contain far more interferon than do the brains of resistant adult animals [83]. However, in neither of these latter experiments were comparisons made of differences in susceptibility to the action of interferon in brains of susceptible and resistant mice. Another pitfall must be considered in animal experiments designed to investigate the relationship between interferon and virus virulence. Most measurements have been made at one particular time after infection. Failure to find interferon late in the course of infection [75] may be misleading because interferon may be synthesized continuously in small amounts, which are constantly being used up [27]. An important contribution was made recently by Baron and Buckler [84] who had the foresight to look for interferon early in the course of infection. They found that interferon appeared in the serum of mice within an hour after the intravenous injection of Newcastle disease virus. The interferon titers reached extremely high levels by 4 hours and declined thereafter. Similar but less dramatic interferon responses were produced by the injection of Sindbis and vaccinia viruses. In chickens [ 18] and rabbits [19], large amounts of interferon or interferon-like inhibitors also appear in the circulation after the intravenous injection of viruses, bacteria or bacterial endotoxins. In each of these experiments the circulating inhibitor had largely disappeared by 24 hours. The cells responsible for synthesizing these interferons have not been identified, although Kono and Ho [85] have presented some tenuous evidence for implicating the reticuloendothelial system. Among other interesting facets of this problem, the question has been posed of the relationship between interferon and endogenous which have similar properties and pyrogen, appear in the serum at about the same time after injection of endotoxins and viruses [85]. AMERICAN

JOURNAL

OF

MEDICINE

InterferonGresser [86] has also found interferon made by circulating leukocytes of children with measles viremia. The presence of interferon in the dermal crusts of vaccinial lesions of man points to its significance in the healing process [87]. However, all these studies have barely scratched the surface of the central problem of the relationship of interferon to host antiviral defenses. Although most investigators have been justifiably cautious in interpreting their data, it is difficult to escape the conclusion that interferon synthesized endogenously during the course of viral infections represents one mechanism that promotes recovery from those infections [74]. Not unexpectedly, attempts have also been made to determine whether exogenous interferon has any therapeutic potentialities. The rationale behind this approach is that endogenous host production may be insufficient to inhibit viral infection or that certain virulent viruses may actually prevent interferon synthesis. If anything, therapeutic trials may be even more difficult to design and execute than studies of endogenous interferon in natural resistance of man. Injection of massive doses to animals is required even for slight degrees of protection against sensitive challenge viruses [88]. More extensive studies must await the availability of concentrated and purified preparations. However, the problem of mass production of this complex protein [29] is a formidable one that is unlikely to be solved in the near future. The one glimmer of hope is the success achieved in a prophylactic trial in man, in which monkey interferon was conclusively shown to inhibit local development of vaccinial lesions [89]. It is my opinion that the only practical application to viral therapeutics in the foreseeable future is the possibility of using interferon-inducing viruses or other substances to bolster natural host defenses. REFERENCES 1. ISAACS, A. and LINDENMANN,J. Virus interference. I. The interferon. II. Some properties of interferon. PTOC.Roy. Sac. London s.B., 147: 285, 268, 1957. 2. SCHLESINGER,R. W. Interference between animal viruses. In : The Viruses : Biochemical, Biological and Biophysical Properties, vol. 3, p. 157. Edited by Burn&, F. M. and Stanley, W. M. New York, 1959. Academic Press, Inc. 3. HENLE, W. Interference phenomena between animal viruses: a review. J. Immunol., 65: 203, 1950. 4. Ho, M. Interferons. New England J. Med., 266: 1258, 1313, 1367, 1962. 5. CHOPPIN, P. W. Multiplication of a myxovirus (SV5) VOL.

38,

MAY

1965

Wagner

6.

7.

8.

9.

10.

11.

12.

13. 14. 15.

16.

17.

18.

19.

20.

21.

22.

23.

24.

735

with minimal cytopathic effects and without interference. Virology, 23: 224, 1964. BALUDA, M. Loss of viral receptors in homologous interference by ultraviolet irradiated Newcastle disease virus. Virology, 7: 315, 1959. CROWELL, R. L. Specific viral interference in HeLa cell cultures chronically infected with Coxsackie B5 virus. J. Bacterial., 86: 517, 1963. JOHNSON,T. C., ANDERSON,D. C. and MCLAREN, L. C. Interference of herpes simplex virus by preinfection of human amnion cells with an attenuated poliovirus. Fed. Proc., 23: 508, 1964. HERMODSSON,S. Action of parainfluenza virus type 3 on synthesis of interferon and multiplication of heterologous virus. Acta path. et microhiol. scandinau., in press. VIL&K, .I. Studies on an interferon from tick-borne encephalitis virus-infected cells. Acta cirol., 6: 144, 1962, PARKMAN,P. D., BUESCHER,E. L. and ARTENSTEIN, M. S. Recovery of rubella virus from army recruits. Proc. Sot. Exper. Biol. & Med., 111 : 225, 1962. SABIN, A. B. et al. Live, orally given poliovirus vaccine. Effects of rapid mass immunization on population under conditions of massive enteric infection with other viruses. J. A. M. A., 173: 1521, 1960. ISAACS, A. Interferon. Ado. Virus Res., 10 : 1, 1963. WAGNER, R. R. The interferons: cellular inhibitors of viral infection. Ann. Rev. Microbial., 17: 285, 1963. ISAACS, A., Cox, R. A. and ROTEM, Z. Foreign nucleic acids as the stimulus to make interferon. Lancet, 2: 113, 1963. HOPPS, H. E., KOHNO, S., KOHNO, M. and SMADEL, J. E. Production of interferon in tissue cultures infected with Rickettsia tsutsugamushi. Burt. Proc., p. 115, 1964. KLEINSCHMIDT,W. J., CLINE, J. C. and MURPHY, E. B. Interferon production induced by statolon. Proc. Nat. Acad. SC. U. S., 52: 741, 1964. YOUNGNER,J. S. and STINEBRING,W. R. Interferon production in chickens injected with Bruce& abortus. Science, 144: 1022, 1964. Ho, M. Interferon-like viral inhibitor in rabbits after intravenous administration of endotoxin. Science, 146: 1472, 1964. STINEBRING,W. R. and YOUNGNER,.J. S. Patterns of interferon appearance in mice injected with bacteria or bacterial endotoxin. Nature, 204: 712, 1964. WAGNER, R. R. Biological studies of interferon. I. Suppression of cellular infection with Eastern equine encephalomyelitis virus. Virolqy, 13: 323. 1961. HERMODSSON,S. and PHILIPSON, L. .4 sensitive method for interferon assay. Proc. Sac. Exper. Biol. & Med., 114: 574, 1963. LINDENMANN,J. and GIFFORD, G. E. Studies on vaccinia virus plaque formation and its inhibition by interferon. I. II. III. Virology, 19: 283, 294, 302, 1963. WAGNER, R. R., LEVY, A. H., SNYDER, R. M., RATCLIFF, G. A. and HYATT, D. F. Biologic properties of two plaque variants of vesicular stomatitis virus. J. Immunol., 91: 112, 1963.

Interferonphysical 25. MERIGAN, T. C. Purified interferons: properties and species specificity. Science, 145 : 811, 1964. 26. PAUCKER, K. and CANTELL, K. Neutralization of interferon by specific antibody. Virology, 18: 145, 1962. 27. PAUCKER,K. Personal communication. 28. WAGNER, R. R. Viral interference. Some considerations of basic mechanisms and their potential relationship to host resistance. Bact. Rev., 24: 151, 1960. 29. LAMPSON,G. P, TYTELL, A. A., NEMES, M. M. and HILLEMAN, M. R. Purification and characterization of chick embryo interferon. Proc. Sot. Exper. Biol. @? Med., 112: 468, 1963. 30. KREUZ. L. E. and LEVY. A. H. Phvsical properties of chick interferon. J. Back, 89 : 462, 1965. _ 31. ROTEM, Z. and CHARLWOOD, P. A. Molecular weights of interferons from different animal species. Nature, 198: 1066, 1963. 32. JUNGWIRTH, C. and BODO, G. Bestimmung des Molekularwichtes von Interferon durch Gelfiltration. B&hem. Ztschr., 339: 382, 1964. 33. WAGNER, R. R. and LEVY, A. H. Interferon as a chemical intermediary in viral interference. Ann. N, Y. Acad. SC., 88: 1308, 1960. 34. MERIGAN, T. C. Personal communication. 35. LOCKHART,R. Z., JR., SREEVALSAN,T. and HORN, B. Inhibition of viral RNA synthesis by interferon. Virologv, 18: 493, 1962. 36. GROSSBERG,S. E. and HOLLAND,J. J. Interferon and viral ribonucleic acid; effect on virus-susceptible and insusceptible cells. J. Zmmunol., 88 : 708, 1962. 37. DE SOMER, P., PRINZIE, A,, DENYS, P. and SCHONNE, E. Mechanism of action of interferon. I. Relationship with viral ribonucleic acid. Virology, 16: 63, 1962. 38. Ho, M. Kinetic considerations of the inhibitory action of an interferon produced in chick cultures infected with Sindbis virus. Virology, I7 : 262, 1962. 39. Ho, M. Effect of an interferon on synthesis of viral ribonucleic acid and plaque formation. Proc. Sot. Exper. Biol. & Med., 112: 511, 1963. 40. PAUCKER,K., CANTELL, K. and HENLE, W. Quantitative studies on viral interference in suspended L cells. III. Effect of interfering virus and interferon on the growth rate of cells. Virology, 17: 324, 1962. 41. LEVY, H. B., SNELLBAKER, L. and BARON, S. Mechanism of action of interferon. Life Sciences, p. 204, 1963. 42. SONNABEND, J. A, An effect of interferon on uninfected chick embryo fibroblasts. Nature, 203: 496, 1964. 43. COCITO, c., DE MAEYER, E. and DE SOMER, P. Synthesis of messenger RNA in neoplastic cells treated in vitro with interferon. Life Sciences, p. 759, 1962, 44. HO, M. and ENDERS, J. F. Further studies on an inhibitor of viral activity appearing in infected cell cultures and its role in chronic viral infections. Virology, 9: 446, 1959. 45. CANTELL, K. and PAUCKER,K. Studies on viral interference in two lines of HeLa cells. Virology, I9 : 81, 1963. 46. ROTEM, Z., BERWALD, Y. and SACHS, L. Inhibition of interferon production in hamster cells trans-

Wagner formed in vitro with carcinogenic hydrocarbons. Virology, 24: 483, 1964. 47. (a) WAGNER, R. R. Unpublished observations. (6) LOCKHART, R. Z., Jr. Variations in the abilities of several lines of L cells to produce and to be affected by interferon. J. Bact., 89: 117, 1965. 48. TAYLOR, J. Inhibition of interferon action by actinomycin. B&hem. &? Biophys. Res. Comm., 14: 447, 1964. 49. LOCKHART, R. Z., JR. The necessity for cellular RNA and protein synthesis for viral inhibition resulting from interferon. Biochem. & Biophys. Res. &mm., 15: 513, 1964. 50. FRIEDMAN,R. M. and SONNABEND,J. A. Inhibition of interferon action by p-fluorophenylalanine. Nature,203: 366, 1964. 51. LEVINE, S. Effect of actinomycin and puromycin dihydrochloride on action of interferon. Virology, 24: 586, 1964. 52. HELLER, E. Enhancement of Chikungunya virus replication and inhibition of interferon production by actinomycin D. Virology, 21: 652, 1963. 53. WAGNER, R. R. Interferon control of viral infection. Trs. A. Am. Physicians, 76: 92, 1963. 54. WAGNER, R. R. Inhibition of interferon biosynthesis by actinomycin D. Nature, 204: 49, 1964. 55. Ho, M. Identification and “induction” of interferon. Bact. Rev., 28: 367, 1964. 56. (a) LEVY, H. B., AXELROD, D. and BARON, S. Personal communication. (6) BURKE, D. C. and BUCHAN, A. Biochem. J., in press, and personal communication. (c) HOLMES, A. W., GILSON, J. and DEINHARDT, F. Inhibition of interferon production by 5-iodo-2 ‘deoxyuridine. Virology, 24: 299, 1964. 57. DE MAEYER-GUIGNARD, J. and DE MAEYER, E. Personal communication. 58. KILBOURNE, E. D., SMART, K. M. and POKORNY, B. A. Inhibition by cortisone of the synthesis and action of interferon. Nature, 190 : 650, 1961. 59. DE MAE~ER, E. and DE MAEYER, .I. Inhibition by 3-methylcholanthrene of interferon formation in rat-embryo cells infected with Sindbis virus. J. Nat. Cam. Inst., 32: 1317, 1964. 60. DE MAEYER, E. and DE MAEYER, J. Two-sided effect of steroids on interferon in tissue culture. Nature, 197: 724, 1963. 61. JACOB, F. and MONOD, J. On the regulation of gene activity. Cold Spring Harbor Symposia Quant. Biol., 26: 193, 1961. 62. WAGNER, R. R. Biological studies of interferon. II. Temporal relationships of virus and interferon production by cells infected with Eastern equine encephalomyelitis and influenza viruses. Virology, 19: 215, 1963. 63. WAGNER, R. R. Cellular resistance to viral infection, with particular reference to endogenous interferon. Bact. Rev., 27: 72, 1963. 64. ISAACS, A. Production and action of interferon. Cold Spring Harbor Symposia Quant. Biol., 27: 343, 1962. 65. GRESSER, I. and ENDERS,J. F. Alteration of cellular resistance to Sindbis virus in mixed cultures of human amnion cells attributable to interferon. Virology, 16 : 428, 1962. 66. SELLERS,R. F. Multiplication, interferon production and sensitivity of virulent and attenuated strains AMERICAN

JOURNAL

or

MEDICINE

Interferon-

67.

68. 69.

70. 71.

72.

73.

74. 75.

76.

77.

78.

of the virus of foot-and-mouth disease. Xature, 198: 1228, 1963. FINTER, N. B. Interferon production and sensitivity of Semliki Forest virus variants. J. Hyg., 62: 337, 1964. LINDENMANN,J. Interferon und inverse Interfcrenz, Ztschr. f. Hyg., 146: 287, 1960. HERMODSSON, S. Inhibition of interferon by an infection with parainfluenza virus type 3. Virology, 20: 333, 1963. HUANG, A. S. Unpublished observations. BALTIMORE, D. and FRANKLIN, R. M. The effect of mengovirus infection on the activity of the DNAdependent RNA polymerase of L-cells. Proc. Nat. Acad. SC. U. S., 48: 1383, 1962. HOLLAND, J. J. Inhibition of host cell macromolecular synthesis by high multiplicities of poliovirus under conditions preventing virus synthesis. J. Molec. Biol., 8: 574, 1964. AURELIAN, L. and ROIZMAN, B. Abortive infection of canine cells by herpes simplex virus. II. The alternative suppression of synthesis of interferon and viral constituents. J. Molec. Biol., in press. BARON, S. Mechanism of recovery from viral infections. Adu. Virus Res., 10: 39, 1963. BARON, S., Du BUY, H., BUCKLER, C. E. and JOHNSON, M. L. Relationship of interferon production to virus growth in Go. Proc. Sac. Exper. Biol. C3 Med., 117: 338, 1964. WHEELOCK, E. F. and DINGLE, J. H. Observations on the repeated administration of viruses to a patient with acute leukemia. l\‘ew England J. Med., 271: 645, 1964. GRESSER, I. and KIARASH, N. Recovery of an interferon-like substance from cerebrospinal fluid. Prod. Sot. Exper. Biol. & Med., 117 : 285, 1964. GRESSER, I. and DULL, H. B. A virus inhibitor in pharyngeal washings from patients with influenza.

VOL.

38,

MAY

1965

Wugm

79.

80.

81. 82.

83.

84.

85.

86.

87.

88.

89.

Aoc. Sot. Enper. Biol. 2 Med.. 1 15 : 192, 1964. BARON, S. and ISAACS, A. Mechanism of recovery from viral infection in the chick embryo. A’ature, 191: 97, 1961. HEINEBERG, H., GOLD, E. and ROBBINS, F. C. Difference in interferon content in tissues of mice of various ages infected with Coxsackie Bl virus. Proc. Sot. Exper. Biol. G? Med., 115: 947, 1964. RUIZ-GOMEZ, J. and SUSA-MARTINE7. J. Personal communication. VAINIO, T., GWATKIN, R. and KOPROWSKI, H. Production of interferon by brains of genetically resistant and susceptible mice infected with West Nile virus. Virology, 14: 385, 1961. VIL&K, J. Production of interferon by newborn and adult mice infected with Sindbis v-irus. Virolqyy, 22: 651, 1964. BARON, S. and BUCKLER, C. E. Circulating interferon in mice after intravenous injection of virus. Science, 141: 1061, 1963. KONO, \i. and Ho, M. The role of the reticuloendothelial system in interferon formation in the rabbit. Virology, 25: 162, 1965. GRESSER, I. Production of interferon by suspensions of human leucocytes. Proc. SOG.Exper. Biol. @ Med., 108: 799, 1961. WHEELOCK, E. F. Interferon in dermal crusts of human vaccinia virus vaccination. Proc. Sac. Exper. Biol. &T Med., 117: 650, 1964. GROSSBERG, S. E., HOOK, E. W. and WAGNER, R. R. Hemorrhagic encephalopathy in chicken embryos infected with influenza virus. III. Viral interference at a distant site induced by prior allantoic infection. J. Zmmunol., 88: 1, 1962. Scientific Committee on Interferon, Report to the Medical Research Council. Effect of interferon on vaccination in volunteers. Lancet, 1: 873, 1962.