Viral and Nonviral Agentsl

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1 BACTERIOLOGICAL REVIEWS, June 1967, p American Society for Microbiology Vol. 31, No. 2 Printed in U.S.A. Various Molecular Species of Interferon Induced by Viral and Nonviral Agentsl THOMAS C. MERIGAN Division of Infectious Disease, Department of Medicine, Stanford University School of Medicine, Palo Alto, California 9435 INTRODUCTION INDUCERS OF INTERFERON PRODUCTION INTERFERON INDUCED BY TRACHOmA-INa.UsION CONJUNCTIVITIS AGENTS STATOLON-INDUCED INTERFERON.14 Mouse Studies.14 Chicken Studies DISCUSSION AND CONCLUDING R MARR E.141 K LITERATURE CITED. 143 INTRODUCTION Interferon can now be best defined as an antiviral material produced by vertebrate cells which is effective intracellularly against a wide variety of viruses in cells of the species in which it is produced. This activity requires time to act on cells in blocking viral replication intracellularly and seems to be associated with a protein. It is usually stable over a wide range of ph and temperature. The material has not yet been purified adequately for study as a protein. However, indirect methods, involving one electrophoresis, molecular-sieve chromatography, and sucrose gradient ultracentrifugation, have been carried out with appropriate known protein standards to evaluate further the nature of the biological activity. Admittedly, results of such studies await validation by more precise standard protein chemical techniques when adequate amounts of purified materials are available. The physical properties of vertebrate interferons are important because they lead to an understanding of the mechanism of action and role of interferon in the cell-virus relationship. Two years ago, it was reported from this laboratory that the major components of chick, mouse, and human interferon as induced by virus were of similar but distinguishably different molecular weights upon cochromatography on G-1 Sephadex (T. C. Merigan, Bacteriol. Proc., p. 115, 1964). This finding permitted the demonstration that a single virus (Chikungunya) could induce 1 A contribution to the symposium on "Interferon", held at the Annual Meeting of the American Society for Microbiology, Los Angeles, Calif., 2 May 1966, under the sponsorship of the Division of Virology with Warren Stinebring as convener. the production of physically distinguishable types of interferon in two different cell species, namely, chick and mouse (18). This confirmed the thesis, based on studies with metabolic inhibitors such as actinomycin, puromycin, etc. (6, 8, 29), that interferon is produced by the host cell genome upon stimulation by the virus. A given cell species was also noted to produce an identical molecule whether stimulated in vivo or in vitro, independent of the nature of the challenge virus (18). More recently it has been observed that the bulk of virus-induced interferons of chick, mouse, and man have similar molecular charge (19, 22) as determined by electrophoretic mobility on polyacrylamide gels. Both in this laboratory and elsewhere, virus-induced chick interferon has been found to be a covalently bonded structure with a molecular weight of approximately 3, and slightly acidic in charge (16, 22). It appears to have disulfide bridges, although the number of polypeptide chains is unknown (3, 22). Chick interferon has been highly purified in several laboratories, and there is general agreement as to its physical properties (2, 16, 22). It has also been shown that, because of these sie and charge similarities, methods for purification are generally interchangeable among the various vertebrate interferons. Virus-induced mouse and chick interferon have indistinguishable behavior on gradient cochromatography on CM- Sephadex (18). Chick, mouse, and human interferon are eluted from the Amberlite resin, XE-64, over the same ph range whether prepared in vivo or in tissue culture (22). Several laboratories have determined a molecular weight of 2, to 38, for the bulk oi 138

2 VOL. 31,1967 INTERFERON INDUCED BY VIRAL AND NONVIRAL AGENTS 139 virus-induced chick interferon by use of either sucrose density gradient ultracentrifugation or Sephadex G-1 gel filtration (11, 14, 15, 18, 24, 25), and the findings in this laboratory with G-1 cochromatography of chick and mouse interferon were confirmed (4). However, several investigators have recently observed multiple molecular species of interferon stimulated by virus infection. Schonne (27) reported that Sindbis virus in tissue culture stimulated the production of rat interferon ranging in molecular weight from 29, to 1,. Ke, Merigan, and Ho (12), using molecular-sieve chromatography, observed two widely differing molecular species of interferon in a sample of serum drawn from a rabbit injected with Newcastle disease virus (NDV), one species having a molecular weight greater than 1, and the other, 42,. J. V. Hallum, J. S. Youngner, and W. R. Stinebring (Bacteriol. Proc., p. 118, 1966) observed a heavier species of interferon (molecular weight, 47,) in serum obtained early in NDV stimulation of interferon production in the mouse. Lampson et al. (17) reported two slightly differing molecular species in mice injected with NDV (molecular weight, 36, and 23,). INDUCERS OF INTERFERON PRODUCTION A number of agents other than virus are now known to induce circulating interferon. These include both whole microorganisms, i.e., bacteria (31), rickettsiae (H. E. Hopps et al., Bacteriol. Proc., p. 115, 1964), and pleuropneumonia-like organisms (W. Stinebring and J. Youngner, personal communication), and extracts derived from living materials, i.e., bacterial endotoxins (7, 28), phytohemagglutinin (3), cyclohexamide (33), and extracts of yeasts such as statolon (13) and helinine (26). Recently, in this laboratory (3a) and independently elsewhere (25a), an interferon-like activity has been observed in mice infected with the protooan Toxoplasma. Evidence has been presented to indicate that the interferon produced by certain of these agents may be present in a preformed state in the cell and released by the inducer, rather than being produced by de novo synthesis as appears to be the case with virus (9, 33). INTERFERON INDUCED BY TRACHOMA-INCLUSION CONjuNCTIvrrIs AGENTS In collaboration with L. Hanna and E. Jawet of the University of California, a new class of trachoma-inclusion conjunctivitis (TRIC) agents has been recently demonstrated which is capable of inducing interferon (2). These obligate intracellular parasites differ from true viruses in having a cell wall, certain enymes, and both ribonucleic acid (RNA) and deoxyribonucleic acid (DNA) in their structure (1). They are sensitive to the action of antibiotics (sulfonamides, tetracyclines, etc.), and appear to have a step in their life cycle involving binary fission. A highly attenuated TRIC agent, LB1, is an interferon inducer both in vivo and in vitro (2) and is sensitive to the action of virus-induced interferon on tissue culture (5). Other workers (23) were unable to induce interferon production with TRIC agents, probably due to the low number of organisms they employed. Good levels of antiviral activity were obtained in this laboratory with 1' 4 ELD5 per monolayer (containing about 16 cells) and 16.2 ELD5 per mouse. The TRIC interferon had all the usual biological characteristics of interferon, including cell species-specific action, acid stability, and trypsin sensitivity. Another TRIC agent (BOUR) which failed to replicate in cell species did not induce interferon despite the demonstration that it could be adsorbed to the cell (2). However, at the time the LB1-induced interferon is harvested in tissue culture, no new TRIC agent has yet appeared. The peak of serum TRIC-induced interferon production is late (6 to 13 hr; see 5a), similar to that of virus (1, 31), Brucella (31), or statolon (21), in contrast to the early (2-hr) peak appearing after endotoxin (28). To characterie the TRIC interferon further, its molecular weight was determined by cochromatography with chick interferon. It emerged from the column before chick interferon and after bovine serum albumin, and its interpolated molecular weight was approximately 5,. NDV induced a species with a molecular weight of 45, in L cells, which does not appear to differ significantly from that observed by others (31) with NDV (molecular weight, 3,) or the TRIC interferon produced in the same cells. TRIC-induced mouse serum interferon was recently chromatographed in this laboratory and was also found to have a molecular weight of 5, (5a). In studies with G-1 Sephadex column chromatography, the approximate molecular weight of the interferons produced in vitro by phytohemagglutinin (molecular weight, 18,; T. C. Merigan and E. Wheelock, unpublished data) and by Rickettsia tsutsugamushi (molecular weight, 3,; H. Hopps and T. C. Merigan, unpublished data) were found to be similar to the virus-induced interferons produced in the same cells.

3 14 MERIGAN STATOLON-INDUCED INTERFERON Mouse Studies Last year, the interferon induced by statolon was investigated (21); as it appears in the serum of the mouse, its molecular weight is approximately 85,. These observations were confirmed by sucrose density gradient ultracentrifugation, and the heavy interferon was also found to have a significantly different molecular charge compared with virus-induced interferon. Independently, Hallum, Youngner, and Stinebring (Bacteriol. Proc., p. 118, 1966) described a species of interferon (molecular weight, 9,) produced by endotoxin in the mouse. Both in vitro (21) and in the spleen of a mouse injected with statolon (21a), statolon induces the production of a material indistinguishable from virus-induced interferon. Table 1 summaries the findings on the molecular characteristics of statolon-induced interferon as it appears in the serum and splenic extracts of mice injected with statolon and mouse tissue culture exposed to statolon, in comparison with virus interferon as it appears in vivo and in tissue culture. All the characteristics were indistinguishable, except for statolon serum interferon which differed both in molecular charge and weight. TABLE 1. Molecular weight and molecular charge of various mouse interferonsa Electro- Type of interferon Inducing agent Mol wt phoretic Rp BACTERIOL. REV. Recently, studies in this laboratory confirmed the findings of Youngner and Stinebring (32) regarding the lack of effect of cyclohexamide on the serum levels of interferon in the mouse after statolon injection. On the other hand, the appearance of the low molecular weight species of interferon in the spleen of the statolon-injected mouse is significantly inhibited by prior administration of cyclohexamide when it is given in dosages (32) shown to block protein synthesis. It is intriguing that Youngner and Stinebring have also observed that the interferon species (molecular weight, 28,) induced by virus in the circulation of the mouse can be inhibited by cyclohexamide blockade of protein synthesis. Chicken Studies Recently, the interferon induced by statolon in the chicken was studied. An intravenous dose of statolon was found which induced an antiviral activity with all the biological characteristics of chick interferon (species specificity, acid stability, and trypsin sensitivity). A significantly higher dose per unit of body weight was required in the chicken than in the mouse. Figure 1 shows the time course of appearance of the antiviral activity in the serum after intravenous injection of statolon in the chicken as compared with the mouse. In contrast to the mouse, much activity is still present in the chicken 18 hr after injection. The molecular weights of the interferon produced Tissue culture... Chikun- 26,.35 gunga virus Serum. Newcastle 26,l.35 disease virus Tissue culture... Statolon 34,.35 Splenic extract... Statolon 3,.36 Serum... Statolon 85,.63 a Molecular weight was measured by G-1 Sephadex column chromatography (2), and molecular charge, determined in polyacrylamide gels at ph 4.3 with respect to a methyl green marker, as described elsewhere (4). 5Although this may be the predominant molecular species in serum obtained 6 hr after NDV administration, recent more detailed analysis of serum reveals at least two more species after NDV injection, one of 45, and one of 85,, which may even predominate in other serum samples (J. V. Hallum, T. C. Merigan, and J. S. Youngner, in preparation). I a he u Y v r HOURS FiG. 1. Time course of production o serum interferon in the mouse and the chick after statolon injection. Interferon is expressed as the reciprocal of the dilution that yields 5% inhibition of vesicular stomatitis virus plaque formation in L cells and chick embryo fibroblast monolayers. One-year-old, single-comb, white leghorn hens and 25-g HA/ICR mice were employed. The dose of statolon used in the mice was.6 mg/g, and in the chicken, 11.6 mg/kg. I a

4 VOL. 31,1967 INTERFERON INDUCED BY VIRAL AND NONVIRAL AGENTS early and late were therefore compared. Figures 2 and 3 present the results of G-1 Sephadex column chromatography of serum interferons appearing 3 and 18 hr after injection of statolon in the chicken. At 3 hr, all of the activity moves with the heaviest of the serum proteins and, by comparison with standards of known molecular weights, behaves as a protein with a molecular weight of 11,. At 18 hr after injection, the bulk of the interferon is associated with the lightest of the serum proteins (molecular weight, 3, to 4,). The molecular charge of this interferon is also indistinguishable from virusinduced chick interferon. In addition, a lesser amount of the heavier species (molecular weight, 11,) is inconstantly noted in chromatograms of the 18-hr material. Ia Lt EFFUENT VOLUME (5 ML/TUBE) FIG. 2. G-1 Sephadex column chromatography of serum interferon appearing 3 hr after the injection of statolon in the chicken. A 1 by 1.5 cm column of G-1 was used as well as a phenol red internal standard which has been described elsewhere (2). 4 UfL - 21 u I U UZ " : A _ / 8 ' ' Ill / II IJ ~ E EFFUENT VOLUME (5 ML/TUBE) FIG. 3. G-1 Sephadex column chromatography of serum interferon appearing 18 hr after the injection of statolon in the chicken. Conditions same as in Fig. 2 g DiscussioN AND CONCLUDING REMARKs These studies indicate that interferon indistinguishable from virus-induced interferon can be induced by statolon both in vitro in mouse cells and in vivo in the chick and mouse. The other two heavier species which have been observed in the serum of the mouse after injection of statolon and bacterial endotoxin (molecular weight, 9,) and TRIC agents (molecular weight, 5,) resemble the two heavier species of rat interferon (molecular weight, 98,5 and 49,3) produced on Sindbis infection of rat cells in tissue culture (27). The precision of the gel filtration technique allows clear discrimination of the three major molecular species (molecular weight, 9,; 5, to 4,; and 3, to 25,), although each could still contain minor components indistinguishable from the major one. The induction of heavy interferon, followed by the appearance of a lighter interferon species when statolon acts in the chicken, is reminiscent of antigen induction of 19S and 7S a-globulin, but the kinetics of the two processes are very different. The heavy and light interferon species appear and disappear much faster than do the immunoglobulins. Figure 4 demonstrates some of the problems that remain in understanding statolon's relation to interferon. First, the heavier species is released quickly into the circulation from an unknown site, perhaps from either white blood cells or tissue reticuloendothelial cells, where it may be already present in a preformed state prior to statolon injection as suggested by Youngner and Stinebring (32). The lighter species is produced both in tissue culture and in the spleen of mice injected with statolon. If the findings in the chick hold in the mouse, later this lighter species may be released from the spleen and appear in the circulation. Interferon, however, cannot always be demonstrated in the spleens of animals injected with interferon-inducing agents; for example, Rytel et al. (26) failed to show antiviral activity in spleen extracts from mice injected with helinine. The major problem remaining is the interrelationship of these biologically indistinguishable species of interferon. It appears that the animal releases varying quantities of each, probably from different sites, in response to different stimuli. Endotoxin (28) and statolon (21) cause the release of the heavy material early, but probably also from different sites (32). However, statolon also causes the production (in the spleen) and release of the lighter form (21a). Virus, on the other hand, may release a small amount of heavier material early and a large amount of lighter

5 142 MERIGAN BACrERIOL. REV. QUESTIONS ABOUT THE INDUCTION OF INTERFERON BY STATOLON STATOLON Monomer-trimer ) Carrier protein (non-covalently or proven by binding or covalently bound] disaggregation studies Two unrelated proteins l proven by isolation g and amino acid analysis FiG. 4. Statolon induction of interferon. SIMILARITIES Both have similar antiviral spectra in vivo and dose response curves in vitro. Both proteins exert their antiviral action through the cell. Both are induced in vivo by the same agent (statolon]. material later (Hallum et al., Bacteriol. Proc., p. 118, 1966). It is possible that any number of other factors, including the previous passage history of a virus, the precise animal strain employed, or the handling of the specimen (acidification or partial purification), may influence the pattern of interferon species observed in a given sample of serum or body fluid. Whether one interferon species represents a precursor of the others or whether they are related as a monomer-trimer or through a carrier protein cannot be determined at this time. They may even be covalently unrelated proteins. A variety of protein disaggregants have been tried in attempting to cause the heavier material to change into the lighter, so far without success (21). An attempt to change lighter into heavier species has also been made. In both the rabbit and the mouse systems, experiments were designed to demonstrate a binding protein in normal serum. Ke et al. (12) incubated virus-induced, light interferon isolated from a rabbit by gel filtration with normal rabbit serum at 38 C, but, on rechromatography of the mixture, only the light species wase seen. Statolon-induced interferon from mouse spleen extracts (21a), which was known to be light, was also mixed with normal mouse serum. After incubation, G-1 chromatography demonstrated only the single light species. In summary, an attempt has been made in this paper to review the multiple molecular species which are defined as interferon in this laboratory. New findings are presented of the results with statolon injection of the chicken. Here, a heavy interferon (molecular weight, 11,) can be demonstrated at early times after injection, and a lighter species (molecular weight, 3,) is seen later which cannot be distinguished from virusinduced interferon. These studies indicate that the molecular nature of interferon depends on the time after stimulation as well as on the inducer, the animal, and the site in the animal examined. Understanding of the relationship of the several different-sied protein molecules with interferon activity would appear to await further

6 VOL. 31, 1967 INTERFERON INDUCED BY VIRAL AND NONVIRAL AGENTS 143 purification and detailed structural characteriation of these species. ACKNOWLEDGMENTS Some of the work reviewed here has been carried out in collaboration with W. J. Kleinschmidt of Lilly Research Laboratories and with L. Hanna and E. Jawet of the University of California. I am grateful to Gertrude Adler, Constance Winget, and Joan Long for technical assistance. This investigation was supported by Public Health Service grant AI and training grant Al from the National Institute of Allergy and Infectious Diseases. LITERATURE CITED 1. BARON, S., AND C. E. BUCKLER Circulating interferon in mice after intravenous injection of virus. Science 141: FANTES, K. H Further purification of chick interferon. Nature 27: FANTES, K. H., AND C. F. O'NEILL Some similarities between a viral inhibitor of plant origin and chick interferon. Nature 23: a. FRESHMAN, M., T. C. MERIGAN, J. REMINGTON, AND I. BROWNLEE In vitro and in vivo antiviral action of an interferon-like substance induced by Toxoplasma gondii. Proc. Soc. Exptl. Biol. Med. 123: HALLUM, J. V., J. S. YOUNGNER, AND W. R. STINEBRING Interferon activity associated with high molecular weight proteins in the circulation of mice injected with endotoxin or bacteria. Virology 27: HANNA, L., T. MERIGAN, AND E. JAWETZ The inhibition of TRIC agents by virus-induced interferon. Proc. Soc. Exptl. Biol. Med. 122: a. HANNA, L., T. C. MERIGAN, AND E. JAWETZ Effect of interferon on TRIC agents and induction of interferon by TRIC agents. Am. J. Ophthalmol., in press. 6. HELLER, E Enhancement of Chikungunya virus replication and inhibition of interferon production by actinomycin. Virology 21: Ho, M Interferon-like viral inhibitor in rabbits after intravenous administration of endotoxin. Science 146: Ho, M., AND M. K. BREINIG Metabolic determinants of interferon formation. Virology 25: Ho, M., AND Y. KONO Effect of actinomycin D on virus and endotoxin-induced interferon-like inhibitors in rabbits. Proc. Nat). Acad. Sci. U.S. 53: JAWETZ, E Agents of trachoma and inclusion conjunctivitis. Ann. Rev. Microbiol. 18: JUNGWIRTH, C., AND G. BODO Bestimmung des Molekulargewichtes von Interferon durch Gelfitration. Biochem. Z. 339: KE, Y., T. MERIGAN, AND M. Ho Heterogeneity of rabbit serum interferons. Nature 211: KLEINSCHMIDT, W. J., J. C. CLINE, AND E. B. MURPHY Interferon production induced by statolon. Proc. Natl. Acad. Sci. U.S. 52: KREUZ, L. E., AND A. H. LEVY Physical properties of chick interferon. J. Bacteriol. 89: LAMPSON, G. P., A. A. TYTELL, M. M. NEMES, AND M. R. HILLEMAN Purification and characteriation of chick embryo interferon. Proc. Soc. Exptl. Biol. Med. 112: LAMPSON, G. P., A. A. TYTELL, M. M. NEMES, AND M. R. HILLEMAN Characteriation of chick embryo interferon induced by a DNA virus. Proc. Soc. Exptl. Biol. Med. 118: LAMPSON, G. P., A. A. TYTELL, M. M. NEMES, AND M. R. HILLEMAN Multiple molecular species of interferons of mouse and rabbit origin. Proc. Soc. Exptl. Biol. Med. 121: MERIGAN, T. C Purified interferons; physical properties and species specificity. Science 145: MERIGAN, T. C., D. GREGORY, AND J. PETRALLI Physical properties of human interferon prepared in vitro and in vivo. Virology 29: MERIGAN, T. C., AND L. HANNA Characteristics of interferon induced in vitro and in vivo by a TRIC agent. Proc. Soc. Exptl. Biol. Med. 122: MERIGAN, T. C., AND W. J. KLEINSCHMIDT Different molecular species of mouse interferon induced by Statolon. Nature 28: a. MERIGAN, T., AND W. KLEINSCHMIDT A second molecular species of mouse interferon in statolon injectedmice. Nature 212 : MERIGAN, T. C., A. C. WINGET, AND C. B. DixoN Purification and characteriation of vertebrate interferon. J. Mol. Biol. 13: MORDHORST, C. H., AND V. REINICKE TRIC (trachoma-inclusion conjunctivitis) agents and interferon. Acta Pathol. Microbiol. Scand. 65: PHILLIPS, A. W., AND P. D. WOOD Some physical properties of chick interferon. Nature 21: ROTEM, Z., AND P. A. CHARLEWOOD Molecular weights of interferons from different animal species. Nature 198: a. RYTEL, M., AND T. JONES Induction of interferon in mice infected with Toxoplasma gondii. Proc. Soc. Exptl. Biol. Med.123: RYTEL, M. W., R. E. SHOPE, AND E. D. K1L- BOURNE An antiviral substance from Penicillium funiculosum. V. Induction of interferon by Helinine. J. Exptl. Med. 123:

7 144 MERIGAN BACTERIOL. REV. 27. SCHONNE, E Properties of rat tumor interferon. Biochim. Biophys. Acta 115: STINEBRING, W. R., AND J. S. YOUNGNER Patterns of interferon appearance in mice injected with bacteria or bacterial endotoxin. Nature 24: WAGNER, R Inhibition of interferon biosynthesis by actinomycin D. Nature 24: WHEELOCK, E. F Interferon-like virusinhibitor induced in human leukocytes by phytohemagglutinin. Science 149: YOUNGNER, J. S., AND W. R. STINEBRING Interferon production in chickens injected with Brucella abortus. Science 144: YOUNGNER, J. S., AND W. F. STINEBRING Comparison of interferon production in mice by bacterial endotoxin and Statolon. Virology 29: YOUNGNER, J. S., W. R. STINEBRING, AND S. E. TAUBE Influence of inhibitors of protein synthesis on interferon formation in mice. Virology 27: