malignant transformation in vitro."5 6 The Kilham rat virus induces a lytic cycle BY CHARLES CHANY AND CARLOS BRAILOVSKY

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1 STIMULATING INTERACTION BETWEEN VIRUSES (STIMULONS) BY CHARLES CHANY AND CARLOS BRAILOVSKY INSTITUT DE RECHERCHES SCIENTIFIQUES SUR LE CANCER, CENTRE NATIONAL DE LA RECHERCHE SCIENTIFIQUE, VILLEJUIF, FRANCE, AND GROUPE DE RECHERCHES SUER LES VIRUS, INSTITUT DE LA SANTA ET DE LA RECHERCHE MADICALE, HOPITAL SAINT VINCENT DE PAUL, PARIS, FRANCE Communicated by Robert J. Huebner, October 14, 1966 During previous investigations on the viral autoinhibition phenomenon' it was observed that interferon was detected in the culture medium of viral-infected cells but not in extracts of these cells. It was suggested that viral-infected cells may contain a substance antagonistic to interferon, a substance which, unlike interferon, diffuses poorly into the surrounding culture medium. Further studies to explore this hypothesis were complicated because of the considerable quantities of interferon produced in most of the cell-virus systems described. The recent observation in our laboratory of a "stimulating effect" on one virus by another virus permitted further investigation of this phenomenons 3 A stimulating interaction between viruses can be defined in the following manner: when two or more antigenically related or unrelated viruses infect the same cell in a certain chronological order, the inducing agent stimulates the multiplication of the challenge virus. Stimulating interaction consequently represents a reverse phenomenon of the interference phenomenon as usually understood by virologists. Several hostvirus relationships of this kind have been observed and reported in the literature.20-22, 24, 29 In this report only the "stimulating interaction" between adenovirus 12 and Kilham's rat virus will be presented in detail. It was postulated that this system might prove useful in the study of the hypothetical factor antagonistic to interferon, since neither adenovirus 12 nor K rat virus produce significant amounts of interferon in rat cells.3 Thus the "antagonistic factor" would not be masked by the presence of interferon. In rat cells adenovirus 12 does not induce the production of virions or even structural antigens in detectable quantities, although T antigens are formed and can be detected in the cell by the use of fluorescent antibody techniques.4' Furthermore, some of the cells in these infected cultures undergo malignant transformation in vitro."5 6 The Kilham rat virus induces a lytic cycle in rat embryonic cells with the production of fully infectious particles and hemagglutinin.7 Both adenovirus and rat virus are DNA viruses and both multiply within the cell nucleus. Material and Methods.-Cell culture system: Rat embryonic fibroblasts (REF): Ten-day-old pregnant Wistar rats were obtained from the breeding colony of the Cancer Institute, Villejuif, France. In some instances rats raised in germ-free conditions were obtained through the courtesy of Dr. Saquet from the laboratory of germ-free animals of the CNRS, Gif sur Yvette, France. In order to avoid external contamination, the gravid uterus was placed in a solution containing Formalin, 30%c; Germibac, 250 ml; water, q.s.p., 51. The embryos obtained after incision of the uterus were then trypsinized and the cells cultured in a slightly modified Eagle's medium plus 10% calf serum in 1-liter Roux bottles. The first subculture was employed for all tests. Human embryonic kidney cells (HEK): Human embryonic kidney cells were trypsinized and ('ltivated in 1-liter Roux bottles with 10% calf serum and Eagle's medium. For cell maintenance the concentration of the serum was reduced to 5%. 87

2 88 MICROBIOLOGY: CHANY AND BRAILOVSKY PROC. N. A. S. Virus strains employed: Adenovirus 12 was obtained from the American Type Culture Collection and propagated in HEK cells. Purified preparations of the virus were examined in the electron microscope by Dr. W. Bernhard and appeared free of contaminating viruses such as AAV.8 9 Kilham's rat virus was obtained through the courtesy of Dr. Wallace Rowe and was propagated in REF cells. This strain was purified and studied in the electron microscope by Breese et al.'0 and by Vasquez and Brailovsky.11 This strain appeared to be free of associated agents by electron microscopy. Vaccinia virus and the Indiana strain of vesicular stomatitis virus were both grown in REF and maintained in this laboratory. Viral titrations: The rat virus was titrated by means of a plaque method previously described.12 Primary rat embryo fibroblasts were cultivated in 60-mm Falcon tissue culture Petri dishes. The viral dilutions were incubated for 2 hr with cells. The cells were then washed and overlaid with twice-concentrated Eagle's medium diluted 1:2 with agarose 1, 50%. The Petri dishes were transferred to airtight containers saturated with air + 5% C02, and incubated for 20 min at +40C in order to obtain complete hardening of the agarose overlay. The cultures were transferred to an incubator at 370C for 3 days. On the third day, the cells were stained with a solution of neutral red 1: 10,000 diluted 1: 2 with a twofold concentrated Eagle's medium and incubated for 48 hr. The excess of neutral red was then removed and the plaques were counted. Vaccinia and vesicular stomatitis viruses were titrated using similar plaque techniques. Adenovirus 12 was titrated by tube dilution techniques employing the tissue culture infectious dose 50 end point in HEK cells. There were eight cell cultures for each dilution and the test was read after 8 days. The TCID50 end point was calculated using the Karber method." Interferon preparation: Interferon was prepared using parainfluenza 1 virus (Sendai) at a multiplicity of 1 in REF cells. After one cycle of infection, interferon was separated from infectious virus by ultracentrifugation and treatment at ph 2. The viral inhibitory activity of the preparation was tested in REF cells, using VSV or Sindbis virus and the 50% plaque-inhibition method.'4 Titration of the stimulating effect of adenovirus or of crude stimulon preparations: The stimulating effect was titrated by inoculating the cell cultures with twofold dilutions of an adenovirus preparation and challenging them simultaneously or 24 hr later with 30 PFU of rat virus. Because of the great stability of the rat virus the same viral suspension was used in all experiments. One stimulating unit was established as that dilution which caused a twofold increase in the number of rat virus plaques. Results.-Simultaneous infection in REF with adenovirus 12 and the rat virus increased the virulence of the rat virus for rat embryonic cells. In doubly infected cell cultures the typical CPE of rat virus appeared earlier than in cells infected with the rat virus alone. Cytopathic effect was not observed in rat cells infected with adenovirus 12 alone. Seven experiments demonstrated increased rat virus infectivity and hemagglutinin titers in doubly infected cultures (Table 1). No hemagglutination was observed in cells infected with adenovirus 12 alone under similar conditions. The TABLE 1 STIMULATION OF RAT N IRUS MULTIPLICATION IN REF CELLS SIMULTANEOUSLY INFECTED WITH ADENOVIRUs TYPE 12 K - - K + Adeno 12 Expt. no. PFU HA PFU HA X X X X X X * 1.6 X X 1(07 Titers are expressed as reciprocal of the dilution. * Expt. 7 was performed with the supernatant after ultracentrifugation.

3 V/OL. 57, 1967 MICROBIOLOGY: CHANY AND BRAILOVSKY 89 TABLE 2 STIMULATING EFFECT OF ADENOVIRUS 12 ON PLAQUE PRODUCTION BY RAT VIRUS Expt. no. Days: Number of PFU's Produced on Rat Cells 1 Rat virus + adeno Change of A ; 4 stimul. medium units 1 Rat virus + ex- " tracts from normal HEK cells 2 Rat virus + adeno " ; 4 stimul. units 2 Rat virus + ex- " tracts from normal HEK cells increase in the HA titer was more apparent than the increase in the infectious titer although the latter was also considered significant. Similar results were obtained when rat virus was partially inactivated by UV irradiation. Since enhancement of multiplication of the rat virus occurred regularly when REF cells were infected simultaneously with adenovirus 12 and the rat virus, it was of interest to determine whether similar enhancement might occur if the rat virus were inoculated after prior infection with the adenovirus. Table 2 indicates that a significant increase in the formation of plaques occurred when the rat virus was inoculated at different intervals, even nine days after infection of the cells by adenovirus 12; therefore, it seemed likely that the enhancing properties observed were not related to a structural antigen of the adenovirus since such antigens are not present in the rat cells after 24 hours.5 Similar effects were not observed in control cell cultures treated with normal cell culture medium or with extracts from uninfected HEK cells. Role of adenovirus 12 multiplicity of infection and its stimulating activity: When the multiplicity of infection of adenovirus 12 was greater than 1, the stimulating activity was markedly reduced or even not detected, whereas enhancement appeared as the adenovirus 12 was diluted (Fig. 1). Detailed analysis of a number of dose-response curves demonstrated the following three types of relationships: (a) As reported previously,3 a linear relationship was observed between the dilution of the inoculum and the stimulating effect of the virus. (b) In most of the experiments the enhancing effect was preceded by a zone in which the stimulating effect was often completely inhibited-in this respect closely resembling the autoinhibition effect of viral multiplication previously described.' (c) Further analysis of this autoinhibitory zone showed that in many instances it contained a low peak of stimulating activity which was located close to the second peak of stimulation (Fig. 1). It would therefore appear that crude adenovirus preparations possess two different stimulating factors together with interferon or other inhibitory substances. The interaction of these different biologic activities may influence the outcome of viral infections. Enhancing effect of infected cell extract on the rat virus: Adenovirus 12 was inoculated in HEK cells at a multiplicity of 1. After 24 hours of incubation the medium

4 go MICROBIOLOGY: CHANY AND BRAILOVSKY PROC. N. A. S. NO PFU NS PfU AA Z0. A~~~~~~ 20 CONTROL CONTROL 0 STIMULON 0. ~ ~~~~ 41b A26i e al 46 32daek izs56 DIL/2 FIG. 2.-Enhancing effect of the supernatant and sediment of adenovirus 12 preparation after FIG. 1.-The dose-response curve of ultracentrifugation. (A) The stimulating effect adenovirus (multiplicity of infection 2 1) of the upper two thirds of the supernatant. (B) on the plaque formation by the rat virus in The stimulating effect of the sediment. (C) The REF. effect of trypsin on the supernatant. was discarded and fresh medium was added. After hours, when cell destruction was almost complete, the preparation was frozen at -20 C. Extractions were performed by grinding frozen cells with sand in a cold room at +40C. During the extraction the preparation was slowly thawed at +4CC and was cleared by centrifugation in a preparative Spinco ultracentrifuge, rotor 40 at 40,000/rpm (120,000 g), for three hours. Figure 2 shows that after ultracentrifugation more stimulating activity was observed in the upper one third of the supernatant (A) than was found in the sediment (B), although 99 per cent of the adenovirus was sedimented. When the supernatant was treated for 30 minutes with 2X-crystallized trypsin (200 'y), the stimulating activity was almost completely destroyed. This finding suggests that enhancement of multiplication of rat virus was not related to a subunit of adenovirus which remained in the supernatant; since antigens A and C have been shown to be resistant to trypsin.15 Furthermore, after treatment of antigen B of adenovirus with trypsin, the capsomers and the hemagglutinin are split, transforming antigen B to antigen C." RNase (100 y) or DNase (100 y) also had no effect on the stimulating activity.3 These data suggest that the stimulating effect may be related to a viral-induced protein present in the cell extract. It should be emphasized that the infected cellular extracts were prepared in HEK cells and were acting in REF cells. Thus the cell species do not appear to be an important factor in the enhancing activity of the preparation. We have proposed that these viral-dependent proteins with viralstimulating effects be called "stimulons," since this designation does not imply a definite mode of action.8 Possible relationship between interferon and stimulon activities: In additional experiments similar results were obtained using the upper two thirds of the supernatants. In these preparations, approximately 99 per cent of infectious adenovirions were

5 VOL 57, 1967 MICROBIOLOGY: CHANY AND BRAILOVSKY 91 NO PFU NO PFU 50-~~~~~~~~~~8 STIMUSON '70-30 INTERFERON STIMULON It dxi U O /^X CONTROL sr - ~ _/ ~ ~ ~ ~ 4 / \ 20- /40 INTERFERON a STIMULON INTERFERON , IF_ - CONTROL 4 b 16 d2 d4 1b 2M6 OIL2 2 e l32 64 % ~8 DIL/2 FIG. 3.-The effect of eight units of stimulon on interferon activity. 010 FIG. 4.-The effect of four units of interferon on stimulating activity. eliminated. We concluded that the small amount of adenovirus present in these preparations could not significantly influence the results since it has been shown that the virions do not multiply in rat cells and they are resistant to trypsin, while the stimulating factor (stimulon) is sensitive to it. The supernatants used in the experiments described below will be referred to as "crude stimulon" preparations. (a) The effect of crude stimulon preparations on the production of interferon: Since the rat virus does not produce interferon in detectable amounts, the effect of stimulon on interferon production was assayed using Sendai virus as the inducing virus. No detectable effect of stimulon preparations on interferon production was observed. (b) Effect of stimulon on interferon-treated cells: Two series of primary REF cells were treated with serial twofold dilutions of interferon overnight. The cells were then washed in order to eliminate unadsorbed interferon. Eight stimulating units of crude stimulon preparation were then added to one of the two series of interferontreated cells. Both series were then inoculated with 30 PFU of rat virus. Figure 3 shows that in the interferon-treated cells plaque formation with rat virus was inhibited. There was a linear relationship between the dilution and the inhibitory activity of the interferon. In the cells treated with interferon and stimulon, the inhibitory activity of interferon was reversed and the number of plaques counted was close to the control levels. In other experiments the cells were treated with four inhibitory units of interferon and challenged the next day with serial twofold dilutions of stimulon preparations. As controls, cells treated with stimulon alone, with interferon alone, or with normal HEK cell extracts were used (Fig. 4). The cells treated with stimulon showed an early autoinhibitory zone. The number of plaques increased and attained a maximum at dilutions of 1: 8-1 :16, following which they decreased in linear fashion with the dilution. In the interferon- and stimulontreated cells the plaque production of the rat virus showed a curve similar to that of cultures treated with stimulon alone but at a lower level (close to the controls).

6 92 MICROBIOLOGY: CHAN Y AND BRAILOVSKY PROC. N. A. S. In addition, the stimulating peak appeared to be shifted to the right, probably because of inhibitors present in the unpurified stimulon preparations. In other experiments the rat virus used as a challenge virus was replaced by other viruses such as vaccinia, herpes, or vesicular stomatitis viruses. In no instance did stimulon induce a change in the protective effect of interferon. On the contrary, an increase in viral inhibition and interferon production was observed when adenovirus 12 was inoculated simultaneously with vaccinia virus. Discussion.-Viral interference is the result of infection of one cell by two antigenically related or unrelated viruses. In most instances the inducing agent inhibits the multiplication of the challenge virus. This inhibitory type of interference has been extensively studied by many virologists. However, the interference phenomenon does not necessarily imply an inhibitory effect. There are examples of the inducing agent stimulating viral multiplication instead of inhibiting the challenge virus. Occasionally, the same viruses may inhibit or stimulate each other, depending on the cell system used, or on the multiplicity of infection. It has been observed in the course of epidemiological studies that patients infected simultaneously with polioviruses and coxsackie A viruses have a more severe disease (more often paralytic) than patients infected by polioviruses alone.16 The association of adenoviral infection with other viral infections such as influenza or measles is also well known."7 18 Some years ago, during an epidemic of adenovirus pneumonia in children with many severe and fatal cases, we postulated that an associated bacterial or viral infection might explain the unusual severity of the disease, for example, infection by measles virus with adenovirus pneumonia. 18 There are also several examples of lack of inhibition or stimulation occurring in vitro when two or more viruses infect a cell. Parainfluenza 3 virus did not inhibit adenovirus which developed in the nuclei of syncytia induced by the parainfluenza 3 virus.'9 Likewise, O'Conor et al. showed that in cells infected with adenovirus 12 and SV40, both viruses were formed in the nucleus and a stimulating effect on adenovirus 12 was observed in doubly infected cells.20 Kumagai et al. reported that NDV produces little or no cytopathic effect in swine testicular cells. 21 However, when the cells were previously exposed to hog cholera virus, a cytopathic effect due to NDV was observed in these cultures. Utilizing these techniques, the authors were able to assay hog cholera virus.2' IFrothingham reported an increase in the plaque size of Sindbis virus when the cells were reinfected by mumps virus.22 In addition, the yield of Sindbis virus was much greater from these doubly infected cell cultures than from cultures infected only with Sindbisvirus. This enhancing propertyseemed to be closely associated with the infectivity of mumps virus and was clearly distinguishable from mumps viral hemagglutinin or hemolysin. Gledhill observed an enhancement of pathogenicity of mouse hepatitis virus by certain leukemia agents such as the Friend and MIoloney viruses. 23 A potentiating effect of K virus on mouse hepatitis virus was observed by Tisdale in weanling mice. 24 The relationship of the stimulating action of viruses and interferon stems from the observation of Lindenmann that productioni of interferon by killed influenza virus was inhibited by reinfection with infectious influenza virus.2) Furthermore, Hermodsson showed that the autoinhibition of parainfluenza 1 was prevented by a strain of parainfluenza 3 virus (PIV-3), and also that the PIV-3 had a stimulating effect on NDV.26 He therefore postulated that PIV-3 acted by inhibiting the

7 VOL. 57, 1967 MICROBIOLOGY: CHANY AND BRAILOVSKY antiviral action of NDV interferon. In a more recent report he showed that UV irradiation of the bovine strain 23 of parainfluenza 3 virus destroyed that part of the viral genome which is responsible for suppression of interferon synthesis, and consequently an increased production of interferon was obtained. Infection with strain 23 stimulated the multiplication of poliovirus in calf cells infected with poliovirus RNA. Our hypothesis that stimulons are coded by the viral genome and not by the cell is based on the observation that stimulons prepared in human kidney cells were active in rat cells. Thus the factor does not appear to be species-specific. Hermodsson's recent observation that UV-irradiated virus lost its enhancing properties may indicate that UV irradiation also destroyed that part of the viral genome responsible for the synthesis of a stimulonlike substance.26 Vilcek and Stancek reported that cells persistently infected with tick-borne encephalitis virus became resistant to the action of exogenous interferon.27 More recently, Valle and Cantrell observed that Sendai virus increased the plaque formation of VSV. The authors suggested that some VSV particles which usually failed to initiate infection because of their sensitivity to interferon were rescued by Sendai viral infection which blocked the formation of interferon.21 It seems very likely, therefore, that some of these observations may also be explained by the presence of stimulating substances which possess an anti-interferon activity. Thus one may theorize that stimulons are produced in many different cell-virus systems. During viral infection the cell produces a variety of new proteins; some of these are involved in the cellular defense mechanism to the multiplication of the virus and are coded by the cellular genome. The viral genome also induces the synthesis of a variety of proteins, some of which are structural antigens of the virus. Because of the marked effectiveness of the cellular defense mechanism it seemed plausible to speculate that viruses possess the ability to induce factors antagonistic to the interferon system. The known antagonistic factors apparently appear very early during the infectious cycle, and the outcome of the infection may well depend on the chronology of their appearance and on their potency. One may postulate two possible mechanisms of inhibition of the interferon system. (1) As shown by Franklin and Baltimore, when Mengo virus causes cell lysis, it may induce a factor which- blocks the synthesis of cellular messenger RNA, thus blocking all cell-dependent protein synthesisa0 Such a factor was apparently identified in the case of myxoviruses.3' (2) The present studies suggest that viruses also induce stimulon synthesis. There may be several different types of stimulons with similar biological activities. Stimulons may even act when the antiviral polypeptide which has been described by Friedman and Sonnabend32 and by Taylor33 is synthesized by the cell. The proviral and anti-interferon factors appear occasionally during fractionation studies of interferon preparations but have not yet been thoroughly studied. It is, however, also possible that stimulon does not act directly on interferon, but by an entirely different and as yet undetermined mechanism. Aside from its theoretical interest, the phenomenon of the stimulating effects of viruses and of stimulons may be of value in enhancing slowly growing or latent viruses found in in vivo and in vitro systems. Thus studies of human hepatitis virus could perhaps benefit from these observations and from those reported by Tisdale.24

8 9494MICROBIOLOGY: CHANY AND BRAILOVSKY The enhancing effect of leukemia viruses on mouse hepatitis was described by Gledhill"3 and could have its counterpart in the well-known observation that vaccinia or other viral infections exacerbate leukemia in humans.3 4 This could also be the underlying mechanism of the suddon appearance of herpes and varicella during viral and bacterial infections. It has recently been shown by various workers that interferonlike inhibitors can be produced in cells by bacteria, mycoplasma, and rickettsii. The observation by Dick et al. that Eperythrozoon coccoide836 can potentiate mouse hepatitis virus suggests that infectious agents other than viruses may also induce the production of stimulating factors. The authors are indebted to Drs. Andr6 Lwoff, Robert Huebner, and Ion Gresser for many pleasant hours of discussion and for their generous help in the preparation of this manuscript. 1 Chany, C., Virology, 13, (1961). 2 Brailovsky, C., and C. Chany, Compt. Rend., 260, (1965). 3 Chany, C., and C. Brailovsky, Compt. Rend., 261, (1965). 4 Pope, J. H., and W. P. Rowe, J. Exptl. Med., 120, (1964). 5 Levinthal, J. D., C. Ahmad-Zadeh, G. Van Hoosier, and J. Trentin, Proc. Soc. Exptl. Biol. Med., 121, (1966). 6 Freeman, A., P. Black, R. Wolford, and R. Huebner, Fed. Proc., 25, 1051 (1966). 7 Kilham, L., and L. Olivier, Virology, 7, (1959). 8 Atchison, R. W., B. C. Casto, and W. McD. Hammon, Science, 149, (1965). 9 Hoggan, M. D., N. R. Blacklow, and W. P. Rowe, these PROCEEDINGS, 55, (1966). 10 Breese, S. S., Jr., A. F. Howatson, and C. Chany, Virology, 24, (1964). 11 Vasquez, C., and C. Brailovsky, Exptl. Mol. Pathol., 4, (1965). 12 Brailovsky, C., Ann. Inst. Pasteur, 110, (1966). 14 Wagner, R. R., Virology, 13, (1961). 13 Karber, G., Arch. Expti. Pathol. Pharmakol., 162, 480 (1931). 15 Valentine, R. C., and H. G. Pereira, J. Mol. Biol., 13, (1965). 16 Melnick, J. L., A. S. Kaplan, E. Zabin, G. Contreras, and N. W. Larkum, J. Exptl. Med., 94, (1951). 17 Chany, C., thesis: "La Pneumopathiea Ad6novirus chezl'enfant," Paris (1957). 18 Chany, C., P. Lepine, M. Lelong, Le Tan Vinh, P. Satge, and G. Virat, Am. J. Hyg., 67, (1958). 19 Chany, C., and K. Cook, Ann. Inst. Pasteur, 98, (1960). 20 O'Conor, T. G., A. S. Rabson, I. K. Berezesky, and F. J. Paul, J. Natl. Cancer Inst., 31, (1963). 21 Kumagai, T., T. Shimizu, S. Ikeda, and M. Matumoto, Arch. Ges. Virus., 14, (1964). 22 Frothingham, T. E., J. Immunol., 94, (1965). 23 Gledhill, A. W., Brit. J. Cancer, 15, (1961). 24 Tisdale, W. A., Proc. Soc. Exptl. Biol. Med., 114, (1963). 25Lindenmann, J., Z. Hyg. Infektionskrankh., 146, (1960). 26 Hermodsson, S., Virology, 20, (1963). 27 Vilcek, J., and D. Stancek, Life Sci., 12, (1963). 28Valle, M., and K. Cantell, Ann. Med. Exptl. Biol. Fenniae, 43, (1965). 29 Kato, N., A. Okada, and F. Ota, Virology, 26, (1965). 30 Franklin, R., and D. Baltimore, Abstract, in Seventeenth Annual Symposium on Fundamental Cancer Research, M. D. Anderson Hospital and Tumor Institute (University of Texas Press, 1963). 31 Isaacs, A., Z. Roten, and K. H. Fantes, Virology, 29, (1966). 32 Friedmann, R., and J. A. Sonnabend, personal communication. 33 Taylor, J., Virology, 25, (1965). PROC. N. 34 Bousser, J., D. Christol, J. Quichaud, Presse Med., 86, (1955). 36 Dick, G. W. A., and J. S. F. Niven, and A. W. Gledhill, Brit. J. Exptl. Pathol., 37, 90 (1956).