Effect of Ammonium Salts on the Interferon-induced Antiviral State in Mouse L Cells

d. gen. Virol. 0978), 4 I, 541-547 Printed in Great Britain 541 Effect of Ammonium Salts on the Interferon-induced Antiviral State in Mouse L Cells By M. J. COMMOY-CHEVALIER, B. ROBERT-GALLIOT A C. CHANY Institut National de la Santd et de la Recherche Mddicale, U.-43 H6pital St Vincent de Paul, 74 avenue Denfert-Rochereau Paris, 75o14, France (Accepted 26 June 1978) SUMMARY The addition of ammonium salts to cells treated with interferon prevents the development of the antiviral state and destroys it when already established. This treatment does not seem to act on the binding of interferon to the cells but blocks a further step of activation on the cell membrane. The anti-interferon effect of ammonium salts is reversible with a complete recovery of the antiviral state. It is postulated that thes(salts may stabilize the interferon-receptor complex and thus prevent the changes in configuration necessary for the establishment and maintenance of its biological functions. INTRODUCTION Previous results have shown that the primary interaction between the interferon molecule and the cell membrane is important in the establishment of the antiviral state and probably in other associated actions of interferon on the cell (Friedman, 1967; Chany et al. 1973; Chany, 1976; Revel et al. 1976). We have postulated that binding of interferon to the cell membrane is a two-step event: first, interferon binds to a non-specific site which probably consists of glycolipids, such as gangliosides (Besan~on & Ankel, 1974; Besangon et al. 1976); and, second, the bound interferon acts on a more specific activator site, probably formed by glycoproteins (Chany et al. 1973). Not much is known about the subsequent intraceuular events which lead finally to the antiviral state. A fortuitous observation revealed that some ammonium salts decrease or abolish the antiviral state induced by interferon. A similar effect of ammonium salts in relation to the action of diphtheria toxin on the cell membrane and, subsequently, on cell metabolism has been reported (Bonventre etal. 1975; Ivins etal. I976; Saelinger etal I976 ). This led us to explore the mechanism by which these salts inhibit interferon action and to draw a parallel between their effect on diphtheria toxin and interferon at the cell membrane level. METHODS Cells and viruses. Mouse L9z 9 and LM cell lines were routinely propagated with Eagle's minimum essential medium (MEM) supplemented with tryptose phosphate broth (2"95 g/l) and IO ~ heat-inactivated newborn calf serum (NCS). For cell maintenance, the serum concentration was reduced to 5 ~. 35-2

542 M.J. COMMOY-CHEVALIER A OTHERS Table I. Effect of different salts on the interferon-#lduced antiviral state* EMC virus yields A Treatment Experiment I Experiment 2 Molarity IF- IF- Salt mm treated treated (NH4)sSO4 4o 20 256-512 (12'5-25)? 64 (t'5) 2o48 4096 ;~ NH4C1 6o I28 (12'5) 1024 256 (6"3) > 4C96 4O 64-128 (3-6) 2048 4-8 Na2SO~ 80 60 2-4 < 2 40 2-4 lo24 < 2 < 2 * After a 5 h treatment of L cells with interferon (IF), the medium was removed and the cells were incubated with different salts for 18 h. The cells were then infected with EMC virus and the virus yield estimated as HA units. t Percentage of control value. :~ = not done. The encephalomyocarditis (EMC) virus, strain MM, was a generous gift from Dr Grossberg and was routinely passaged in L cells. The yield of virus was expressed either by its haemagglutinating (HA) titre with human (O +) red cells or as plaque-forming units (p.f.u.)/o- 5 ml. Interferon production and assay. Mouse interferon was produced in LM cells induced by Newcastle disease virus (V) as described by Chany & Vignal (1968). The preparations were assayed in terms of inhibition of the yield of EMC virus in cultures challenged at a multiplicity of infection (m.o.i.) of IO p.f.u./cell. The virus was removed after I h and the cells were incubated with medium for 8 h at 37 C. The virus was expressed either as HA or p.f.u. The standard error of the titration was measured after IO independent estimations of the same suspension and was + o.i t log for P < o.oi. The preparations used contained 200o to 5ooo mouse research reference units with asp. act. of IO s units/rag protein. Salts. The different neutral salts employed in these experiments were purchased from Merck, and diluted as indicated for each experiment in MEM supplemented with calf serum (5 }/o)- Antiserum and neutralization tests. Interferon-specific antiserum was produced by immunizing a rabbit with purified interferon according to the technique described by Fauconnier (1967). Before use, however, the crude serum was fractionated with (NH~)~SO4 according to Anfinsen et al. (I974). At a dilution of I/3 o, the serum inhibited 2000 reference units of mouse interferon. Trypsin treatment. Twice crystallized trypsin was obtained from Sigma Laboratories, and the pancreatic inhibitor of Kiinitz (Iniprol) was obtained from Choay Laboratories (Paris, France). Radioactive labelling. The effect of NH~C1 on cellular RNA and protein synthesis was estimated as follows: interferon and NH4CI were added to confluent monolayers of L cells and incubated for 5 h at 37 C in the presence of ZH amino-acids (1-amino acid mixture 3H, New England Nuclear) at a concentration of 4 #Ci/ml; or 3H-5-uridine (sp. act. 24 Ci/ mmol, C.E.A., Saclay, France) at a concentration of z #Ci/ml. The cells were washed twice with cold Earle's salt solution, and TCA (IO ~) was added for I h at I C. The precipitate was washed with TCA (5 ~) and dissolved in ammonia. Both the acid-soluble and insoluble fractions were counted in Bray's scintillator fluid.

Interferon action and ammonium salts 543 I I l I I I I i virus 107 O u2 d. "d > lo 6 l0 s I [ I I t I I 0 10 20 30 40 50 60 70 Concentration of NH4C1 (mm) Fig. I. Dose-response effect between increasing concentrations of NH4C1 and the decrease in the interferon-induced antiviral state. NH4CI was added to the cells 5 h after interferon for an 18 h incubation period./--i, virus+ salt; 0---0, IF + salt. RESULTS Effect of different ammonium salts on the interferon-induced antiviral state Different ammonium salts were assayed for their effect on the interferon-induced antiviral state. The experimental procedure was as follows: L cells were incubated for 5 h with 2ooo units of mouse interferon. The interferon was then removed and different salt solutions were added at various concentrations. All the salts were diluted in MEM supplemented with 5 ~ NCS. The same medium was used without the salts in the control series. After 18 h at 37 C, the medium was removed and the cells were challenged with EMC virus. The yield of challenge virus was estimated by the HA titre. Similar results were obtained in terms of infectious virus yield. As shown in Table I, the results suggest that the anti-interferon effect of the salts is related to the NH4 + ion, since the only neutral salt which did not carry the biological activity was the one lacking the ammonium ion, namely Na2SO~. Effect of NH4Cl Dose-response experiments were performed with increasing concentrations of NH4C1 from IO mm to 7O mm. In one set of experiments, interferon and the different concentrations of NH4C1 were added simultaneously to the cells and the mixture was kept for 5 h at 37 C. Then the medium was removed and the antiviral state estimated using EMC virus under the

544 M. J. COMMOY-CHEVALIER A OTHERS Table 2. Effect of IOO mn-nh4c1 on interferon binding to the membrane of L cells* Experiment I 2 3 Salt treatment --~ ~ Tempera- Virus titre (p.f.u. x Io-5/o.5 ml) Time ture ~ ~, (min) ( C) IF-treated NH~CI 15 I 12 2100 15 21 oo NH~C1 15 37 4"9 5"5 82o 7oo NH4C1 120 37 4"7 4"6 1400 I ooo * L cells were treated with NH~CI as shown and then interferon (IF) was added for 30 min at I C. The mixture was removed and the cells were transferred to 37 C for I6 h in presence of MEM+ 5 ~ NCS and then challenged with EMC virus. conditions described in Methods. In parallel sets of experiments, the cells were treated with interferon for 5 h at 37 C. The interferon was then removed and NH4C1 was added at different concentrations for an I8 h incubation period prior to infection with EMC virus. In the case of simultaneous addition of interferon and the salts, the establishment of the antiviral state diminished in proportion to the salt concentration used (data not shown). When the salt was added 5 h after interferon, the already established antiviral state decayed in the same manner (Fig. 0- In order to check for a possible toxic effect of NH4C1 on RNA and protein synthesis, the incorporation of 3H-uridine or ~H-amino-acid was studied. In spite of a small increase of uridine uptake in the acid-soluble fraction, no change in RNA incorporation was observed at the different concentrations used. Protein synthesis decreased slightly with NHaC1, perhaps in part because there was an increase of protein synthesis in the presence of interferon (data not shown). Effect of NH4Cl on interferon binding and activation of the antiviral state Having studied the mechanism by which NH4CI affected interferon action, we then explored the binding of interferon to the cells and the development and decay of the antiviral state. In the absence of pure and labelled interferon preparations, the binding of interferon to the cells can only be estimated by indirect methods. First, we used an approach described by Friedman (~967). The cells were incubated at I C or at 37 C with NH4C1 (Io ram) for I5 min or 2 h. Interferon was added and maintained in contact with the cells for 3 min at I C. The salt and interferon were then removed and the cells washed and transferred to 37 C for I6 h in the presence of MEM supplemented with 5 ~ NCS. After removal of medium, the cells were challenged with EMC virus and the antiviral state estimated as described in the Methods. The results of the three experiments showed that pre-treatment of the cells with NH4C1 for two different time periods and at two temperatures did not modify the establishment of the antiviral state when interferon was added to the cells (Table 2). Another way of exploring indirectly the effect of NH4C1 on interferon binding was also based on experiments previously reported by Friedman 0967). Interferon was bound at

InterJeron action and ammonium salts 545 Table 3. Recovery of response to interferon in cells after removal of NH~Cl* Treatment IF Virus yield (p.f.u. x lo-5/o.5 ml) r A. Challenge virus at 7 h Challenge virus at I6 h z_z. r" r Experiment I Experiment 2 Experiment I Experiment 2 NH4C1- NH4C1- NH4CI- NH4C1- treated treated treated treated 230 4-2 IOO 5-8 I I 3-I 2-7 76oo 8ooo 6ooo 8ooo 6ooo 62oo 34oo 44oo * Interferon (IF) and NH4C1 (50 mm) were added simultaneously to the cells which were incubated for 7 h at 37 C. One half of the cultures was immediately infected after the removal of interferon-salt mixture. With the remainder, the mixture was removed and the cells were incubated with MEM + 5 ~ NCS for a further 16 h before challenge with EMC virus. I C to the cell membrane in the presence or absence of NH4C1. After a 30 rain incubation period, in one set of experiments, the cells were washed with medium. In another set, the cells were trypsinized and both sets were transferred to 37 C. The antiviral state developed in the presence and absence of the salt and disappeared in both cases after trypsinization, Thus, the interferon which bound to the cells in presence of NH4C1 was normally accessible to trypsin (data not shown). It is known that the interferon bound to cells at I C can be neutralized by immune serum specific to interferon (Dianzani & Baron, I977). When such an experiment was done with untreated ceils or with cells treated with NH4CI, the antibody neutralized most of the membrane-bound interferon (unpublished data). Recovery of interferon sensitivity of the cells after removal of NH4Cl When NH~C1 was added simultaneously with interferon to the cells, the development of antiviral protection was arrested. Similarly, when NH~C1 was added 5 h after interferon, the already established antiviral state decayed rapidly. It was thus important to determine whether or not inhibition of development of the interferon effect (and similarly decay of the effect) was reversible. Therefore cells were incubated with interferon and NH4C1 (5o mm) for 7 h. After removal of the mixture, they were challenged with EMC virus. In a parallel set of experiments, after 7 h and the removal of both components, the cells were incubated for a further 16 h in fresh medium with 5 ~ NCS at 37 C, and were then challenged with EMC virus. In the presence of the salt for 7 h, the interferon-induced antiviral state diminished about Ioo-fold, whereas after a further I6 h of incubation, in the absence of NH4C1, the whole of the previous antiviral state was re-established (Table 3)- We can conclude, therefore, that the salt does not induce a significant celluiar injury, and that its effect is reversible. DISCUSSION The data reported here clearly show that ammonium salts modify the cellular response to interferon. It is likely that this modification involves a regulatory step located on the cell membrane. RNA synthesis is not significantly affected in cells treated with both interferon and ammonium chloride, and the slight decrease of protein synthesis which was observed probably cannot explain the lack of development of the antiviral protection. In addition, NH4C1 depresses the established antiviral state after a 5 h incubation period with interferon. As previously shown, blockage of protein synthesis at that stage has the opposite effect of

546 M. J'. COMMOY-CHEVALIER A OTHERS increasing the antiviral protection of the cells, probably by inhibiting a regulatory step leading to its decay (Chany et al. ~971 ; Lab & Koehren, 1976 ). The salt has no detectable effect on the replication of the challenge virus. The action of ammonium salts on interferon-treated cells is very reminiscent of that obtained by Bonventre et ai. 0975) and Saelinger et al. (1976) for diphtheria toxin. At the cell membrane level, they maintain toxin in a state accessible to antitoxin neutralization. Toxin 12.5[ is biologically inactive, provided that NH4C1 is maintained in the medium, in spite of the normal binding of the toxin (cp. in our case, interferon) to the ceils and the unchanged uptake by pinocytosis. Furthermore, after elimination of the salt, the fragment of toxin penetrates into the cell and its full activity is expressed. The mechanism by which NH4C1 exerts its protective effect is not yet certain. Von Hippel & Wong (1964) suggested that ammonium salts could behave like protein stabilizers by elevating the temperature necessary for configurational transition. It is therefore conceivable that ammonium salts stabilize the interferon-receptor complex. This, in turn, might inhibit the conformational change which the interferon (or toxin) molecule has to undergo for activation, thus blocking the interactions between binding and activator sites. It has been shown that cooperation between these two sites is necessary since the free movements of membrane constituents in the plane of the membrane have to be respected in order to obtain the antiviral state. Such free movements can be blocked from the inside by inhibitors of the cytoskeletal system (Bourgeade & Chany, 1976), and from the outside by lectins (Pauloin et al. I977). In both cases the development of antiviral resistance is blocked, or, when already established, rapidly decayed. Although no changes in penetration of amino acids were observed, the slight increase in uridine uptake could indicate that modifications of cell-membrane permeability may occur and could perhaps partially explain the results presented here. In addition, our unpublished data show that changes in the osmolarity of the salt medium are not involved in a decrease of interferon action. Our results point out once more the importance of the functional integrity of the cell membrane for interferon action. They support our previously proposed model of a specific cell membrane bound two component receptor system for interferon (Chany et al. 1973). We are grateful for the skilful assistance of C. Nicoletta and the able secretarial support orb. Le Mire and C. Girard. This work was partly supported by grants from the D.G.R.S.T. and I.N.S.E.R.M. REFERENCES ANFINSEN, C. B., BOSES, S., CORLEY, L. & GURARI-ROTMAN, D. (I974). Partial purification of human interferon by affinity chromatography. Proceedings of the National Academy of Sciences of the United States of America 7 x, 3139-3142. BESANCON, F. & ANKEL, It. (I974). Binding of interferon to gangliosides. Nature, London 252, 478-48o. BESANCON, r., ANGEL, H. & BASO, S. 0976). Specificity and reversibility of interferon ganglioside interaction. Nature, London 259, 576-578. BONVENTRE, ~P. F., SAELINGER, C. B., IVINS, B., WOSCINSKI, C. & AMORINI, M. (1975). Interaction of cultured mammalian cells with (125 I) diphtheria toxin. Infection and Immunity xx, 675-684. BOURGEADE, M.F. & CHANY, C. (1976). Inhibition of interferon action by cytochalasin B, colchicine, and vinblastine. Proceedings of the Society for Experimental Biology and Medicine x53, 5Ol-5O4. IqANY, C. (I976). Membrane-bound interferon specific cell receptor system: role in the establishment and amplification of the antiviral state. Biomedicine 24, I48-157. CHANY, C. & VlGNAL, M. (I968). Etude du m6canisme de l'6tat r6fractaire des cellules 5 la production d'interf6ron, apr~s inductions r6p6t6es. Comptes Rendus Itebdomadaires des Sdances de l'acaddmie des Sciences 267, 1798-I8oo.

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