Interferon action: Role of membrane gangliosides

VIROLOGY 72, 486-493 (1976)

Interferon

Action:

Role of Membrane

Gangliosides

V. E. VENGRIS, F. H. REYNOLDS, JR., M. D. HOLLENBERG,’ AND P. M. PITHA Oncology Center, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21205; and ‘Division of Clinical Pharmacology, Departments of Pharmacology and Medicine, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21205 Accepted March 23,1976 The antiviral activity of human (fibroblast + leukocyte) and mouse fibroblast interferon was neutralized by preincubation with ganglioside before application to cells. Ganglioside mixtures were as effective as pure gangliosides, with no particular ganglioside specificity observed for neutralization of human interferon. Ganglioside-agarose derivatives removed human interferon from solution; interferon was eluted with buffers containing urea, sodium dodecyl sulfate, and mercaptoethanol. Ganglioside-deficient transformed mouse cell lines were relatively insensitive to interferon action. Treatment of such cells with ganglioside led to an increase in membrane ganglioside content and in two of three cell lines to an increase in cell sensitivity to mouse interferon; in mouse SW AL/N and TAL/N cells, GM2, GT,, and a mixture of crude gangliosides were effective whereas GM, and GD,, were without effect. The interferon sensitivity of untransformed cells, which failed to take up appreciable ganglioside, was not increased by treatment with exogenous ganglioside; the sensitivity of one GM,-deticient transformed cell line (K-Balb/C3T3) was not increased by pretreatment with ganglioside. These results indicate an interaction between gangliosides and several types of interferon and suggest a role for membrane gangliosides in the antiviral action of interferon. INTRODUCTION

Although the mechanism by which interferon induces an antiviral state in the cell is not completely understood, the membrane has been implicated as a primary site of interferon action (Friedman, 1967; Berman and Vilcek, 1974; Ankel et al., 1973; Knight, 1974; and Vengris et al., 1975). Recently, it has been observed that mouse interferon binds to gangliosides (Besancon and Ankel, 1974). Since ganglioside GM, can function as a specific membrane receptor to mediate the action of cholera toxin (choleragen) (Cuatrecasas, 1973; Holmgren et al., 1975; van Heyningen, 1974), it is not unreasonable to suggest, in view of the interferon-ganglioside interaction, that endogenous membrane gangliosides may also play a role in * Author to whom reprint requests should be addressed.

the binding and action of interferon. This work explores further the relationship between gangliosides and interferon action in normal human fibroblasts and in ganglioside-deficient transformed mouse embryo fibroblasts (Mora et al ., 1969,197l; Brady and Mora, 1970; Brady and Fishman, 1974). It is demonstrated that ganglioside-agarose derivatives can effectively remove human interferon from solution, and that biologically active interferon can subsequently be eluted from the ganglioside-agarose derivatives. When incubated with ganglioside solutions before application to cells, human interferon is also neutralized. It is further shown that, whilst ganglioside-deficient mouse fibroblasts are markedly insensitive to interferon action, the sensitivity of these cells to the antiviral action of mouse interferon can be partially restored by incorporating exogenous ganglioside into the cell mem486

Copyright 8 1976 by Academic Press, Inc. All rights of reproduction in any form reserved.

INTERFERON

GANGLIOSIDE

brane. These data further implicate a role for membrane gangliosides in the action of interferon. MATERIALS

AND METHODS

Chemicals. Poly(I*C) complex was from P. L. Biochemicals, Inc. (mol wt >2 x lo5 molar extinction coefficient of 4.5 X lo3 at 260 nm). The complex was dissolved in 0.01 M phosphate buffer containing 0.15 M NaCl at a concentration of 10 mglml. A mixture and individually purified gangliosides were obtained from Supelco, Bellefonte, Pa. Both poly(IC) and gangliosides were applied to cells in phosphate buffered saline solution (PBS), 0.02 M sodium phosphate buffer, pH 7.2, and 0.14 M sodium chloride. Cell cultures and virus. Human foreskin Iibroblast cells (HFC) (passages 5-15) and mouse L cells were grown in Eagle’s minimal essential medium (MEM), supplemented with 5% fetal bovine serum (FBS). Balb/c-3T3 (Aaronson and Todaro, 1968) and mouse C243-3 cells were grown in McCoy’s 5a medium, supplemented with 10% FBS. Ganglioside-deficient cell lines, SVSAL/N and TAL/N (passage >200) (Brady and Fishman, 1974), and K Balb/c3T3 cells (Aaronson and Weaver, 1971) were grown in MEM supplemented with 10% FBS. Cloned vesicular stomatitis virus (VSV), New Jersey serotype, was propagated in DEAE-dextran (lOpg/ml) treated (mouse) L cells infected at low multiplicity and harvested 24 hr later, when the titer was 10’ plaque-forming units/ml (PFU/ml). Induction and assay of interferon. Human interferon used (5 x lo4 units/ml) was induced in HFC by poly(1. C) (100 pg/ml) following sequential addition of cycloheximide (50 Fg/ml) and actinomycin D (1 pgl ml) (Vilcek and Havel, 1973; Reynolds and Pitha, 1974). Mouse interferon used (3 x lo4 units/ml) was produced in C243-3 cells (Oie et al., 1972) using a complex of poly(IC) (100 wglml) with DEAE-dextran (30 bglml) as an inducer. Interferon was assayed either colorimetrically (Finter, 1969), employing VSV as a challenge virus, or by the reduction in VSV yield after a single replication cycle.

487

INTERACTION

Interferon activity is given in research reference units, using the NIH standards of human and mouse interferons as reference; one unit of this material (50% reduction in VSV yield) titrated in our assay as 0.3 interferon unit. Interaction of gangliosides with interferon and cells. Interferon and ganglio-

sides (equal volumes) were incubated in MEM or PBS at 37” for 1 hr before application to cells. In order to determine if the binding of gangliosides to the cells affects VSV yield or the induction of interferon by poly(IC), HFC were incubated overnight with a solution of gangliosides in maintenance medium before VSV challenge or poly (IX!) treatment. To measure the effect of ganglioside uptake on the antiviral activity of interferon, cell monolayers were incubated with varying concentrations of ganglioside in PBS for 1 hr at 37”, were washed free of ganglioside solution, and were then incu bated for another 30 min in maintenance medium before treatment with interferon. Affinity chromatography. Interferon was chromatographed on columns (1 x 5 cm) of a ganglioside mixture covalently linked at the sialic acid residue via a poly Glysine spacer arm to agarose (Cuatrecasas et al., 1973). The derivative was a generous gift of Dr. I. Parikh. The content of bound ganglioside was approximately 0.5 mg/ml of gel. Chromatography was done in PBS, and interferon was eluted with PBS containing 6 M urea, 0.1% w/v sodium dodecyl sulfate, and 0.1 M 2-mercaptoethanol. The reductive denaturation of interferon followed by renaturation in the presence of 50% FBS was performed as previously described (Stewart, 1974; Reynolds and Pitha, 1975). Binding

of ‘“51-labeled

cholera

toxin.

Uptake of GM, ganglioside by HFC, Balb/ c-3T3, and SVSAL/N cells was measured indirectly using lz51-labeled cholera toxin as the indicator of GM, incorporation into the membrane (Hollenberg et al., 1974). Cholera toxin (Lot No. 0172) was from Dr. C. E. Miller, SEATO Cholera Research Program. Confluent cell monolayers were incubated with ganglioside GM, (10 @g/ml) for

488

VENGRIS ET AL.

40 min at 37”, and then rinsed and incubated further for 1 hr at 37” with 1 ml of ganglioside-free buffer. The binding of 1251labeled choleragen (6.5 &i/pg) was then measured at 24” in fresh buffer (final vol 470 ~1). Unlabeled choleragen (834 rig/ml) was added 10 min prior to the addition of 1251-labeledcholeragen (2.8 x lo5 cpm in 50 ~1). After equilibrating 40 min at 24”, monolayers were rinsed with ice-cold buffer (4 x 2 ml), solubilized in 0.4 ml of 0.2N NaOH, neutralized with 0.4 ml of 0.2 N HCl, and radioactivity was measured by crystal scintillation counting (efficiency, 85%). The “specific” binding of choleragen was obtained by subtracting the total radioactive uptake from the amount bound in the presence of an excess unlabeled choleragen. RESULTS

Inhibition of Antiviral Activity feron by Gangliosides

of Inter-

When human fibroblast interferon was incubated (1 hr at 37”) with a ganglioside mixture before being applied to cells, the antiviral effect was neutralized (Fig. 1). Neutralization was dependent on the concentration of ganglioside, with halfmaximal neutralization of 10,000 units of interferon at 50 pg/ml, and maximal neutralization (99.9%) at 300 pug/ml of crude ganglioside. The rate of neutralization of human fibroblast interferon by gangliosides was rapid, with maximal inactivation achieved after a 30-min preincubation period (Fig. 2). Human leukocyte interferon (10,000 units) could also be completely neutralized by preincubation with gangliosides (500 pglml). If the ganglioside-interferon mixture was not allowed sufficient time to interact (Fig. 2) or if the mixture was highly diluted with buffer before application to cells (data not shown), the antiviral effect of interferon was not neutralized. The crude ganglioside mixture was as effective as the individual purified gangliosides in neutralizing human fibroblast interferon (Table 1). Preincubation with N-acetyl-neuraminic acid had no effect on the antiviral activity of human interferon.

FIG. 1. The inactivation of human fibroblast interferon by ganglioside. Human fibroblast interferon (10,000 units/ml) and different amounts of gangliosides were preincubated for 1 hr at 37”. The mixtures were then incubated on HFC overnight at 37”. Following washing, cells were infected with VSV at m.o.i. 10. Virus was collected after 15 hr and titered on L cells. O-O, VSV yield with ganglioside alone; O-O, interferon and ganglioside mixture.

MINUTES

OF F+RElNCU0ATlON

FIG. 2. Kinetics of human interferon inactivation by ganglioside. A mixture of human tibroblast interferon (1000 units) and 100 pg/ml of a ganglioside mixture in a total volume of 1 ml were preincubated for various times (30 set to 60 min). The mixture was then applied to HFC and incubated overnight at 37”; cells were then washed and infected with VSV. Virus in the fluids was harvested after 15 hr and titered on L cells.

INTERFERON

GANGLIOSIDE TABLE

INHIBITION

Interferon (units/ml)

0 104 103 102

OF ANTIVIRAL

ACTIVITY

489

INTERACTION

1

OF HUMAN FIBROBLA~T GANGLIOSIDE~

INTERFERON

BY PREINCUBATION

WITH

VSV yieldb (log,,,) Control

N-Acetyl neuraminic acid

Ganglioside mixture

GT,

G&a

GM,

GM?

7.89

7.34 4.30 3.84 4.15

7.38 6.30 7.66 7.15

7.41 6.00 7.15 6.78

7.20 6.34 7.70 7.68

7.17 6.73 7.47 7.15

7.75 6.83 7.83 7.81

4.30 3.83 4.26

’ Interferon, at the concentration shown above, was incubated with 100 pg/ml of ganglioside mixture or isolated ganglioside for 1 hr at 37”, and then incubated with HFC overnight (37”); cells were then washed and infected with VSV and virus in the fluids was assaved 15 hr later. DPFU/ml.

Binding of Human Fibroblast to Ganglioside-Agarose

Interferon

When solutions containing human fibroblast interferon were passed over ganglioside-agarose columns (Cuatrecasas et al., 1973), over 90% of the antiviral activity was retained on the column (Fig. 3). Interferon could be eluted with buffers containing urea. Optimal elution (approx 80% recovery) was obtained with PBS containing 6 M urea, 0.1% w/v SDS, and 0.1 M 2mercaptoethanol (Fig. 3). These concentrations of urea, mercaptoethanol, and SDS, however, inactivate the human interferon. The antiviral activity in the eluate could, therefore, be assayed only after renaturation of interferon in the presence of 50% fetal bovine serum (Reynolds and Pitha, 1975). Treatment of Human Gangliosides

Fibroblasts

with

The antiviral effect of human fibroblast interferon was not affected by pretreatment of HFC with exogenous gangliosides; lo4 units of interferon caused the same decrease in VSV yield (3.5 log) on cells treated (1 hr at 37”) or untreated with gangliosides. Also, the incubation of HFC with gangliosides did not affect either the poly(IC)-stimulated induction of an antiviral state in these cells or the production of interferon. The same interferon titer (1024 U/ml) observed in the absence of gangliosides was obtained in HFC which were incubated with exogenous ganglio-

60 70 n‘0 ; 60

1 0 2

4

6

I 6 IO 12 14 16 16 20 22 24 26 26 30 FRACTION NUMBER

FIG. 3. Chromatography of human fibroblast interferon on columns of ganglioside-agarose. Human tibroblast interferon 100,000units (5 ml) was applied to the column (1 x 5 cm) which was washed with 15 ml of PBS; l-ml fractions were collected. The arrow denotes the addition of buffers containing urea for elution of bound interferon. -------, Elution with 0.5 M urea in PBS (10% recovery); U-0, elution with 6 M urea, 0.1 M 2-mercaptoethanol, and 0.1% SDS in PBS (80% recovery).

sides (100 pg/ml) oyernight and then treated with poly(IC) (100 pg/ml for 1 hr at 37”). The presence of gangliosides (100 pg/ml) in the medium during the time of poly(IC) (100 pg/ml for 1 hr at 37”) induced interferon production was without any effect an the induction of an antiviral state of the

490

VENGRIS ET AL.

producer cells or on the antiviral activity of the produced interferon (1024 units in the presence or absence of gangliosides); these results indicate that, under these conditions, gangliosides are not able to neutralize interferon action. In contrast, preincubation of 10,000 units of human interferon with 100 pg/ml of ganglioside neutralized more than 70% of the antiviral activity (Fig. 1). Treatment of HFC with gangliosides was also without any detectable effect on VSV replication. Cells incubated with different levels of exogenous gangliosides for 24 hr before viral infection were as sensitive to VSV replication as control cells, Furthermore, direct mixing of VSV with gangliosides before infection did not have any effect on virus absorption or replication in HFC (data not shown). Treatment of Ganglioside-Deficient Cells with Gangliosides

Mouse

Transformed mouse cells, which are deficient in some of the more complex gangliosides, were much less sensitive to the antiviral effect of the exogenous mouse interferon than were the nontransformed Balb/c-3T3 cells which contain the normal complement of gangliosides (Table 2). Both the SV40-transformed cells (SVSAL/ N) and spontaneously transformed cells (TAL/N, p > 200) are deficient in ganglioside GM, (Brady and Fishman, 1974). SVSAL/N cells have the lowest sensitivity to mouse interferon; 100 times more interferon was required to reduce VSV replication in these cells than in nontransformed Balb/c-3T3 cells. The Balb/c-3T3 cells transformed by Kirsten strain of MSV which have appreciable amounts of ganglioside GM, but lack GM, (Fishman et al., 1974) were only 25 times less sensitive to interferon than the parent cell line. The incubation of ganglioside-deficient mouse cells with exogenous ganglioside GM, led to the membrane uptake of ganglioside, as indicated by the increase in the cellular binding of 1251-labeledcholeragen (Table 3). Little or no increase in the amount of GM, was detected under similar conditions with untransformed cells. Independent studies have confirmed that the

TABLE 2 INCREASE OF SENSTIVITY OF GANGLKKXDE-DEFICIENT MOUSE CELLS TO MOUSE INTERFERON BY INCUBATION WITH VARIOUS GANGLIOSIDE~

Gangliosides

Interferon* (units/ml) Balbl c-3T3

Mixture GT,, GM,, GD,,, GM,

SVSNAL/ TAL/ N

1 1

K BY

100 12

50 25

25 25

25 25 25

25 25 50

-

GT,, GM, GT, G&t

100

50

-

GM,

100

50

-

25

25

-

GM,

a Cell cultures were preincubated at 37” for 1 hr with gangliosides (10 pg/ml), then washed thoroughly and incubated with interferon overnight; medium was then aspirated, cells were washed and infected with VSV for virus yield assay. The assay of interferon sensitivity was performed by an investigator unaware of the previous treatment of cells with ganglioside. * The lowest concentration of interferon necessary to reduce VSV yield by 50%. TABLE 3 EFFECT OF CELL TREATMENT WITH GANGLIOSIDE GM, ON [‘Z511C~~~~~~~~~ BINDINGS

Cells

Tr&nenet 1

SVS AL/N

-

+ Balb/c 3T3 HFC

+ -

+

[1*511Choleragen boun$fdm) ?

470 " 80 3170f 360 1250 + 340

13702 230 2050k 100 2690 + 360

a Cells were treated with ganglioside GM, (10 Kg/ ml) for 40 min at 37”, washed, and the binding of [‘2511choleragen was determined as described in the Methods. The mean values k SEM of triplicate measurements are given.

uptake of GM, reflects the incorporation of the other gangliosides into transformed cells (Moss et al., 1976). The binding of choleragen reflects only the incorporation of ganglioside into the plasma membrane and not into the cell as a whole. In view of the incorporation of ganglioside into cells, the question arose as to whether the sensitivity of the ganglioside-

INTERFERON

GANGLIOSIDE

deficient cells to interferon could be increased by preincubation with exogenous gangliosides. Cells were, therefore, incubated (1 hr at 37”) either with a mixture of gangliosides or with purified gangliosides before being exposed to interferon. It can be seen in Table 3 that ganglioside treatment (10 kg/ml) increased the sensitivity of the SVS AL/N and TAL/N cells to interferon two- to fourfold. No further increase in the sensitivity of the cells was obtained when the concentration of gangliosides was increased to 100 /*g/ml. The mixture of gangliosides gave the highest increase in sensitivity. In SVS AL/N and TAL/N cells, treatment with gangliosides GT, and GM, increased the sensitivity to interferon, while GD,, and GM, were without effect. K-Balb/c-ST3 cells showed no measurable increase in sensitivity to interferon after treatment with exogenous gangliosides; as with HFC, the interferon sensitivity of Balb/c-3T3 cells, which contain a full complement of gangliosides, was not affected by preincubation with exogenous gangliosides. Under no conditions, however, could treatment with exogenous gangliosides increase the sensitivity of the ganglioside-deficient cells to that of the untransformed cell lines. DISCUSSION

The present study reveals that at least two kinds of human interferon (fibroblast and leukocyte) interact with gangliosides, so as to confirm and extend similar observations with mouse interferon (Besancon and Ankel, 1974). While mouse interferon appears to interact preferentially with ganglioside GM2, no such specificity is observed for human interferon; the terminal galactose residue does not, therefore, contribute significantly to the interaction of gangliosides with human interferon. The facts that the agarose-ganglioside derivatives, which are coupled via the ganglioside sialic acid residue, bind interferon avidly, and that N-acetyl-neuraminic acid itself does not neutralize human interferon indicate that that the sialic acid residue may not participate in the gangliosideinterferon interaction. The contributions of the ceramide residue and the three

INTERACTION

491

sugar residues of ganglioside GM, (glucosyl, galactosyl, and N-acetylgalactosaminyl) remain to be evaluated. The binding of human interferon to immobilized ganglioside is particularly strong; only solutions containing urea caused the elution of antiviral activity. While the contribution of the spacer ligand (poly-Llysine) to the binding was not examined in the present study, it is likely that hydrophobic interactions between gangliosides and interferon are involved, as has been suggested for the concanavalin-A-interferon interaction (Davey et al., 1974). The chromatography of human interferon on columns of immobilized gangliosides may provide a novel approach to its purification. Whereas the present study demonstrates a strong interaction between gangliosides and human interferon, the role played in interferon action by endogenous membrane ganglioside remains an open question. Clearly, in the transformed mouse cell lines lacking the more complex gangliosides, mouse interferon is markedly less active than in untransformed cells. In addition, the incorporation of gangliosides into SVS AL/N and TAL/N cells increases the cell sensitivity to mouse interferon; no increase in interferon sensitivity on exposure to ganglioside was detected in those cells which failed to take up ganglioside, as indicated by increased choleragen-binding. It may be particularly significant in this regard that GMp, which is thought to interact preferentially with mouse interferon (Besancon and Ankel, 1974), does increase the sensitivity of mouse cells (SVS AL/N and TAL/N) to interferon. Nonetheless, two observations in the present work suggest that the gangliosides so far examined may not function as the only part of endogenous interferon receptors. First, the presence of ganglioside in the medium at a concentration which neutralizes interferon by preincubation, did not prevent a poly(IC)-induced antiviral state in human cells producing interferon; anti-interferon antibody does block interferon action on interferon-producing cells in similar experiments (Vengris et al., 1975). Second, the lack of spe-

492

VENGRIS ET AL.

ties specificity in the ganglioside-interferon interaction indicates that the species specificity of interferon, if determined on the cell surface (Chany et al., 1973) has to be governed by components other than gangliosides. Thus, while the present work shows that several kinds of interferon can interact with gangliosides, further work is warranted to determine if membrane gangliosides play a regulatory role in interferon action. The use of transformed cell lines with different cell surface glycolipids (Hakomori, 1975) will prove of value in such studies. ACKNOWLEDGMENTS We want to thank Dr. A. H. Owens, Jr., for continuous interest and encouragements, and Dr. I. Parikh for the gift of agarose-immobilized gangliosides. This work was supported by grants from National Institutes of Health (AI 10944-03) and the American Cancer Society, Maryland Division (7411). M.D.H. is an investigator of the Howard Hughes Medical Institute, and P.M.P. is a Leukemia Society Scholar. REFERENCES AARONSON,S. A., and TODARO,G. J. (1968). Development of 3T3-like lines from Balb/c mouse embryo cultures: Transformation susceptibility to SV40. J. Cell. Physiol. 72, 141-148. AARONSON, S. A., and WEAVER, C. A. (1971). Characterization of Murine sarcoma virus (Kirsten) transformation of mouse and human cells. J. Gen . Viral. 13, 245-252. ANKEL, H., CHANY, C., GALLIOT, B., CHEVALIER, M. J., and ROBERT M. (1973). Antiviral effect of interferon covalently bound to sepharose. Proc. Nat. Acad. Sci. USA 70, 2360-2363. BERMAN, B., and VILCEK, J. (1974). Cellular binding characteristics of human interferon. Virology 57, 378-386. BESANCON,F., and ANKEL, H. (1974). Binding of interferon to gangliosides. Nature (London) 252, 478-480. BRADY, R. O., and MORA, P. J. (1970). Alteration in ganglioside pattern and synthesis in SV40- and polyoma virus-transformed mouse cell lines. Biochim. Biophys. Actu 218, 308-319. BRADY, R. O., and FISHMAN, P. H. (1974). Biosynthesis of glycolipids in virus-transformed cells. B&him. Biophys. Acta 355, 121-148. CHANY, C., GREGOIRE, A., VIGNAL, M., LEMAITREMENCUW, J., BROWN, P., BESANCON,F., JUAREZ,

H., and CAB~INGENA,R. (1973). Mechanism of in-

terferon uptake in parental and somatic monkeymouse hybrid cells. Proc. Nat. Acad. Sci. USA 70, 557-561. CUATRECASAS,P. (1973). Gangliosides and membrane receptors for cholera toxin. Biochemistry 12, 3558-3566. CUATRECASAS,P., PARIKH, I., and HOLLENBERG, M. D. (19731.Affinity chromatography and structural analysis of Vibrio chof erae enterotoxin-ganglioside agarose and the biological effects of ganglioside-containing soluble polymers. Biochemistry 12, 4253-4264. DAVEY, M. W., HUANG, J. W., SULKOWSKI, E., and CARTER,W. A. (1974). Hydrophobic interaction of human interferon with concanavalin A-agarose. J. Biol. Chem. 249, 6345-6355. FINTER, N. B. (1969). Dye uptake method of assessing viral cytopathogenicity and their application to interferon assays. J. Gen. Vi&. 5, 419-427. FISHMAN, P. H., BFUDY, R. O., BRADLEY, R. M., AARONSON, S. A., and TODARO, G. J. (1974). Absence of a specific ganglioside galactosyltransferase in mouse cells transformed by murine sarcoma virus. Proc. Nat. Acad. Sci. USA 71, 298-301. FRIEDMAN, R. (1967). Interferon binding: The first step in establishing of antiviral activity. Science 156, 1760-1761. HAKOMORI, S. (1975). Structures and organization of cell surface of cell surface glycolipids. Dependency on cell growth and malignant transformation. Biochim. Biophys. Acta 417, 55-89. HOLLENBERG, M. D., FISHMAN, P. H., BENNETT, V., and CUATRECASAS, P. (1974). Cholera toxin and cell growth: Role of membrane gangliosides. Proc. Nat. Acad. Sci. USA 71, 4224-4228. HOLMGREN, J., LONNROTH, I., MANEISON, J. E., and SVENNERHOLM, L. (1975). Interaction of cholera toxin and membrane GM, ganglioside of small intestine. Proc. Nat. Acad. Sci. USA 72, 25202524. KNIGHT, E. (1974). Interferon-sepharose: Induction of the antiviral state. Biochem. Biophys. Res. Commun. 56, 860-864. MORA, P. T., BRADY, R. O., BRADLEY, R. M., and MCFARLAND, V. W. (1969). Gangliosides in DNA virus-transformed and spontaneously transformed tumorigenic mouse cell lines. Proc. Nat. Acad. Sci. USA 63, 1290-1296. MORA, P. T., CUMAR, F. A., and BRADY, R. 0. (1971). A common biochemical change in SV40 and polyoma virus transformed mouse cells coupled to control of cell growth in culture. Virology 46,60-72. Moss, J., FISHMAN, P. H., MANGENIELLO, V. C., VAUGHAN, M., and BRADY, R. 0. (1976). Functional incorporation of gangliosides into intact cells: Induction of choleragen responsiveness. Proc. Nat. Acad. Sci. USA 73, 1034-1037. OIE, H. K., GAZDAR, A. F., BUCKLER, C. E., and BARON, S. (1972). High interferon producing line

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GANGLIOSIDE

of transformed murine cells. J. Gen. Viral. 17, 107-10s. REYNOLDS, F. H., JR., and PITHA, P. M. (1974). Induction of interferon and its messenger RNA in human tibroblasts. Biochem. Biophys. Res. Commun. 59, 1023-1030. REYNOLDS, F. H., JR., and PITHA, P. M. (1975). Molecular weight study of human tibroblast interferon. Biochem. Biophys. Res. Commun. 65, 107112. STEWART, W. E., II (1974). Distinct molecular species of interferons. virology 61, 80-86. STEWART, W. E., II, DECLERCQ, E., and DESOMER, I. P. (1972). Recovery of cell-bound interferon. Virol-

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ogy 10, 707-712. VAN HEYNINGEN, W. E. (1974). Gangliosides as membrane receptors for tetanus toxin, cholera toxin and serotonin. Nature (London) 249, 415417. VENGRIS, V. E., STOLLAR, B. D., and PITHA, P. M. (1975). Interferon externalization by producing cell before induction of antiviral state. Virology 65, 410-417. VILCEK, J., and HAVELL, E. A. (1973). Stabilization of interferon messenger RNA activity by treatment of cells with metabolic inhibitors and lower ing of the incubation temperature. Proc. Nat. Acad. Sci. USA 70, 3909-3913.