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Interferon externalization by producing cell before induction of antiviral state
VIROLOGY
65, 410-417 (1975)
Interferon
Externalization
by Producing Antiviral
V. E. VENGRIS,’
Cell Before
Induction
of
State
B. D. STOLLAR,2
AND
P. M. PITHA.’
The Johns Hopkins University School of Medicine, Oncology Center, Baltimore, Maryland 21205, ’ and, 7hfts University School of Medicine, Department of Biochemistry and Pharmacology, Boston, Massachusetts 02111’ Accepted Janwry
13,1975
The mechanism by which poly(I. C) induces the antiviral state in human fibroblast cells was studied using antisera which were selectively reactive with the inducer [poly(I. C) 1,the product (human fibroblast interferon), and the fibroblast cell surface. Anti-interferon serum completely neutralized the antiviral effect of human fibroblast interferon. Furthermore, when added to the medium of the cells in which interferon synthesis had already been induced by poly(I-C), interferon antiserum prevented the intracellular antiviral state from developing. Antibodies specific to double stranded RNA inhibited the antiviral activity of poly(1.C) whether it was in solution or bound to the cells, but the same antibodies were without effect when added after the induction of the antiviral state was initiated. Treatment with serum directed against the cell surface of human fibroblasts failed to inhibit the antiviral activity of poly (1.C) or of human interferon in these cells. These results indicate that the antiviral effect of poly(1.C) is interferon mediated, and that for the development of the antiviral state, interferon has to interact with the external part of the cellular membrane of the producing cell. INTRODUCTION
1968). This raises another question as to whether the antiviral activity of poly(1. C) is interferon mediated, or if these two phenomena are due to the different mechanisms which could be separated. The availability of antisera capable of reacting specifically with the inducer, the product and the cell should facilitate studies on the mode of antiviral action of poly(1.C). In this work, we describe the use of sera which react specifically with human fibroblast interferon, double stranded (ds) RNA, and the cell surfaces of human fibroblasts. The correlation between interferon induction and the antiviral effect and further implications for the mechanism of interferon action are discussed.
Human cells, exposed to the complex of polyinosinic and polycytidylic acids [poly(I.C)], developed resistance to direct virus challenge and simultaneously released interferon into the medium (Field et al., 1967). The mechanism by which poly(1 .C!) induces the antiviral state and interferon production has not been completely clarified. We have shown that in human cells, poly(1. C) induces the formation of interferon-specific mRNA, which can be translated with fidelity in the heterologous cell system (Reynolds and Pitha, 1974). However, it has not yet been determined whether the induction of the antiviral state and interferon production require the mere contact of poly(1 .C) with the cellular membrane or the uptake of the inducer into the cell (Bausek and Merigan, 1969; Pitha and Pitha, 1973). In several cell systems, poly(1. C) induces an antiviral state without the production of any detectable exogenous interferon (Field et al.,
MATERIALS
410 Copyright 0 1975 by Academic Press, Inc. All rights of reproduction in any form reserved.
AND METHODS
Polynucleotides. Poly(1.C) L. Biochemicals, Inc.) had weight higher than 2 x lo5 extinction coefficient of 4.5 x
complex (P. a molecular and a molar 10s at 260 nm
INTERFERON
AND ANTIVIRAL
in 0.01 M sodium phosphate, pH 7.0. The complex was dissolved in 0.01 M phosphate buffer containing 0.15 M NaCl at concentration 10 mg/ml. One preparation of poly(1. C) was used for all experiments. Cells and virus. Human foreskin diploid fibroblast cells (HFC), passages 9-15 [high interferon producers (Vilcek and Havell, 1973)] and human diploid fibroblast cells (HFC-M) were used in all studies. They were grown in Eagle’s minimal essential medium (MEM) supplemented with 2 mM glutamine and enriched with 5% fetal bovine serum (FBS), and were maintained in the same medium containing 2% FBS. Cells were confluent 6 days after seeding, and most experiments were carried out on 12-day-old cultures. Vesicular stomatitis virus (VSV), New Jersey serotype, was propagated in DEAE-dextran (10 pg/ml) treated (mouse) L cells infected at low multiplicity and harvested 24 hr later when the titer was lo8 plaque forming units (PFU/ml). Assays for antiviral activity. HFC were incubated for various lengths of time with a solution of poly(1.C) (100 pg/ml or 25 &ml) in maintenance medium. Resistance to virus infection and interferon titers were measured calorimetrically, (Finter, 1969) employing VSV as a challenge virus (Pitha et al., 1974). The activity is given in research reference units, using NIH standard of human interferon as a reference. The antiviral effect of poly(1. C) on a single step infection was determined by virus yield at 15 hr postinfection. Antisera to human interferon. Antibodies were induced in 12-wk-old white Leghorn roosters by weekly injections (intravenously via jugular vein) of unpurified interferon (5 ml, 4800 units/ml) produced in HFC by poly(1.C) (Vilcek and Havell, 1973). Beginning on the 60th day after injection, chickens were bled and serum collected for antibody assay. Pooled sera were inactivated by heating at 56°C for 30 min. The neutralization titer of the antisera was proportional to the dilution of the interferon which was assayed; 1/64dilution completely neutralized 12 units of human interferon. Antibody titer is expressed in units; 1 unit is the dilution of antisera
STATE
INDUCTION
411
which is able to neutralize completely 12 units of human interferon; both colometric and virus yield assays produced similar antibody titer. Control sera were prepared by injecting roosters with medium from uninduced cells under identical conditions to those described above. Antibody to HFC interferon prepared in rabbits was a gift of Dr. K. Paucker from the Medical College of Pennsylvania in Philadelphia; 1/3000 dilution of this sera neutralized 12 units of human fibroblast interferon. The y-globulin fraction of sheep antisera to partially purified human leukocyte interferon was a gift of Dr. C. B. Anfinsen of the National Institutes of Health; 1/;00dilution of this fraction completely neutralized 100 units of human leukocyte interferon (Anfinsen et al., 1974), but was without any effect on human fibroblast interferon (Vengris, unpublished observations; Baron, personal communication). Antisera to HFC. Rabbits were immunized by injection of 1 x lo* HFC (in complete Freund’s adjuvant) into the footpad. Two, three, and four weeks later, they received intraperitoneal injections- of the same cell dose without adjuvant. Five weeks after the start of immunization, rabbits were bled and the serum was heat inactivated. Antibody titer was determined using the chromium release (“‘Cr) cytotoxicity test (Wigzell, 1965); 1/64dilutions of this serum increased radioactive chromium release by 35%, as compared to the control serum. Antisera to ds polynucleotides. Rabbits were immunized with poly(A.U)-methylated bovine serum albumin (BSA) complexes, as previously described (Schwartz and Stollar, 1969). RESULTS
Effect of anti-interferon serum. The kinetics of the appearance of interferon and the antiviral state in HFC treated with poly(1 .C) for 15 min are shown in Fig. 1. Interferon was not detected in the medium until 2 hr after poly(1 .C) removal; however, at that time the cells had already reached their complete antiviral state. Previously, we have shown that’ at this time,
412
VENGRIS,
STOLLAR
mRNA specific for human interferon can be detected (Reynolds and Pitha, 1974). Chicken or rabbit antisera to human fibroblast interferon were used to examine the correlation between the antiviral effect of poly(1 .C) and the induction of interferon. After induction, cells were incubated for an addional 6 or 20 hr in maintenance medium containing a sufficient amount of anti-interferon serum to neutralize any interferon produced and released into the medium (Table 1). When poly(I.C)-
FIG. 1. Kinetics of interferon and antiviral state appearance. HFC (3 x lo6 cells) were treated with solution of poly(1. C) (100 &ml) for 15 min at 37°C; poly (1.C) was then removed, cells washed and incubated in maintenance medium. Medium was collected at the indicated times and assayed for interferon, and cells were washed and infected with VW for virus yield assay. (04) VSV yield. (04) Interferon titer. TABLE
AND
PITHA
induced extracellular interferon was neutralized by the presence of antisera to human fibroblast interferon, the antiviral state was not induced. Within the limits described in the next section, this effect was independent of the exposure time to poly(1. C) and the time of incubation of the antisera was HFC; complete inhibition of the antiviral state was observed with exposure time as short as 2 hr after induction with poly(1. C). Sera from nonimmunized animals were without any effect, as was a control serum prepared against medium from uninduced cells. Additionally, the anti-interferon sera did not inhibit virus replication in HFC. The sheep antiserum (y-globulin fraction) to human leukocyte interferon (which partially binds human fibroblast interferon but does not neutralize its antiviral activity), when studied under identical conditions, was without any effect. In addition, the antiserum to human fibroblast interferon did not inhibit the induction of the antiviral state of poly(1 .C) in mouse L cells. For the inhibitory effect to be achieved, the interferon antiserum had to be present in the medium from the time when interferon starts to be released into the medium. Pretreatment of HFC with the anti-human interferon serum for 1 hr before or during exposure of the cells to poly(1 .C) affected neither interferon production nor 1
INHIBITION OF THE ANTIVIRAL ACTIVITY OF POLY(I . C) BY ANTISERUM TO HUMAN FIBROBLAST INTERFERON Poly(1. C)
Incubation
medium
VSV yield (PFU/ml) b
a -
+ + +
MEM Interferon MEM Interferon Serum*
antiserum antiserum
4.0 3.8 8.2 1.2 2.5
x x x x x
10’ 10’ 10’ 10’ 10’
1.5 1.4 6.2 5.6 9.6
x x x x x
Antibody** titer
Interferon** (units/ml)
10’ 10’ 108 10’ lo3
a
0
D
b
<4 t4 96 <4 24
<4 <4 384 <4 384
32 2 -
200 10 -
aHFC(M) were treated with poly(1.C) (100 pglml) for 1 hr at 37°C; poly(1.C) was removed, cells washed and incubated in MEM containing chicken anti-interferon serum for an additional 20 hr. Medium was collected and assayed for interferon and antibody titer; cells were washed and infected with VSV for virus yield assay. b HFC were treated with poly(1 .C) (100 &ml) for 15 min at 37”C, poly(1.C) was removed and cells incubated in MEM containing rabbit anti-interferon serum for 6 hr. * Serum from nonimmunized rabbits or chickens injected with medium from uninduced cells. ** In the medium at the end of the incubation period.
INTERFERON
AND ANTIVIRAL
STATE
413
INDUCTION
with multiple virus hit (2 hr postinfection) will register as an infectious center in 24 hr. Under conditions where the spread of interferon should be minimal [l hr after poly(1 .C) induction, no detectable interferon in the medium (Fig. l)], 88% of the cells (percentage of cells in antiviral state = loo-percentage of infected cells; the number of infectious centers of untreated controls were taken as 100%) were in antiviral state (Table 2). When assayed 6 hr postinduction, when a substantial amount of extracellular interferon was produced (Table l), 98% of the cells was found to be in antiviral state. Thus, under our conditions, a maximum of only 10% of the cells are poor or noninterferon producers. The number of cells producing virus increased substantially when the cells after induction were incubated in the presence of antiinterferon antisera for either 1 hr (64% infected in the presence of antisera and 12% in the controls) or 6 hr (42% infected with antiserum and 2.5% in the controls) postinduction (Table 2). Should the interferon antiserum prevent the establishment of the antiviral state only in the population of cells which are nonproducers, while the antiviral state in the producing cells would not be affected, virus yield in noninduced cells should be ten times higher than in poly(1 .C) induced cells incubated in the presence of anti-interferon antiserum. However, only three- to fourfold reduction
the appearance of the antiviral state (192 units of interferon and 1.4 x lo4 PFU/ml in the presence of anti-interferon serum and 192 units of interferon and 2 x lo4 PFU/ml in the controls). The next question we asked was whether anti-interferon antiserum prevented the establishment of the antiviral state of the interferon-producing cells, or whether it only blocked the spread of interferon from the small population of cells producing interferon to nonproducers. It was recently shown that in the cat cells producing high levels of interferon, one-third of the cell population was poor or noninterferon producers (Rodgers and Merigan, 1974). To test the fraction of interferon-producing cells after poly(1. C) treatment in the population of HFC, we measured the number of cells in antiviral state on a single cell basis. The untreated cells and cells exposed to poly(1 .C) (15 min at 37°C) were incubated with medium or with the interferon antiserum either for 1 or 6 h, and then infected with high multiplicity of VSV. Two hours after infection, cells were trypsinized and the number of cells producing virus was measured as infectious centers on L cells in 24 hr. As shown in Table 2, the efficiency of infectious centers formation under these conditions is very low (5%). This is partially caused by low plating efficiency (10%) of human fibroblast cells used (12day-old cultures), and probably only cells TABLE
2
THE ANTIVIRAL EFFECT OF POLY(I C) ASSAYED BY INFECTIOUS CENTERS AT DIFFERENT TIMES POSTINDUCTION’ Poly(1. C)
+ + -
Treatment
0 Interferon 0 Interferon
antiserum antiserum
Number of infectious centersC
Est. p+* 1 hr 10-2.2” lo- 1.49 lo- 1.32 10-L 30
6hr 10-2.92 IO- 1.67 10-1.33 IO- *.a0
1 hr
6hr
28 158 230 250
6 105 237 249
DHFC were incubated in MEM or treated with poly(1. C) (100 &ml) for 15 min; poly(1. C) was removed, cells washed and incubated in MEM or MEM containing rabbit anti-interferon antiserum for an additional 1 or 6 hr. The cells were then infected with WV (m.o.i. 10) and 2 hr later trypsinized; 1000 cells were plated over monolayer of mouse L cells; plaques were read 24 hr later. bEst. p+-the estimated portion of the cells yielding virus: it was calculated by dividing the number of responses (plaques) from five plates by the number of cells plated. c The numbers represent the total amount of plaques in five plates.
414
VENGRIS,
STOLLAR
AND
PITHA
in the virus yield was observed (Table 1). pg poly (1.C) when the reagents were Thus, these data indicated that the abol- preincubated before addition to cells. ishment of the antiviral effect in poly(1. C) When antiserum was added to the cells induced .cells by anti-interferon antisera which were pretreated with poly(1 .C) at was due to the direct inhibition of the 37°C and in which the production of interantiviral state in the interferon producing feron and the antiviral state had already been initiated, no alteration of antiviral cells. However, since only unpurified or par- state was observed. However, when the cells were treated with poly(1 .C) at 4°C tially human interferon (human interferon purified to homogenicity is not available as [where the inducer binds to the cellular yet) was used for immunization, an objec- membrane, but the induction does not tion could be raised that the observed occur (Bausek and Merigan, 1969)], and then with the antiserum, the antiviral effect was due to the presence of antibodies to the inducer or to cell proteins in the effect of poly(1 .C) was again blocked anti-interferon antiserum. To examine this (Table 3). Thus, the antiserum reacted not possibility, we studied the effect of both only with poly(1 .C) in solution, but also antiserum against ds polynucleotides and with the inducer which was bound to the antiserum against HFC on the induction of cellular membrane, and thereby altered the antiviral properties of poly(1. C). interferon and the antiviral state in HFC. Having neutralized poly(1. C) which was Effect of antiserum against polynucleobound to the cells, but which had not tides. Serum from rabbits immunized with reacted with the triggering site for intercomplexes of poly(A. U) and methylated feron induction, we were able to determine BSA reacted specifically with ds polyribonucleotide-containing molecules (Schwartz the minimal period during which poly(1. C) and Stollar, 1969). Poly(I. C), when reacted had to be associated with the cells in order with this antiserum, lost the ability to in- to either initiate antiviral effect or reach a duce both interferon and the antiviral state protective site. When the cells were treated in HFC (Fig. 2). Quantitative equivalence with a solution of poly(1 .C) and then precipitation occurred between 0.1 ml of washed, a 1-min exposure was sufficient to the antisera and 25 pg of poly(1. C), and induce the antiviral state and interferon this amount of antiserum was also able to production (Fig. 3A). This is in aggreement completely abolish the antiviral effect of 25 TABLE
3
EFFECT OF ANTIBODIES TO DOUBLE POLYNUCLEOTIDES ON THE INTERFERON POLY(I. C)”
Treatment
FIG. 2. Inhibition of the antiviral activity of poly(1.C) by antiserum to ds polynucleotides. HFC-M (3 x lo5 cells) were treated with the mixture of poly(1. C) and given volume of the antiserum to ds polynucleotides (preincubated at 37°C for 30 min) for 60 min at 37°C. The mixture was removed, cells were washed and incubated in maintence medium for an additional 20 hr; then interferon activity was assayed in collected medium, cells were washed and infected with VSV for virus yield assay. (0-O) VSV yield. (04) Interferon titer.
Poly(1. C) (37 or 4°C) Poly(1 .C) and antibodies (preincubated) Antibodies Poly(1 .C) (37°C) - antibodies Poly(1 .C) (4°C) 4 antibodies
Interferon (units/ml)
STRANDED INDUCTION
BY
VSV yield (PFU/ml)
192 <4
6.4 x lo3 4.5 x 10’
<4 96
6.0 x 10’ 1.8 x 10’
<4
8.0 x lo6
“HFC were treated with poly(1.C) (25 &ml) for 30 min (37 or 4”C), or poly(I.C), preincubated with 0.1 ml of antibodies for 30 min (37”C), washed and incubated with maintenance medium, or medium containing 0.1 ml of antibodies to ds polynucleotides, for an additional 30 min. Antiviral activity was assayed 20 hr later.
INTERFERON
AND ANTIVIRAL
STATE
415
INDUCTION
Fro. 3. Rate of induction of interferon and the antiviral state by poly(I .C) in human fibroblast cells. HFC-M (3 x lo5 cells) were treated with poly(1.C) (25 pg/ml) at 37°C for given periods of time. The inducer was then washed and cells were incubated in maintenance medium (A), or medium containing 0.1 ml of anti-ds polynucleotide antibodies (B). After 24 hr, interferon and antiviral activity were assayed as described in the Materials and Methods section. (0-O) VSV yield. (O-O) Interferon titer.
with our previous findings (Pitha et al., 1972). However, when poly(1.C) was washed off the cells and then the antiserum to ds polynucleotides was added to neutralize the unreacted poly(1. C) associated with the cellular membrane, the results were different (Fig. 3B). To create the full antiviral state in these cells, a 5-min preexposure to poly(1 .C) was required; the interferon production showed repeatedly a delay which was not observed under previous conditions. These results show that in a very short time, an amount of poly(1. C) sufficient to start, but not complete, the induction of interferon and the antiviral state associates with the cellular membrane. However, the bound inducer needs additional time for full interaction with the actual induction site or for the penetration. Effect of antisera directed against the cell surface of HFC. Antiserum to HFC contained antibodies for surface components of these cells, as shown by membrane immunofluorescence (indirect method using fluorescein labeled goat antirabbit globulin). The antiserum was also cytotoxic to HFC in the presence of guinea pig complement, as measured by isotope release from cells previously labeled with radioactive chromium ( 51Cr). Seventy-five percent of the cells were lysed at a 1:lO dilution. The addition of the antiserum (1:lO dilution) to HFC, before or after treatment with poly(1. C), did not affect its antiviral activity (Table 4). Also, 1:2 dilution of this sera used under identical conditions to those described was without any effect. The antisera to cell surfaces of
competent cells blocked the transforming activity of DNA and helped to demonstrate the existence of specific binding sites on these cells for single stranded DNA (Tomasz and Beiser, 1965). The treatment of HFC with anti-HFC serum, either at 37 or 4°C (not shown), before or simultaneously with interferon, did not inhibit the action of human fibroblast interferon in these cells (Table 4). DISCUSSION
The present results indicate that in HFC, the antiviral activity of poly(1. C) is interferon mediated. In the HFC system, at TABLE
4
EFFECT OF ANTISERA DIRECTED AGAINST THE CELL SURFACE OF HUMAN FIBROBLAST CELLS= Treatment 0 Poly(1 ‘C) Antiserum Poly(I. C) Interferon Antiserum Antiserum
- poly(1 .C) + antiserum - interferon + interferon
Interferon (units/ml) <4 384 384 384 96 96 96
VSV yield (PFU/ml) 3.2 2.0 3.2 3.0 3.6 2.8 3.4
x x x x x x x
10’ 10’ 10’ 10’ 10’ 10’ 10’
DHFC were treated with antiserum (1:lO dilution) against HFC surfaces either before (1 hr at 37°C) or after (6 hr at 37%) exposure to poly(1.C) (100 rg/ml, 15 min. 37°C). Interferon and antiviral activity were assayed 6 hr after the start of poly(1. C) treatment, as described in Methods. To assay the effect on the activity of human fibroblast interferon, the cells were either treated with the antisera (1 hr at 37”C), before exposure to interferon (6 hr at 37”C), or with antisera and interferon simultaneously (6 hr at 37°C).
416
VENGRIS,
STOLLAR
least 88% of the cells were producing interferon. When the induced interferon was neutralized by antiserum, cells which produced interferon did not develop the antiviral state. The effect appears to be specific for interferon since antiserum directed against the cell surface of HFC did not inhibit the establishment of the antiviral state. Additionally, serum which reacted with poly(1 .C) was without any effect when added after interferon induction was initiated. The requirement of interferon synthesis for the induction of the antiviral state by nonreplicating Newcastle disease virus has been previously suggested (Dianzani et al., 1970). This finding may have further implications for the mechanism of interferon action. The possibility that antiserum would neutralize interferon at the site internal to the cellular membrane cannot be completely eliminated. However, if the uptake of antibodies occurs (Liebeskind et al., 1971), it may be expected to be a time dependent process and the inhibitory effect of the antiserum should increase with time. The observed effect, on the other hand, was time independent. Furthermore, it is generally believed that the antibodies do not enter living cells. Thus, our results lead us to suggest that in order to induce the antiviral state, interferon has to be associated with the external part of the cellular membrane, even in the cell in which it is produced. It was recently suggested that for biological activity, penetration of interferon was not required (Ankel et al., 1973), and binding of mouse interferon to the gangliosides was demonstrated (Besancon and Ankel, 1974). The existence of the interferon-specific membrane receptor site has been postulated (Friedman, 1967; Chany et al., 1973; Berman and Vilcek, 1974). It was suggested that the receptor is probably a protein molecule, since the binding of interferon can be destroyed by mild trypsin treatment (Chany et al., 1973; Berman and Vilcek, directed 1974). However, antibodies against the cell surface of HFC (sensitive to interferon in tissue culture) failed to inhibit the antiviral effect of interferon in these cells. The results may indicate either that
AND
PITHA
the antibodies to the receptor site were not present in our serum (receptor site may form only a very small part of the cell surface proteins), or that the lack of the effect was caused by a capping of the antigens on the cell membrane (Edidin, 1972). This would change the redistribution of the membrane receptor sites (Nicolson, 1972). However, the fact that no inhibition was observed when cells were treated with the anti-HFC serum at a temperature at which the capping is restricted, revealed the latter explanation less probable. The study of the interaction between poly(I. C) and anti-ds serum revealed some interesting insights into the mechanism of the antiviral effect. The ability of the antibody to inhibit the antiviral activity of poly(1. C) bound to the cell surface confirms our previous observation (Pitha et al., 1974; Harper and Pitha, 1973) that the initial interaction of poly(1. C) with the cell membrane is a process separable from the subsequent events leading to the induction of interferon. For the induction of both the antiviral state and interferon, mere contact of the inducer with the external cellular membrane is not sufficient. Additional time is required for induction to occur, or for the bound inducer to translocate to an internal site. The antiviral state is induced in a much shorter time than detectable amounts of interferon, but thus may represent only a difference in our ability to quantitate these two effects. In summary, the results presented here lead to the suggestion that interferon must interact with the outside of the cellular membrane in order to induce the antiviral state in cells. In addition, they support the hypothesis that the antiviral effects of poly(1. C) are mediated through interferon. ACKNOWLEGMENTS We wish to thank Drs. S. Baron, D. Brown, M. Colvin, and J. DeMaeyer-Guignard for helpful comments during the course of this work, Drs. C. Anfinsen and K. Paucker for the generous gift of antisera, and Dr. A. H. Owens, Jr. for continuous interest and encouragements. The work was supported by Grants from NIAID (2ROlAI 10944-03) and NSF (G.P.
INTERFERON
AND ANTIVIRAL
37937). Dr. V. E. Vengris is a postdoctoral trainee of NCI. REFERENCES ANFINSEN,C. B., BOSE, S., CORLEY,L., and GURARIROTMAN,D. (1974). Partial purification of human interferon by affinity chromatography. Proc. Nat. Acad. Sci. USA 71, 3139-3142. 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.
BAUSEK, G. H. and MERIGAN, T. C. (1969). Cell interaction with a synthetic polynucleotide and interferon production in oitro. Virology 39,491-498. 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, 478480. CHANY, C., GREGOIRE,A. VIGNAL, M., LEMAITREMENCUIT,J., BROWN,P., BESANCON,F., JUAREZ,H., and CASSINGENA, R. (1973). Mechanism of interferon uptake in parental and somatic monkey-mouse hybrid cells. Proc. Nat. Acad. Sci. USA 70, 557-561. DIANZANI, F., GAGNONI, S., BUCKLER, C. E., and BARON, S. (1970). Studies of the induction of interferon system by nonreplicating Newcastle disease virus. Proc. Sot. Exp. Biol. Med. 133,325-328. EDIDIN, M. (1972). In “Membrane Research” (C. F. Fox, ed.), pp. 15-26. (Academic Press, New York). FIELD, A. K., TYTELL, A. A., LAMPSON,G. P., and HILLEMAN,M. R. (1967). Inducers of interferon and host resistance. II. Multistranded synthetic polynucleotide complexes. Proc. Nat. Acad. Sci. USA 58, 1004-1010. FIELD, A. K., TYTELL, A. A., LAMPSON,G. P., and HILLEMAN,M. R. (1968). Inducers of interferon and host resistance. V. In vitro studies. Proc. Nat. Acad. Sci. USA 61, 340-346.
FINTER,N. B. (1969). Dye uptake method for assessing viral cytopathogenicity and their application to
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interferon assay. J. Gen. Virol. 5. 419-427. FRIEDMAN,R. (1967). Interferon binding: The first step in establishing of antiviral activity. Science 156, 1760-1761. HARPER,H. D. and PITHA, P. M. (1973). Effect of concanavalin A on interferon induction by poly IC. Biochem. Biophys. Res. Commun. 53, 1220-1226. LIEBESKIND,D. S. P., Hsu, K. C., ERLANGER, B. F., and BEISER,S. M. (1971). Selective inhibition of transformed cells in cell culture by anti-thymidine antibodies. Natiue New Biol. 234, 127-128. NICOLSON,G. L. (1972). In “Membrane Research” (C. F. Fox, ed.), pp. 53-70. Academic Press, New York. PITHA, P. M., MARSHALL,L. N., and CARTER,W. A. (1972). Interferon induction: Rate of cellular attachment of poly IC. J. Gen. Viral. 15, 89-92. PITHA, P. M. and PITHA, J. (1973). Interferon induction site: Poly IC on solid substrate carriers. J. Gen. Virol. 21, 31-37. PITHA, P. M., HARPER,H. D., and PITHA, J. (1974). Dependence of interferon induction on cell membrane integrity. Virology 59, 40-50. REYNOLDS,F. H., JR. and PITHA, P. M. (1974). The induction of interferon and its messenger RNA in human fibroblasts. Biochem. Biophys. Res. Commun. 59, 1023-1030. ROM;ERS,R. and MERIGAN,T. C. (1974). Interferon 57, production by individual cells. Virology 467-474. SCHWARTZ,E. F. and STOLLAR,B. D. (1969). Antibodies to polyadenylate-polyuridylate copolymers as reagents for double stranded RNA and DNARNA hybrid complexes. Biochem. Biophys. Res. Commun. 35, 115-120. TOMASZ,A. and BEISTER,S. M. (1965). Relationship between the competence antigen and the competence activator substance in pneumococci. J. Bacteriol. 90, 1226-1232. VILCEK, J. and HAVELL, E. A. (1973). Production of high-titered interferon in cultures of human diploid cells. Proc. Nat. Acad. Sci. USA 70, 3909-3913. WIGZELL,H. (1965). Quantitative titrations of mouse H-2 antibodies using Cr-51-labeled target cells. Transplantation 3, 423-431.
65, 410-417 (1975)
Interferon
Externalization
by Producing Antiviral
V. E. VENGRIS,’
Cell Before
Induction
of
State
B. D. STOLLAR,2
AND
P. M. PITHA.’
The Johns Hopkins University School of Medicine, Oncology Center, Baltimore, Maryland 21205, ’ and, 7hfts University School of Medicine, Department of Biochemistry and Pharmacology, Boston, Massachusetts 02111’ Accepted Janwry
13,1975
The mechanism by which poly(I. C) induces the antiviral state in human fibroblast cells was studied using antisera which were selectively reactive with the inducer [poly(I. C) 1,the product (human fibroblast interferon), and the fibroblast cell surface. Anti-interferon serum completely neutralized the antiviral effect of human fibroblast interferon. Furthermore, when added to the medium of the cells in which interferon synthesis had already been induced by poly(I-C), interferon antiserum prevented the intracellular antiviral state from developing. Antibodies specific to double stranded RNA inhibited the antiviral activity of poly(1.C) whether it was in solution or bound to the cells, but the same antibodies were without effect when added after the induction of the antiviral state was initiated. Treatment with serum directed against the cell surface of human fibroblasts failed to inhibit the antiviral activity of poly (1.C) or of human interferon in these cells. These results indicate that the antiviral effect of poly(1.C) is interferon mediated, and that for the development of the antiviral state, interferon has to interact with the external part of the cellular membrane of the producing cell. INTRODUCTION
1968). This raises another question as to whether the antiviral activity of poly(1. C) is interferon mediated, or if these two phenomena are due to the different mechanisms which could be separated. The availability of antisera capable of reacting specifically with the inducer, the product and the cell should facilitate studies on the mode of antiviral action of poly(1.C). In this work, we describe the use of sera which react specifically with human fibroblast interferon, double stranded (ds) RNA, and the cell surfaces of human fibroblasts. The correlation between interferon induction and the antiviral effect and further implications for the mechanism of interferon action are discussed.
Human cells, exposed to the complex of polyinosinic and polycytidylic acids [poly(I.C)], developed resistance to direct virus challenge and simultaneously released interferon into the medium (Field et al., 1967). The mechanism by which poly(1 .C!) induces the antiviral state and interferon production has not been completely clarified. We have shown that in human cells, poly(1. C) induces the formation of interferon-specific mRNA, which can be translated with fidelity in the heterologous cell system (Reynolds and Pitha, 1974). However, it has not yet been determined whether the induction of the antiviral state and interferon production require the mere contact of poly(1 .C) with the cellular membrane or the uptake of the inducer into the cell (Bausek and Merigan, 1969; Pitha and Pitha, 1973). In several cell systems, poly(1. C) induces an antiviral state without the production of any detectable exogenous interferon (Field et al.,
MATERIALS
410 Copyright 0 1975 by Academic Press, Inc. All rights of reproduction in any form reserved.
AND METHODS
Polynucleotides. Poly(1.C) L. Biochemicals, Inc.) had weight higher than 2 x lo5 extinction coefficient of 4.5 x
complex (P. a molecular and a molar 10s at 260 nm
INTERFERON
AND ANTIVIRAL
in 0.01 M sodium phosphate, pH 7.0. The complex was dissolved in 0.01 M phosphate buffer containing 0.15 M NaCl at concentration 10 mg/ml. One preparation of poly(1. C) was used for all experiments. Cells and virus. Human foreskin diploid fibroblast cells (HFC), passages 9-15 [high interferon producers (Vilcek and Havell, 1973)] and human diploid fibroblast cells (HFC-M) were used in all studies. They were grown in Eagle’s minimal essential medium (MEM) supplemented with 2 mM glutamine and enriched with 5% fetal bovine serum (FBS), and were maintained in the same medium containing 2% FBS. Cells were confluent 6 days after seeding, and most experiments were carried out on 12-day-old cultures. Vesicular stomatitis virus (VSV), New Jersey serotype, was propagated in DEAE-dextran (10 pg/ml) treated (mouse) L cells infected at low multiplicity and harvested 24 hr later when the titer was lo8 plaque forming units (PFU/ml). Assays for antiviral activity. HFC were incubated for various lengths of time with a solution of poly(1.C) (100 pg/ml or 25 &ml) in maintenance medium. Resistance to virus infection and interferon titers were measured calorimetrically, (Finter, 1969) employing VSV as a challenge virus (Pitha et al., 1974). The activity is given in research reference units, using NIH standard of human interferon as a reference. The antiviral effect of poly(1. C) on a single step infection was determined by virus yield at 15 hr postinfection. Antisera to human interferon. Antibodies were induced in 12-wk-old white Leghorn roosters by weekly injections (intravenously via jugular vein) of unpurified interferon (5 ml, 4800 units/ml) produced in HFC by poly(1.C) (Vilcek and Havell, 1973). Beginning on the 60th day after injection, chickens were bled and serum collected for antibody assay. Pooled sera were inactivated by heating at 56°C for 30 min. The neutralization titer of the antisera was proportional to the dilution of the interferon which was assayed; 1/64dilution completely neutralized 12 units of human interferon. Antibody titer is expressed in units; 1 unit is the dilution of antisera
STATE
INDUCTION
411
which is able to neutralize completely 12 units of human interferon; both colometric and virus yield assays produced similar antibody titer. Control sera were prepared by injecting roosters with medium from uninduced cells under identical conditions to those described above. Antibody to HFC interferon prepared in rabbits was a gift of Dr. K. Paucker from the Medical College of Pennsylvania in Philadelphia; 1/3000 dilution of this sera neutralized 12 units of human fibroblast interferon. The y-globulin fraction of sheep antisera to partially purified human leukocyte interferon was a gift of Dr. C. B. Anfinsen of the National Institutes of Health; 1/;00dilution of this fraction completely neutralized 100 units of human leukocyte interferon (Anfinsen et al., 1974), but was without any effect on human fibroblast interferon (Vengris, unpublished observations; Baron, personal communication). Antisera to HFC. Rabbits were immunized by injection of 1 x lo* HFC (in complete Freund’s adjuvant) into the footpad. Two, three, and four weeks later, they received intraperitoneal injections- of the same cell dose without adjuvant. Five weeks after the start of immunization, rabbits were bled and the serum was heat inactivated. Antibody titer was determined using the chromium release (“‘Cr) cytotoxicity test (Wigzell, 1965); 1/64dilutions of this serum increased radioactive chromium release by 35%, as compared to the control serum. Antisera to ds polynucleotides. Rabbits were immunized with poly(A.U)-methylated bovine serum albumin (BSA) complexes, as previously described (Schwartz and Stollar, 1969). RESULTS
Effect of anti-interferon serum. The kinetics of the appearance of interferon and the antiviral state in HFC treated with poly(1 .C) for 15 min are shown in Fig. 1. Interferon was not detected in the medium until 2 hr after poly(1 .C) removal; however, at that time the cells had already reached their complete antiviral state. Previously, we have shown that’ at this time,
412
VENGRIS,
STOLLAR
mRNA specific for human interferon can be detected (Reynolds and Pitha, 1974). Chicken or rabbit antisera to human fibroblast interferon were used to examine the correlation between the antiviral effect of poly(1 .C) and the induction of interferon. After induction, cells were incubated for an addional 6 or 20 hr in maintenance medium containing a sufficient amount of anti-interferon serum to neutralize any interferon produced and released into the medium (Table 1). When poly(I.C)-
FIG. 1. Kinetics of interferon and antiviral state appearance. HFC (3 x lo6 cells) were treated with solution of poly(1. C) (100 &ml) for 15 min at 37°C; poly (1.C) was then removed, cells washed and incubated in maintenance medium. Medium was collected at the indicated times and assayed for interferon, and cells were washed and infected with VW for virus yield assay. (04) VSV yield. (04) Interferon titer. TABLE
AND
PITHA
induced extracellular interferon was neutralized by the presence of antisera to human fibroblast interferon, the antiviral state was not induced. Within the limits described in the next section, this effect was independent of the exposure time to poly(1. C) and the time of incubation of the antisera was HFC; complete inhibition of the antiviral state was observed with exposure time as short as 2 hr after induction with poly(1. C). Sera from nonimmunized animals were without any effect, as was a control serum prepared against medium from uninduced cells. Additionally, the anti-interferon sera did not inhibit virus replication in HFC. The sheep antiserum (y-globulin fraction) to human leukocyte interferon (which partially binds human fibroblast interferon but does not neutralize its antiviral activity), when studied under identical conditions, was without any effect. In addition, the antiserum to human fibroblast interferon did not inhibit the induction of the antiviral state of poly(1 .C) in mouse L cells. For the inhibitory effect to be achieved, the interferon antiserum had to be present in the medium from the time when interferon starts to be released into the medium. Pretreatment of HFC with the anti-human interferon serum for 1 hr before or during exposure of the cells to poly(1 .C) affected neither interferon production nor 1
INHIBITION OF THE ANTIVIRAL ACTIVITY OF POLY(I . C) BY ANTISERUM TO HUMAN FIBROBLAST INTERFERON Poly(1. C)
Incubation
medium
VSV yield (PFU/ml) b
a -
+ + +
MEM Interferon MEM Interferon Serum*
antiserum antiserum
4.0 3.8 8.2 1.2 2.5
x x x x x
10’ 10’ 10’ 10’ 10’
1.5 1.4 6.2 5.6 9.6
x x x x x
Antibody** titer
Interferon** (units/ml)
10’ 10’ 108 10’ lo3
a
0
D
b
<4 t4 96 <4 24
<4 <4 384 <4 384
32 2 -
200 10 -
aHFC(M) were treated with poly(1.C) (100 pglml) for 1 hr at 37°C; poly(1.C) was removed, cells washed and incubated in MEM containing chicken anti-interferon serum for an additional 20 hr. Medium was collected and assayed for interferon and antibody titer; cells were washed and infected with VSV for virus yield assay. b HFC were treated with poly(1 .C) (100 &ml) for 15 min at 37”C, poly(1.C) was removed and cells incubated in MEM containing rabbit anti-interferon serum for 6 hr. * Serum from nonimmunized rabbits or chickens injected with medium from uninduced cells. ** In the medium at the end of the incubation period.
INTERFERON
AND ANTIVIRAL
STATE
413
INDUCTION
with multiple virus hit (2 hr postinfection) will register as an infectious center in 24 hr. Under conditions where the spread of interferon should be minimal [l hr after poly(1 .C) induction, no detectable interferon in the medium (Fig. l)], 88% of the cells (percentage of cells in antiviral state = loo-percentage of infected cells; the number of infectious centers of untreated controls were taken as 100%) were in antiviral state (Table 2). When assayed 6 hr postinduction, when a substantial amount of extracellular interferon was produced (Table l), 98% of the cells was found to be in antiviral state. Thus, under our conditions, a maximum of only 10% of the cells are poor or noninterferon producers. The number of cells producing virus increased substantially when the cells after induction were incubated in the presence of antiinterferon antisera for either 1 hr (64% infected in the presence of antisera and 12% in the controls) or 6 hr (42% infected with antiserum and 2.5% in the controls) postinduction (Table 2). Should the interferon antiserum prevent the establishment of the antiviral state only in the population of cells which are nonproducers, while the antiviral state in the producing cells would not be affected, virus yield in noninduced cells should be ten times higher than in poly(1 .C) induced cells incubated in the presence of anti-interferon antiserum. However, only three- to fourfold reduction
the appearance of the antiviral state (192 units of interferon and 1.4 x lo4 PFU/ml in the presence of anti-interferon serum and 192 units of interferon and 2 x lo4 PFU/ml in the controls). The next question we asked was whether anti-interferon antiserum prevented the establishment of the antiviral state of the interferon-producing cells, or whether it only blocked the spread of interferon from the small population of cells producing interferon to nonproducers. It was recently shown that in the cat cells producing high levels of interferon, one-third of the cell population was poor or noninterferon producers (Rodgers and Merigan, 1974). To test the fraction of interferon-producing cells after poly(1. C) treatment in the population of HFC, we measured the number of cells in antiviral state on a single cell basis. The untreated cells and cells exposed to poly(1 .C) (15 min at 37°C) were incubated with medium or with the interferon antiserum either for 1 or 6 h, and then infected with high multiplicity of VSV. Two hours after infection, cells were trypsinized and the number of cells producing virus was measured as infectious centers on L cells in 24 hr. As shown in Table 2, the efficiency of infectious centers formation under these conditions is very low (5%). This is partially caused by low plating efficiency (10%) of human fibroblast cells used (12day-old cultures), and probably only cells TABLE
2
THE ANTIVIRAL EFFECT OF POLY(I C) ASSAYED BY INFECTIOUS CENTERS AT DIFFERENT TIMES POSTINDUCTION’ Poly(1. C)
+ + -
Treatment
0 Interferon 0 Interferon
antiserum antiserum
Number of infectious centersC
Est. p+* 1 hr 10-2.2” lo- 1.49 lo- 1.32 10-L 30
6hr 10-2.92 IO- 1.67 10-1.33 IO- *.a0
1 hr
6hr
28 158 230 250
6 105 237 249
DHFC were incubated in MEM or treated with poly(1. C) (100 &ml) for 15 min; poly(1. C) was removed, cells washed and incubated in MEM or MEM containing rabbit anti-interferon antiserum for an additional 1 or 6 hr. The cells were then infected with WV (m.o.i. 10) and 2 hr later trypsinized; 1000 cells were plated over monolayer of mouse L cells; plaques were read 24 hr later. bEst. p+-the estimated portion of the cells yielding virus: it was calculated by dividing the number of responses (plaques) from five plates by the number of cells plated. c The numbers represent the total amount of plaques in five plates.
414
VENGRIS,
STOLLAR
AND
PITHA
in the virus yield was observed (Table 1). pg poly (1.C) when the reagents were Thus, these data indicated that the abol- preincubated before addition to cells. ishment of the antiviral effect in poly(1. C) When antiserum was added to the cells induced .cells by anti-interferon antisera which were pretreated with poly(1 .C) at was due to the direct inhibition of the 37°C and in which the production of interantiviral state in the interferon producing feron and the antiviral state had already been initiated, no alteration of antiviral cells. However, since only unpurified or par- state was observed. However, when the cells were treated with poly(1 .C) at 4°C tially human interferon (human interferon purified to homogenicity is not available as [where the inducer binds to the cellular yet) was used for immunization, an objec- membrane, but the induction does not tion could be raised that the observed occur (Bausek and Merigan, 1969)], and then with the antiserum, the antiviral effect was due to the presence of antibodies to the inducer or to cell proteins in the effect of poly(1 .C) was again blocked anti-interferon antiserum. To examine this (Table 3). Thus, the antiserum reacted not possibility, we studied the effect of both only with poly(1 .C) in solution, but also antiserum against ds polynucleotides and with the inducer which was bound to the antiserum against HFC on the induction of cellular membrane, and thereby altered the antiviral properties of poly(1. C). interferon and the antiviral state in HFC. Having neutralized poly(1. C) which was Effect of antiserum against polynucleobound to the cells, but which had not tides. Serum from rabbits immunized with reacted with the triggering site for intercomplexes of poly(A. U) and methylated feron induction, we were able to determine BSA reacted specifically with ds polyribonucleotide-containing molecules (Schwartz the minimal period during which poly(1. C) and Stollar, 1969). Poly(I. C), when reacted had to be associated with the cells in order with this antiserum, lost the ability to in- to either initiate antiviral effect or reach a duce both interferon and the antiviral state protective site. When the cells were treated in HFC (Fig. 2). Quantitative equivalence with a solution of poly(1 .C) and then precipitation occurred between 0.1 ml of washed, a 1-min exposure was sufficient to the antisera and 25 pg of poly(1. C), and induce the antiviral state and interferon this amount of antiserum was also able to production (Fig. 3A). This is in aggreement completely abolish the antiviral effect of 25 TABLE
3
EFFECT OF ANTIBODIES TO DOUBLE POLYNUCLEOTIDES ON THE INTERFERON POLY(I. C)”
Treatment
FIG. 2. Inhibition of the antiviral activity of poly(1.C) by antiserum to ds polynucleotides. HFC-M (3 x lo5 cells) were treated with the mixture of poly(1. C) and given volume of the antiserum to ds polynucleotides (preincubated at 37°C for 30 min) for 60 min at 37°C. The mixture was removed, cells were washed and incubated in maintence medium for an additional 20 hr; then interferon activity was assayed in collected medium, cells were washed and infected with VSV for virus yield assay. (0-O) VSV yield. (04) Interferon titer.
Poly(1. C) (37 or 4°C) Poly(1 .C) and antibodies (preincubated) Antibodies Poly(1 .C) (37°C) - antibodies Poly(1 .C) (4°C) 4 antibodies
Interferon (units/ml)
STRANDED INDUCTION
BY
VSV yield (PFU/ml)
192 <4
6.4 x lo3 4.5 x 10’
<4 96
6.0 x 10’ 1.8 x 10’
<4
8.0 x lo6
“HFC were treated with poly(1.C) (25 &ml) for 30 min (37 or 4”C), or poly(I.C), preincubated with 0.1 ml of antibodies for 30 min (37”C), washed and incubated with maintenance medium, or medium containing 0.1 ml of antibodies to ds polynucleotides, for an additional 30 min. Antiviral activity was assayed 20 hr later.
INTERFERON
AND ANTIVIRAL
STATE
415
INDUCTION
Fro. 3. Rate of induction of interferon and the antiviral state by poly(I .C) in human fibroblast cells. HFC-M (3 x lo5 cells) were treated with poly(1.C) (25 pg/ml) at 37°C for given periods of time. The inducer was then washed and cells were incubated in maintenance medium (A), or medium containing 0.1 ml of anti-ds polynucleotide antibodies (B). After 24 hr, interferon and antiviral activity were assayed as described in the Materials and Methods section. (0-O) VSV yield. (O-O) Interferon titer.
with our previous findings (Pitha et al., 1972). However, when poly(1.C) was washed off the cells and then the antiserum to ds polynucleotides was added to neutralize the unreacted poly(1. C) associated with the cellular membrane, the results were different (Fig. 3B). To create the full antiviral state in these cells, a 5-min preexposure to poly(1 .C) was required; the interferon production showed repeatedly a delay which was not observed under previous conditions. These results show that in a very short time, an amount of poly(1. C) sufficient to start, but not complete, the induction of interferon and the antiviral state associates with the cellular membrane. However, the bound inducer needs additional time for full interaction with the actual induction site or for the penetration. Effect of antisera directed against the cell surface of HFC. Antiserum to HFC contained antibodies for surface components of these cells, as shown by membrane immunofluorescence (indirect method using fluorescein labeled goat antirabbit globulin). The antiserum was also cytotoxic to HFC in the presence of guinea pig complement, as measured by isotope release from cells previously labeled with radioactive chromium ( 51Cr). Seventy-five percent of the cells were lysed at a 1:lO dilution. The addition of the antiserum (1:lO dilution) to HFC, before or after treatment with poly(1. C), did not affect its antiviral activity (Table 4). Also, 1:2 dilution of this sera used under identical conditions to those described was without any effect. The antisera to cell surfaces of
competent cells blocked the transforming activity of DNA and helped to demonstrate the existence of specific binding sites on these cells for single stranded DNA (Tomasz and Beiser, 1965). The treatment of HFC with anti-HFC serum, either at 37 or 4°C (not shown), before or simultaneously with interferon, did not inhibit the action of human fibroblast interferon in these cells (Table 4). DISCUSSION
The present results indicate that in HFC, the antiviral activity of poly(1. C) is interferon mediated. In the HFC system, at TABLE
4
EFFECT OF ANTISERA DIRECTED AGAINST THE CELL SURFACE OF HUMAN FIBROBLAST CELLS= Treatment 0 Poly(1 ‘C) Antiserum Poly(I. C) Interferon Antiserum Antiserum
- poly(1 .C) + antiserum - interferon + interferon
Interferon (units/ml) <4 384 384 384 96 96 96
VSV yield (PFU/ml) 3.2 2.0 3.2 3.0 3.6 2.8 3.4
x x x x x x x
10’ 10’ 10’ 10’ 10’ 10’ 10’
DHFC were treated with antiserum (1:lO dilution) against HFC surfaces either before (1 hr at 37°C) or after (6 hr at 37%) exposure to poly(1.C) (100 rg/ml, 15 min. 37°C). Interferon and antiviral activity were assayed 6 hr after the start of poly(1. C) treatment, as described in Methods. To assay the effect on the activity of human fibroblast interferon, the cells were either treated with the antisera (1 hr at 37”C), before exposure to interferon (6 hr at 37”C), or with antisera and interferon simultaneously (6 hr at 37°C).
416
VENGRIS,
STOLLAR
least 88% of the cells were producing interferon. When the induced interferon was neutralized by antiserum, cells which produced interferon did not develop the antiviral state. The effect appears to be specific for interferon since antiserum directed against the cell surface of HFC did not inhibit the establishment of the antiviral state. Additionally, serum which reacted with poly(1 .C) was without any effect when added after interferon induction was initiated. The requirement of interferon synthesis for the induction of the antiviral state by nonreplicating Newcastle disease virus has been previously suggested (Dianzani et al., 1970). This finding may have further implications for the mechanism of interferon action. The possibility that antiserum would neutralize interferon at the site internal to the cellular membrane cannot be completely eliminated. However, if the uptake of antibodies occurs (Liebeskind et al., 1971), it may be expected to be a time dependent process and the inhibitory effect of the antiserum should increase with time. The observed effect, on the other hand, was time independent. Furthermore, it is generally believed that the antibodies do not enter living cells. Thus, our results lead us to suggest that in order to induce the antiviral state, interferon has to be associated with the external part of the cellular membrane, even in the cell in which it is produced. It was recently suggested that for biological activity, penetration of interferon was not required (Ankel et al., 1973), and binding of mouse interferon to the gangliosides was demonstrated (Besancon and Ankel, 1974). The existence of the interferon-specific membrane receptor site has been postulated (Friedman, 1967; Chany et al., 1973; Berman and Vilcek, 1974). It was suggested that the receptor is probably a protein molecule, since the binding of interferon can be destroyed by mild trypsin treatment (Chany et al., 1973; Berman and Vilcek, directed 1974). However, antibodies against the cell surface of HFC (sensitive to interferon in tissue culture) failed to inhibit the antiviral effect of interferon in these cells. The results may indicate either that
AND
PITHA
the antibodies to the receptor site were not present in our serum (receptor site may form only a very small part of the cell surface proteins), or that the lack of the effect was caused by a capping of the antigens on the cell membrane (Edidin, 1972). This would change the redistribution of the membrane receptor sites (Nicolson, 1972). However, the fact that no inhibition was observed when cells were treated with the anti-HFC serum at a temperature at which the capping is restricted, revealed the latter explanation less probable. The study of the interaction between poly(I. C) and anti-ds serum revealed some interesting insights into the mechanism of the antiviral effect. The ability of the antibody to inhibit the antiviral activity of poly(1. C) bound to the cell surface confirms our previous observation (Pitha et al., 1974; Harper and Pitha, 1973) that the initial interaction of poly(1. C) with the cell membrane is a process separable from the subsequent events leading to the induction of interferon. For the induction of both the antiviral state and interferon, mere contact of the inducer with the external cellular membrane is not sufficient. Additional time is required for induction to occur, or for the bound inducer to translocate to an internal site. The antiviral state is induced in a much shorter time than detectable amounts of interferon, but thus may represent only a difference in our ability to quantitate these two effects. In summary, the results presented here lead to the suggestion that interferon must interact with the outside of the cellular membrane in order to induce the antiviral state in cells. In addition, they support the hypothesis that the antiviral effects of poly(1. C) are mediated through interferon. ACKNOWLEGMENTS We wish to thank Drs. S. Baron, D. Brown, M. Colvin, and J. DeMaeyer-Guignard for helpful comments during the course of this work, Drs. C. Anfinsen and K. Paucker for the generous gift of antisera, and Dr. A. H. Owens, Jr. for continuous interest and encouragements. The work was supported by Grants from NIAID (2ROlAI 10944-03) and NSF (G.P.
INTERFERON
AND ANTIVIRAL
37937). Dr. V. E. Vengris is a postdoctoral trainee of NCI. REFERENCES ANFINSEN,C. B., BOSE, S., CORLEY,L., and GURARIROTMAN,D. (1974). Partial purification of human interferon by affinity chromatography. Proc. Nat. Acad. Sci. USA 71, 3139-3142. 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.
BAUSEK, G. H. and MERIGAN, T. C. (1969). Cell interaction with a synthetic polynucleotide and interferon production in oitro. Virology 39,491-498. 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, 478480. CHANY, C., GREGOIRE,A. VIGNAL, M., LEMAITREMENCUIT,J., BROWN,P., BESANCON,F., JUAREZ,H., and CASSINGENA, R. (1973). Mechanism of interferon uptake in parental and somatic monkey-mouse hybrid cells. Proc. Nat. Acad. Sci. USA 70, 557-561. DIANZANI, F., GAGNONI, S., BUCKLER, C. E., and BARON, S. (1970). Studies of the induction of interferon system by nonreplicating Newcastle disease virus. Proc. Sot. Exp. Biol. Med. 133,325-328. EDIDIN, M. (1972). In “Membrane Research” (C. F. Fox, ed.), pp. 15-26. (Academic Press, New York). FIELD, A. K., TYTELL, A. A., LAMPSON,G. P., and HILLEMAN,M. R. (1967). Inducers of interferon and host resistance. II. Multistranded synthetic polynucleotide complexes. Proc. Nat. Acad. Sci. USA 58, 1004-1010. FIELD, A. K., TYTELL, A. A., LAMPSON,G. P., and HILLEMAN,M. R. (1968). Inducers of interferon and host resistance. V. In vitro studies. Proc. Nat. Acad. Sci. USA 61, 340-346.
FINTER,N. B. (1969). Dye uptake method for assessing viral cytopathogenicity and their application to
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interferon assay. J. Gen. Virol. 5. 419-427. FRIEDMAN,R. (1967). Interferon binding: The first step in establishing of antiviral activity. Science 156, 1760-1761. HARPER,H. D. and PITHA, P. M. (1973). Effect of concanavalin A on interferon induction by poly IC. Biochem. Biophys. Res. Commun. 53, 1220-1226. LIEBESKIND,D. S. P., Hsu, K. C., ERLANGER, B. F., and BEISER,S. M. (1971). Selective inhibition of transformed cells in cell culture by anti-thymidine antibodies. Natiue New Biol. 234, 127-128. NICOLSON,G. L. (1972). In “Membrane Research” (C. F. Fox, ed.), pp. 53-70. Academic Press, New York. PITHA, P. M., MARSHALL,L. N., and CARTER,W. A. (1972). Interferon induction: Rate of cellular attachment of poly IC. J. Gen. Viral. 15, 89-92. PITHA, P. M. and PITHA, J. (1973). Interferon induction site: Poly IC on solid substrate carriers. J. Gen. Virol. 21, 31-37. PITHA, P. M., HARPER,H. D., and PITHA, J. (1974). Dependence of interferon induction on cell membrane integrity. Virology 59, 40-50. REYNOLDS,F. H., JR. and PITHA, P. M. (1974). The induction of interferon and its messenger RNA in human fibroblasts. Biochem. Biophys. Res. Commun. 59, 1023-1030. ROM;ERS,R. and MERIGAN,T. C. (1974). Interferon 57, production by individual cells. Virology 467-474. SCHWARTZ,E. F. and STOLLAR,B. D. (1969). Antibodies to polyadenylate-polyuridylate copolymers as reagents for double stranded RNA and DNARNA hybrid complexes. Biochem. Biophys. Res. Commun. 35, 115-120. TOMASZ,A. and BEISTER,S. M. (1965). Relationship between the competence antigen and the competence activator substance in pneumococci. J. Bacteriol. 90, 1226-1232. VILCEK, J. and HAVELL, E. A. (1973). Production of high-titered interferon in cultures of human diploid cells. Proc. Nat. Acad. Sci. USA 70, 3909-3913. WIGZELL,H. (1965). Quantitative titrations of mouse H-2 antibodies using Cr-51-labeled target cells. Transplantation 3, 423-431.