VIROLOGY
92,
324-330 (1979)
Isolation of a Subspecies of Murine Interferon Antigenically Human Leukocyte Interferon EDWARD Department
of Microbiology,
Related to
A. HAVELL’
New York University
School of Medicine,
New York, New York 10016
Accepted August 21, 1978 Various preparations of virus-induced murine tissue culture interferon were found to exhibit some antiviral activity in cultures of human and bovine cells. The heterologous antiviral activity of these murine interferons on both human and bovine cultures was shown to be due to an antigenically distinct murine interferon subspecies. Isolation of the murine interferon species responsible for the heterologous antiviral activities was accomplished by means of antibody affinity chromatography using anti-human leukocyte interferon to selectively remove this interferon species from the bulk of murine antiviral activity. The isolated species, while only accounting for l-2% of homologous antiviral activity of the unfractionated interferon, was responsible for all the heterologous human and bovine antiviral activity. The antiviral activities of this minor murine interferon component on both murine and human cells were neutralized by antiserum against leukocyte interferon as well as by antiserum against mouse L cell interferon. INTRODUCTION
The degree of antiviral activity on cells of heterologous species has proven a useful parameter for differentiating interferons produced by the same species. Human interferons derived from cells of lymphoid origin differ markedly from interferons produced by nonlymphoid cells in their ability to render cells of other species resistant to virus replication. Human leukocyte (Le) interferon exhibits relatively more antiviral activity on bovine and porcine cells than human fibroblast (F) interferon (Gresser et al., 1974; Havell et al., 1977), whereas F interferon has been reported to be more active on rabbit cells than Le interferon (Desmyter et al., 1968). These two human interferons also differ in their physicochemical and antigenic properties (Stewart et al., 1974; Havell et al., 1975a) and available evidence suggests that these interferons are coded for by different genes (Cavalieri et al., 1977). Thus, the capacity of the two human interferons to render varying degrees of virus resistance to cells of other species is probably determined by the pri’ Present address: Trudeau Instit,ute, Inc., P. 0. Box 59, Saranac Lake, N.Y. 12983. 324 0042-6822/79/020324-07$02.00/O Copyright 0 1979by Academic Press, Inc. All rights of reproduction in any form reserved.
mary structures of the two interferon molecules. While it is not understood what inherent property of an interferon molecule is responsible for antiviral activity on cells of certain species, reports in the literature seem to suggest that human interferons generally exert relatively more antiviral activity on cells of other species than interferons derived from other species do in human cells (Finter, 1973). The notable exception to this generalization are murine interferons which have been reported to exhibit varying degrees of antiviral activity on human cells while human interferons were determined to be at best only marginally active on murine cells (Levy-Koenig et al., 1970; Bodo et al., 1971; Samuel and Farris, 1977). In this study, we have established that a minor interferon component present in virus-induced murine tissue culture interferon preparations is responsible for the heterologous antiviral activity on human and bovine cells. This murine interferon molecule is of interest from an evolutionary standpoint inasmuch as it possesses a common antigenic determinant(s) with human Le interferon.
DISTINCT MATERIALS
AND
MURINE
INTERFERON
METHODS
Cells. The murine L-929 cell line used in these studies has been maintained in this laboratory for several years and was originally obtained from Dr. J. S. Youngner, University of Pittsburgh. A strain of human skin fibroblast cells trisomic for chromosome 21, designated GM-258, was obtained from the Mammalian Genetic Mutant Cell Repository (Camden, N. J.) . A line of bovine kidney cells (MDBK) was provided by Dr. P. Sehgal (Rockefeller University). All cells were propagated in Eagle’s minimal essential medium (MEM) supplemented with 5% fetal bovine serum. Interferons. The Newcastle disease virus (NDV)-induced murine C-243-3 interferon was the kind gift of Dr. M. G. Tovey (Institut de Recherches Scientifiques sur le Cancer, Villejuif, France) and was prepared as previously described (Tovey et al., 1974). The NDV-induced murine L-929 interferon was prepared by inducing confluent monolayers of cells with NDV (Hickman strain) at an m.o.i. of 10. After a 1-hr adsorption period, the cultures were washed 3 x with MEM and then reincubated with medium containing 0.5% fetal bovine serum for an additional 30 hr, after which the interferoncontaining medium was collected. The mouse mengo virus interferon was a gift of Dr. E. Knight (DuPont, Wilmington, Del.), and was prepared according to his published procedure (Knight, 1975). All virusinduced interferon preparations were acidified at pH 2.0 for 5 days and then centrifuged at 100,000 g/hr to remove all residual VhS.
Human leukocyte (Le) interferon was prepared by Dr. K. Cantell (Helsinki, Finland) and human fibroblast (F) interferon was prepared by inducing FS-4 skin fibroblasts by the superinduction method of Havell and VilEek (1972). Interferon assays. Interferon assays were done by the micromethod of Armstrong (1971) as modified by Havell and Vilcek (1972) using murine (L-929), human (GM258), or bovine (MDBK) cells and vesicular stomatitis virus (VSV). Comparisons of the homologous and heterologous antiviral activities of the various interferon preparations were done simultaneously. The titer
SUBSPECIES
325
of the human leukocyte standard (G-023901-527) was about 20 times its assigned value when titrated on GM-258 cells. The murine interferon standard (G-002-904-511) gave titers equivalent to its designated antiviral activity on the murine L-929 cells.
Anti-interferon sera and affinity chromatography. The neutralization assays of interferon activities on homologous and heterologous cells with the various anti-interferon sera were performed as previously described (Have11 et al., 1975a). The rabbit anti-murine L cell interferon globulin (A-L Inf. No. 12,3/17/72) was obtained from the Research Resources Branch of the National Institute of Allergy and Infectious Diseases (Bethesda, Md.). The anti-human F interferon serum was prepared by immunization of rabbits as described earlier (Havell et al., 1975a) using purified human F interferon (>107 units/mg protein) as the antigen. The sheep anti-human Le interferon antibody used in the neutralization assays and in the antibody affinity chromatography procedures was the kind gift of Drs. D. GurariRotman and C. B. Anfinsen. The coupling of the anti-Le interferon antibodies to CnBr-activated Sepharose 4B (Pharmacia, Uppsala, Sweden) and the affinity chromatography procedures were essentially as described (Havell et aE.,1975a).
CH-Sepharose 4B Sorbent chromatography. Sorbent chromatography of murine interferon on CH-Sepharose 4B (Pharmacia, Uppsala, Sweden) was done according to the method of Davey et al., (1976). The murine interferon sample to be placed on the column was dialyzed against 0.05 M sodium acetate, pH 5.0 (E. buffer), for 18 hr at 4”. The sample was placed on a column (1.5 x 10 cm) equilibrated with E. buffer at 4’. The column was eluted sequentially with E. buffer, followed by 0.02 M sodium phosphate, pH 7.4 (El), and finally with 0.02 M sodium phosphate containing 0.05 M NaCl, pH 7.4 (E2). One-milliliter fractions were collected and then immediately assayed for antiviral activities on homologous and heterologous cells. RESULTS
Three . preparations of virus-induced murine tissue culture interferons were simul-
326
EDWARD
A. HAVELL
taneously assayed for their antiviral activities on murine L-929, human GM-258, and bovine MDBK cell cultures (Table 1). All three murine interferons were active on both human GM-258 and bovine MDBK cultures. The ratio of the murine to human antiviral activities was 128:l for both L cell interferon preparations and 338:l for the NDV-induced C-243-3 cell interferon. A human leukocyte interferon, while inactive on murine cells, showed a ratio of human to bovine antiviral activity which was in a similar range as observed with the murine interferons. A quite unexpected finding came from an experiment in which the heterologous human antiviral activity of a murine interferon preparation was found to be neutralized not only by homologous anti-murine interferon serum but also by antiserum against human Le interferon. Antiserum specific for human F interferon did not neutralize the heterologous activity of this murine interferon on human GM-258 cells (Table 2). The results obtained with the specific anti-human interferon sera and the demonstration that the various murine interferons exhibited similar human to bovine antiviral activity ratios as did the human leukocyte interferon suggested the existence of a murine interferon which resembled human leukocyte interferon both antigenically and in expression of antiviral activity on bovine cells. It was reasoned that if the antiviral activity of the murine interferon was neutralized by the specific anti-human Le serum, then it should be possible to isolate the murine interferon component bearing the common antigenic determinant(s) with the human Le interferon by means of antibody affinity chromatography. The C-243-3 murine inTABLE
terferon preparation was passed through a column consisting of anti-human Le interferon y-globulin covalently coupled to a Sepharose 4B matrix. In previous experiments, this chromatographic technique enabled the identification and quantitation of antigenically distinct interferon subspecies present in various human interferon preparations (Berg et al., 1975; Have11 et al., 1975a). Two fractions of interferon activity were isolated after passing the original (applied) murine C-243-3 interferon through the anit-human Le interferon column (Table 3). The murine antiviral activity which was specifically bound (retained fraction) by the immobilized antibody could only be eluted by lowering the pH of the column. The retained fraction represented only about 1.5% of the murine antiviral activity present in the original interferon preparation. However, this fraction contained all of the human and bovine antiviral activity recovered from the affinity column. While the ratio of the murine to human antiviral activity for the bulk of the murine interferon which passed unretained through the column was >40,000, this ratio for the specifically bound interferon component was 26. In a similar affinity chromatography TABLE
2
THE EFFECT OF SPECIFIC INTERFERON NEUTRALIZING SERA ON THE ANTIVIRAL ACTIVITIES OF MURINE AND HUMAN INTERFERONS IN HUMAN GM-258 CELLS Interferon-neutralizmg serum
Neutralizing Murine (C-243-3)
Anti-murine Anti-human Anti-human
Le F
titer for interferon HumanLe
40 200 t25
~25 6,500 t25
Human F
~25 l,f=)
1
ANTIVIRAL ACTIVITIES OF VARIOUS MURINE INTERFERON PREPARATIONS ON CELLS OF DIFFERENT SPECIES Antiviral activity (units/ml) on cells Inducing virus Cell source of interferon Murine
(L-929) Hum28iGM-
C-243-3 L-929 L-929 Leukocyte
(human)
Newcastle disease Newcastle diesase Mouse mengo Sendai
1,300,OOO 32,800 32,800 >4
3,840 256 256 2,048
Bovine (MDBK) 60 24 8 48
DISTINCT
MURINE
INTERFERON
procedure using a column of anti-human F interferon y-globulin, none of the murine antiviral activity was retained by the antibody. These findings demonstrate that a minor species of murine interferon (1) shows antigenic homology with human Le interferon and (2) is responsible for the heterologous antiviral activities on human and bovine cells. Antibody neutralization assays were performed on the antiviral activities of the original murine interferon and the two fractions of antiviral activity separated by means of affinity chromatography with neutralizing antisera specific for murine, human Le, and human F interferons. The antisera and interferon mixtures were assayed both on murine and human cells to determine the degree of neutralization achieved by each antiserum for the homologous and heterologous antiviral activities (Table 4). The anti-murine interferon serum neutralized the homologous antiviral TABLE
3
AFFINITY CHROMATOGRAPHY OF MURINE INTERFERON ON ANTI-HUMAN LEUKOCYTE INTERFERON ANTIBODY COLUMN Interferon
Fraction
Applied“ Unretained Retained*
activity
on
Murine (L-929)
Human (GM-258)
Bovine (MDBK)
1,309,ooo 1,309,ooo 25,600
3,640 ~32 980
60 43 10
a C-243-3 murine interferon applied in phosphatebuffered saline (pH 7.4) to column equilibrated in same buffer. * Antiviral activity specifically retained by column and eluted by pH 2.5 solution.
327
SUBSPECIES
activities of the three interferon preparations, whereas the anti-human Le interferon serum neutralized only the homologous antiviral activity of the murine interferon species retained by the affinity column. The anti-mm-me and anti-human Le interferon sera neutralized the heterologous human antiviral activities present both in the original murine interferon and the subspecies isolated by affinity chromatography. The degree of neutralization by the two antisera for the heterologous antiviral activity of the original murine interferon was lower than that achieved for the isolated murine interferon fraction possessing all the heterologous antiviral activities. One possible explanation for the lower neutralizing titers obtained with the original murine interferon preparation is that the interferon component comprising the bulk of homologous antiviral activity, while inactive on human cells, may interfere with the expression of antiviral activity by the minor murine species on human cells and, therefore, proportionately more neutralizing antibody would be required to neutralize the heterologous human activity in the original murine interferon. The anti-human F serum did not neutralize the antiviral activities of any one of the three murine interferons. The subspecies of murine interferon which was bound to the anti-Le interferon antibody column was chromatographed on a CH Sepharose 4B column in an attempt to further resolve the homologous and heterologous antiviral activities. This chromatographic technique has been reported to separate interferons based on differences in hydrophobicity (Davey et al., 1976) and
TABLE 4 NEUTRALIZATION ASSAYS OF THE ANTIVIRAL ACTIVITIES OF THE AFFINITY CHROMATOGRAPHY SEPARATED MURINE INTERFERONS BY SPECIFIC INTERFERON ANTISERA Interferon-neuNeutralization titer of antiserum on tralizing antiserum Murine (L-929) Human (GM-258) Original” Anti-mu&e Anti-human Anti-human
Le F
13,ooo ~25
92,
324-330 (1979)
Isolation of a Subspecies of Murine Interferon Antigenically Human Leukocyte Interferon EDWARD Department
of Microbiology,
Related to
A. HAVELL’
New York University
School of Medicine,
New York, New York 10016
Accepted August 21, 1978 Various preparations of virus-induced murine tissue culture interferon were found to exhibit some antiviral activity in cultures of human and bovine cells. The heterologous antiviral activity of these murine interferons on both human and bovine cultures was shown to be due to an antigenically distinct murine interferon subspecies. Isolation of the murine interferon species responsible for the heterologous antiviral activities was accomplished by means of antibody affinity chromatography using anti-human leukocyte interferon to selectively remove this interferon species from the bulk of murine antiviral activity. The isolated species, while only accounting for l-2% of homologous antiviral activity of the unfractionated interferon, was responsible for all the heterologous human and bovine antiviral activity. The antiviral activities of this minor murine interferon component on both murine and human cells were neutralized by antiserum against leukocyte interferon as well as by antiserum against mouse L cell interferon. INTRODUCTION
The degree of antiviral activity on cells of heterologous species has proven a useful parameter for differentiating interferons produced by the same species. Human interferons derived from cells of lymphoid origin differ markedly from interferons produced by nonlymphoid cells in their ability to render cells of other species resistant to virus replication. Human leukocyte (Le) interferon exhibits relatively more antiviral activity on bovine and porcine cells than human fibroblast (F) interferon (Gresser et al., 1974; Havell et al., 1977), whereas F interferon has been reported to be more active on rabbit cells than Le interferon (Desmyter et al., 1968). These two human interferons also differ in their physicochemical and antigenic properties (Stewart et al., 1974; Havell et al., 1975a) and available evidence suggests that these interferons are coded for by different genes (Cavalieri et al., 1977). Thus, the capacity of the two human interferons to render varying degrees of virus resistance to cells of other species is probably determined by the pri’ Present address: Trudeau Instit,ute, Inc., P. 0. Box 59, Saranac Lake, N.Y. 12983. 324 0042-6822/79/020324-07$02.00/O Copyright 0 1979by Academic Press, Inc. All rights of reproduction in any form reserved.
mary structures of the two interferon molecules. While it is not understood what inherent property of an interferon molecule is responsible for antiviral activity on cells of certain species, reports in the literature seem to suggest that human interferons generally exert relatively more antiviral activity on cells of other species than interferons derived from other species do in human cells (Finter, 1973). The notable exception to this generalization are murine interferons which have been reported to exhibit varying degrees of antiviral activity on human cells while human interferons were determined to be at best only marginally active on murine cells (Levy-Koenig et al., 1970; Bodo et al., 1971; Samuel and Farris, 1977). In this study, we have established that a minor interferon component present in virus-induced murine tissue culture interferon preparations is responsible for the heterologous antiviral activity on human and bovine cells. This murine interferon molecule is of interest from an evolutionary standpoint inasmuch as it possesses a common antigenic determinant(s) with human Le interferon.
DISTINCT MATERIALS
AND
MURINE
INTERFERON
METHODS
Cells. The murine L-929 cell line used in these studies has been maintained in this laboratory for several years and was originally obtained from Dr. J. S. Youngner, University of Pittsburgh. A strain of human skin fibroblast cells trisomic for chromosome 21, designated GM-258, was obtained from the Mammalian Genetic Mutant Cell Repository (Camden, N. J.) . A line of bovine kidney cells (MDBK) was provided by Dr. P. Sehgal (Rockefeller University). All cells were propagated in Eagle’s minimal essential medium (MEM) supplemented with 5% fetal bovine serum. Interferons. The Newcastle disease virus (NDV)-induced murine C-243-3 interferon was the kind gift of Dr. M. G. Tovey (Institut de Recherches Scientifiques sur le Cancer, Villejuif, France) and was prepared as previously described (Tovey et al., 1974). The NDV-induced murine L-929 interferon was prepared by inducing confluent monolayers of cells with NDV (Hickman strain) at an m.o.i. of 10. After a 1-hr adsorption period, the cultures were washed 3 x with MEM and then reincubated with medium containing 0.5% fetal bovine serum for an additional 30 hr, after which the interferoncontaining medium was collected. The mouse mengo virus interferon was a gift of Dr. E. Knight (DuPont, Wilmington, Del.), and was prepared according to his published procedure (Knight, 1975). All virusinduced interferon preparations were acidified at pH 2.0 for 5 days and then centrifuged at 100,000 g/hr to remove all residual VhS.
Human leukocyte (Le) interferon was prepared by Dr. K. Cantell (Helsinki, Finland) and human fibroblast (F) interferon was prepared by inducing FS-4 skin fibroblasts by the superinduction method of Havell and VilEek (1972). Interferon assays. Interferon assays were done by the micromethod of Armstrong (1971) as modified by Havell and Vilcek (1972) using murine (L-929), human (GM258), or bovine (MDBK) cells and vesicular stomatitis virus (VSV). Comparisons of the homologous and heterologous antiviral activities of the various interferon preparations were done simultaneously. The titer
SUBSPECIES
325
of the human leukocyte standard (G-023901-527) was about 20 times its assigned value when titrated on GM-258 cells. The murine interferon standard (G-002-904-511) gave titers equivalent to its designated antiviral activity on the murine L-929 cells.
Anti-interferon sera and affinity chromatography. The neutralization assays of interferon activities on homologous and heterologous cells with the various anti-interferon sera were performed as previously described (Have11 et al., 1975a). The rabbit anti-murine L cell interferon globulin (A-L Inf. No. 12,3/17/72) was obtained from the Research Resources Branch of the National Institute of Allergy and Infectious Diseases (Bethesda, Md.). The anti-human F interferon serum was prepared by immunization of rabbits as described earlier (Havell et al., 1975a) using purified human F interferon (>107 units/mg protein) as the antigen. The sheep anti-human Le interferon antibody used in the neutralization assays and in the antibody affinity chromatography procedures was the kind gift of Drs. D. GurariRotman and C. B. Anfinsen. The coupling of the anti-Le interferon antibodies to CnBr-activated Sepharose 4B (Pharmacia, Uppsala, Sweden) and the affinity chromatography procedures were essentially as described (Havell et aE.,1975a).
CH-Sepharose 4B Sorbent chromatography. Sorbent chromatography of murine interferon on CH-Sepharose 4B (Pharmacia, Uppsala, Sweden) was done according to the method of Davey et al., (1976). The murine interferon sample to be placed on the column was dialyzed against 0.05 M sodium acetate, pH 5.0 (E. buffer), for 18 hr at 4”. The sample was placed on a column (1.5 x 10 cm) equilibrated with E. buffer at 4’. The column was eluted sequentially with E. buffer, followed by 0.02 M sodium phosphate, pH 7.4 (El), and finally with 0.02 M sodium phosphate containing 0.05 M NaCl, pH 7.4 (E2). One-milliliter fractions were collected and then immediately assayed for antiviral activities on homologous and heterologous cells. RESULTS
Three . preparations of virus-induced murine tissue culture interferons were simul-
326
EDWARD
A. HAVELL
taneously assayed for their antiviral activities on murine L-929, human GM-258, and bovine MDBK cell cultures (Table 1). All three murine interferons were active on both human GM-258 and bovine MDBK cultures. The ratio of the murine to human antiviral activities was 128:l for both L cell interferon preparations and 338:l for the NDV-induced C-243-3 cell interferon. A human leukocyte interferon, while inactive on murine cells, showed a ratio of human to bovine antiviral activity which was in a similar range as observed with the murine interferons. A quite unexpected finding came from an experiment in which the heterologous human antiviral activity of a murine interferon preparation was found to be neutralized not only by homologous anti-murine interferon serum but also by antiserum against human Le interferon. Antiserum specific for human F interferon did not neutralize the heterologous activity of this murine interferon on human GM-258 cells (Table 2). The results obtained with the specific anti-human interferon sera and the demonstration that the various murine interferons exhibited similar human to bovine antiviral activity ratios as did the human leukocyte interferon suggested the existence of a murine interferon which resembled human leukocyte interferon both antigenically and in expression of antiviral activity on bovine cells. It was reasoned that if the antiviral activity of the murine interferon was neutralized by the specific anti-human Le serum, then it should be possible to isolate the murine interferon component bearing the common antigenic determinant(s) with the human Le interferon by means of antibody affinity chromatography. The C-243-3 murine inTABLE
terferon preparation was passed through a column consisting of anti-human Le interferon y-globulin covalently coupled to a Sepharose 4B matrix. In previous experiments, this chromatographic technique enabled the identification and quantitation of antigenically distinct interferon subspecies present in various human interferon preparations (Berg et al., 1975; Have11 et al., 1975a). Two fractions of interferon activity were isolated after passing the original (applied) murine C-243-3 interferon through the anit-human Le interferon column (Table 3). The murine antiviral activity which was specifically bound (retained fraction) by the immobilized antibody could only be eluted by lowering the pH of the column. The retained fraction represented only about 1.5% of the murine antiviral activity present in the original interferon preparation. However, this fraction contained all of the human and bovine antiviral activity recovered from the affinity column. While the ratio of the murine to human antiviral activity for the bulk of the murine interferon which passed unretained through the column was >40,000, this ratio for the specifically bound interferon component was 26. In a similar affinity chromatography TABLE
2
THE EFFECT OF SPECIFIC INTERFERON NEUTRALIZING SERA ON THE ANTIVIRAL ACTIVITIES OF MURINE AND HUMAN INTERFERONS IN HUMAN GM-258 CELLS Interferon-neutralizmg serum
Neutralizing Murine (C-243-3)
Anti-murine Anti-human Anti-human
Le F
titer for interferon HumanLe
40 200 t25
~25 6,500 t25
Human F
~25 l,f=)
1
ANTIVIRAL ACTIVITIES OF VARIOUS MURINE INTERFERON PREPARATIONS ON CELLS OF DIFFERENT SPECIES Antiviral activity (units/ml) on cells Inducing virus Cell source of interferon Murine
(L-929) Hum28iGM-
C-243-3 L-929 L-929 Leukocyte
(human)
Newcastle disease Newcastle diesase Mouse mengo Sendai
1,300,OOO 32,800 32,800 >4
3,840 256 256 2,048
Bovine (MDBK) 60 24 8 48
DISTINCT
MURINE
INTERFERON
procedure using a column of anti-human F interferon y-globulin, none of the murine antiviral activity was retained by the antibody. These findings demonstrate that a minor species of murine interferon (1) shows antigenic homology with human Le interferon and (2) is responsible for the heterologous antiviral activities on human and bovine cells. Antibody neutralization assays were performed on the antiviral activities of the original murine interferon and the two fractions of antiviral activity separated by means of affinity chromatography with neutralizing antisera specific for murine, human Le, and human F interferons. The antisera and interferon mixtures were assayed both on murine and human cells to determine the degree of neutralization achieved by each antiserum for the homologous and heterologous antiviral activities (Table 4). The anti-murine interferon serum neutralized the homologous antiviral TABLE
3
AFFINITY CHROMATOGRAPHY OF MURINE INTERFERON ON ANTI-HUMAN LEUKOCYTE INTERFERON ANTIBODY COLUMN Interferon
Fraction
Applied“ Unretained Retained*
activity
on
Murine (L-929)
Human (GM-258)
Bovine (MDBK)
1,309,ooo 1,309,ooo 25,600
3,640 ~32 980
60 43 10
a C-243-3 murine interferon applied in phosphatebuffered saline (pH 7.4) to column equilibrated in same buffer. * Antiviral activity specifically retained by column and eluted by pH 2.5 solution.
327
SUBSPECIES
activities of the three interferon preparations, whereas the anti-human Le interferon serum neutralized only the homologous antiviral activity of the murine interferon species retained by the affinity column. The anti-mm-me and anti-human Le interferon sera neutralized the heterologous human antiviral activities present both in the original murine interferon and the subspecies isolated by affinity chromatography. The degree of neutralization by the two antisera for the heterologous antiviral activity of the original murine interferon was lower than that achieved for the isolated murine interferon fraction possessing all the heterologous antiviral activities. One possible explanation for the lower neutralizing titers obtained with the original murine interferon preparation is that the interferon component comprising the bulk of homologous antiviral activity, while inactive on human cells, may interfere with the expression of antiviral activity by the minor murine species on human cells and, therefore, proportionately more neutralizing antibody would be required to neutralize the heterologous human activity in the original murine interferon. The anti-human F serum did not neutralize the antiviral activities of any one of the three murine interferons. The subspecies of murine interferon which was bound to the anti-Le interferon antibody column was chromatographed on a CH Sepharose 4B column in an attempt to further resolve the homologous and heterologous antiviral activities. This chromatographic technique has been reported to separate interferons based on differences in hydrophobicity (Davey et al., 1976) and
TABLE 4 NEUTRALIZATION ASSAYS OF THE ANTIVIRAL ACTIVITIES OF THE AFFINITY CHROMATOGRAPHY SEPARATED MURINE INTERFERONS BY SPECIFIC INTERFERON ANTISERA Interferon-neuNeutralization titer of antiserum on tralizing antiserum Murine (L-929) Human (GM-258) Original” Anti-mu&e Anti-human Anti-human
Le F
13,ooo ~25
