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Synthesis and biological characterization of a covalent conjugate between interferon and ricin toxin B chain
VIROLOGY 123,457-460
(1982)
Synthesis and Biological Characterization of a Covalent Conjugate Interferon and Ricin Toxin 6 Chain
PAUL ANDERSON’ Department
AND
between
JAN VIL~EK
of Microbiology, New York University School of Medicine, 550 First Avenue, New York, New York 10016 Received June 22, 1982; accepted August 9, 1982
Human interferon (IFN)-d has been covalently conjugated to ricin toxin B chain (RTB) using the heterobifunctional crosslinking reagent, N-succinimidyl-3(2-pyridyldithio)propionate (SPDP). Conjugate molecules (RTB:IFN) were purified by preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and assayed for antiviral activity on human GM-258 fibroblasts in the presence or absence of competing concentrations of galactose, the specific sugar employed by RTB for cell surface binding. While the antiviral activity of native IFN-a was unchanged in the presence of galactose, the antiviral activity of RTB:IFN was markedly diminished in the presence of galactose at concentrations ranging from 1 to 100 n&f. These studies demonstrate that IFN-(Y can exert an antiviral effect on human fibroblasts after binding to the receptor of another ligand molecule (i.e., RTB).
ways be necessary for the expression of biological activity. In this paper, we describe the construction of an intermolecular conjugate between RTB and IFN (RTB:IFN) which possesses IFN activity mediated through the receptor for RTB. Our data indicate that in conjugated form IFN can exert its antiviral effect after initially binding to the cell surface receptor of another ligand. This may be useful as a method to target IFN to specific tissues or to increase the effective number of IFN molecules bound to cells. Procedures used for the propagation of human fibroblast cell strains (FS-4 and GM258) have been described (7). IFN titers were determined by a semimicro method in human FS-4 cells using mouse encephalomyocarditis virus as previously described (8). Titers of IFN-(U are expressed in international units using human IFN research standard G-023-901-527 provided by the National Institute of Allergy and Infectious Diseases. Partially purified human IFN-a was obtained from Key Interferon; the preparation contained approximately 1 X lo6 units
The binding of IFN to specific membrane receptors elicits a characteristic biological response in susceptible cells (1-3). It is not clear, however, whether receptor binding is in itself sufficient, or even necessary for the induction of the antiviral state. In the case of some polypeptide toxins (e.g., ricin and diptheria toxin), it has recently been shown that their toxic action can be expressed without binding of the toxin to its usual cell surface receptor. Thus, intermolecular conjugates between the ricin toxin A chain and either epiderma1 growth factor (4) or human chorionic gonadotropin (5) have been shown to mediate A chain toxicity via the receptor for the conjugate ligand. More recently, a conjugate between the ricin toxin B chain (RTB) and insulin has been shown to mediate insulin biological activity via the ritin receptor (6). Experiments such as these suggest that conjugate molecules may alter the receptor specificity of their biologically active components. Furthermore, they suggest that specific receptor occupancy may not ali To whom reprint
requests should be addressed.
457
0042-6822/82/160457-04$02.00/O
Copyright 0 1982by AcademicPress,Inc. All riehta of reproductionin any form reserved.
458
SHORT
COMMUNICATIONS
and 1 mg protein/ml. Antiserum to IFN(Ywas prepared as previously described (9). The covalent coupling of IFN and RTB was effected with the aid of the heterobifunctional crosslinking reagent, Nsuccinimidyl- 3(2 - pyridyldithio)propionate (SPDP). Human IFN-a was treated with 20 mM recrystallized iodoacetamide (Pierce Chem. Co.) for 18 hr at 22” to block free sulfhydryls. Amino-terminal nitrogens were then blocked by reaction with 10 mM citraconic anhydride (Sigma Chem. Co.) for 30 min at 22” at pH 6.9 (6, 10). IFN was then reacted with a twofold molar excess of SPDP (Pierce Chem. Co.) for 30 min at 22’. This solution was made 0.2 M in glycine (Miles Lab., Inc.), incubated for 15 min, dialyzed against 10 mM HCl for 6 hr at 22’, then further dialyzed against phosphatebuffered saline (PBS), pH 7.4 for 18 hr at 4”. The resulting IFN derivative (IFN-PDP) was stored at -70” for up to 30 days without loss of activity. Ricin toxin (RCA-60) was obtained from E-Y Laboratories. Toxin subunits were separated by treatment with 5% p-mercaptoethanol and 0.5 M galactose overnight at 22”. Following adsorption to DEAE-cellulose, toxin subunits were eluted with a linear NaCl gradient (O-O.15 M) as described by Olsnes and Pihl (12). Gradient elution produced two protein peaks, one containing RTB, the other RCA-60 which apparently reformed during the elution procedure. Pooled fractions from the RTB peak contained a single Coomassie blue-stainable band migrating at approximately 30,000 daltons on SDS-PAGE. Purified RTB (1 mg/ml in PBS, pH 7.4) was treated with 50 m&f dithiothreitol (Calbiochem-Behring Corp.) for 4 hr at 22”. Reduced RTB was separated from dithiothreitol by chromatography on Sephadex G-25 (fine) equilibrated with deoxygenated PBS containing 1 miV ethylenediaminetetraacetic acid. Fractions eluting in the void volume were pooled and immediately reacted with IFN-PDP at a weight ratio of 1.0 for 72 hr at 4” or 12 hr at 22”. Reaction mixtures containing RTB:IFN were purified by preparative SDS-PAGE according to the method of Laemmli (12).
Gel slices (2 mm) were eluted into MEM containing human serum albumin (1 mg/ ml) overnight at room temperature. Eluates were assayed for IFN activity (Fig. 1). Native IFN-a is resolved into two major components which migrate at approximately 16,000 and 20,000 daltons (slices 35-42, Fig. 1) as has been reported previously (13). In addition, there is a peak of IFN activity which migrates at approximately 50,000 daltons (slices 18-20, Fig. 1). This is precisely the position at which RTB:IFN would be expected to migrate based on the molecular weights of its component parts. Gel slices obtained from regions containing unreacted RTB (M, = 30,000, slices 26-28), the presence of which was established by the inclusion of actracer lz51-RTB, showed no antiviral tivity in this assay. RTB binds to galactose residues present on the surface of toxin-susceptible cells (14). We have verified that the binding of lz51-RTB to human GM-258 fibroblasts is greatly reduced in the presence of competing concentrations of galactose (Fig.
10
ib Gel Slice
30
40
FIG. 1. SDS-PAGE of RTB:IFN. RTB:IFN, prepared as described in the text, was adjusted to contain 0.1% SDS, 6% glycerol, and 0.01% bromphenol blue. After 15-30 min at room temperature, samples were applied to a 3-mm SDS-polyacrylamide slab gel containing 5% acrylamide in the stacking gel and a gradient of lo-16% acrylamide in the separating gel. Electrophoresis was for 3-4 hr at 60 mA. Following electrophoresis, gel slices (2 mm) were eluted into MEM containing HSA (1 mg/ml) and assayed for antiviral activity (0) as described in the text. The positions of molecular weight markers (0) relative to the gel slices were determined by Coomassie blue staining. The migratory positions of purified RCA-60 and RTB are indicated by arrows.
SHORT
[Goloctose]
459
COMMUNICATIONS
, mM
FIG. 2 (A). Effect of various concentrations of galactose on ‘*sI-RTB binding. Purified RTB was iodinated with the aid of chloramine-T under the following reaction conditions: 80 ~1 RTB (1 mg/ml in PBS), 10 pl i*‘I (1 mCi) in 0.5 A4 phosphate buffer, pH 7.5, 12 ~1 Me&SO, 20 ~1 chloramine-T (2.5 mg/ml in 0.5 M phosphate buffer, pH 7.5). After 25 set at room temperature, 20 pl sodium metahisulfite (5 mg/ ml in 0.5 it4 phosphate buffer) was added to stop the reaction. Following the removal of free ‘%I on Sephadex G-25, iZ51-RTB (4.5 pg/ml; 1.2 X lo6 cpm) was added to confluent monolayers of GM-258 fibroblasts for 60 min at 37” in the presence of increasing concentrations of galactose in MEM. Cells were washed four times, solubilized in 1% SDS, and counted for iz51 cpm in a Beckman gamma counter. Specific binding averaged 95% of total binding which in the absence of free galactose was 7.5 X lo5 cpm per monolayer. (B) Effect of galactose on RTB:IFN biological activity. RTB:IFN (approx. 100 units/ml in MEMHSA, from fractions 18-20, Fig. 1) or native IFN (approx. 50 units/ml in MEM-HSA, from fractions 37-42, Fig. 1) were applied to confluent monolayers of GM-258 fibroblasts for 60 min at 37” in the presence of increasing concentrations of galactose. Cells were then washed three times with MEM, overlaid with MEM containing FBS (lo%, v/v) and incubated for 16 hr before challenge with vesicular stomatitis virus (VSV) at an m.o.i. of 2-3. Virus was absorbed for 1 hr, the cells were washed extensively, and the culture fluid was harvested after one cycle of replication (9 hr). Virus titers were determined by a plaque assay in Vero cells. The graph indicates reduction in VSV yield produced by native IFN-(Y (0) or by RTB:IFN (0) in the presence of various concentrations of free galactose. The VSV yield in control cultures not treated with IFN was 8.5 X 10s PFU/ml.
2A). Similarly, the antiviral activity of both native IFN (eluted from gel slices 3542, Fig. 1) and RTB:IFN (eluted from gel slices 18-20, Fig. 1) were measured in the absence or presence of competing galac-
tose. As shown in Fig. 2B, both native IFN and RTB:IFN exert antiviral activity on human GM-258 fibroblasts. In the presence of galactose, however, the antiviral effect of RTB:IFN is markedly diminished, whereas that of native IFN is unaltered. It is interesting that when the activity of RTB:IFN is maximally inhibited by galactose, a significant amount of antiviral activity remains (0.5 log reduction at lOO150 m&f galactose). This may result from the dissociation of RTB:IFN in the presence of cells to liberate free IFN which then binds to its native receptor. Alternatively, some of the RTB:IFN may directly bind to the IFN receptor without first binding to cell surface galactose residues. The ability of antiserum to IFN-LX to neutralize the antiviral activity of free IFN and RTB:IFN is shown in Table 1. The fact that activity of RTB:IFN is abolished after treatment with this antiserum excludes the possibility that a residual toxic effect of RTB:IFN is responsible for the inhibition of virus replication. Although it is clear that RTB:IFN possesses an altered receptor specificity for IFN biological activity, the molecular mechanism by which this occurs is uncertain. Both RCA-60 (15) and RTB (14) are known to be internalized after binding to the cell surface. While the fate of the internalized ligand has not been established, TABLE
1
NEUTRALIZATION OF RTB:IFN ANTISERUM TO IFN-d IFN preparation IFN-(U (fraction RTB:IFN
39)
(fraction
19)
BY
Antiserum
IFN titer
None Anti-IFN-cY
128 <4
None Anti-IFN-cu
64 <4
a Samples eluted from the indicated gel slice (see Fig. 1) were incubated in the presence or absence of antiserum to IFN-a for 60 min at 37’. Antiserum to IFN-o, prepared in rabbits as described previously (9), was used at a final dilution of 1:lOO. IFN titers were determined as described in the text, using FS4 fibroblasts and vesicular stomatitis virus.
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proteolytic degradation of internalized RTB could not be detected after a 3-br incubation with mouse leukemia cells (14). IFN bound to the receptor of its conjugate ligand (RTB) may be directly internalized whereupon it may interact with intracellular components to induce an antiviral state. We have previously shown that certain primary amines known to inhibit the internalization of some receptor-binding ligands also inhibit the antiviral activity of native IFNs (16). Circumstantial support for the idea that surface-bound native IFN is internalized also comes from studies employing radiolabeled IFN-a (2) or IFN--, (3) in which the maximum binding obtained at 37” is significantly greater than that obtained at 4”. These results are compatible with a mechanism involving the internalization of surface-bound IFN with the subsequent recycling of internalized receptors back to the plasma membrane. We cannot rule out the possibility that after the initial binding of RTB:IFN to a cell surface galactose residue, the IFN portion of the conjugate must also interact with the IFN receptor in order to induce the antiviral state. If RTB:IFN is internalized via the RTB receptor, it is possible that upon sequestration within an endocytic vesicle, the IFN portion of the conjugate binds to its proper receptor which could conceivably be present in the same vesicle. Alternatively, RTB:IFN may first bind to a cell surface galactose residue via its RTB component after which it may also bind to the IFN receptor while still on the cell surface. Any of these proposed mechanisms would allow the targeting of IFN to cells possessing a high number of receptors for RTB. The targeting of IFN to specific cell types would require the use of different conjugate ligands depending on the selected target tissue. We have also constructed an intermolecular conjugate between IFN-a and the serum glycoprotein, az-macroglobulin (Tycko, Anderson, Vilcek, and Maxfield, manuscript in preparation). It too was shown to exert IFN bio-
logical activity which was mediated through the receptor for the conjugate ligand (LYEmacroglobulin). The interaction of CQmacroglobulin with its cell surface receptor has been well characterized (27). A comparison of the biological activities of RTB:IFN and a2-macroglobulin:IFN may help to differentiate between the putative mechanisms listed above. ACKNOWLEDGMENTS We thank Benjamin Tycko and Frederick Maxfield for help in the preparation of interferon conjugates, and Michele Cassano for typing the manuscript. This work was supported by USPHS Grants AI-07057 and AI-12948. REFERENCES 1. AGUET, M., Nature (London) 284,459-4610980). 2. BRANCA, A. A., and BAGLIONI, C., Nature (Lendon), 294, 768-770 (1981). 3. ANDERSON, P., YIP, Y. K., and VILCEK, J., J. Biol. Chem., in press. 4. CAWLEY. D. B.. HERSCHMAN, H. R., GILLILAND, D. G.; and COLLIN, R. J.; Cell 22, 563-570 (1980). 5. OELTMANN, T. N., and HEATH, E. C., J. Biol. &em. 254, 1028-1032 (1979). 6. ROTH, R. A., MADDUX, B. A., WONG, K. Y., IWAMOTO, Y., and GOLDFINE, I. D., J. Biol. Chm. 256, 5350-5354 (1981). 7. HAYES, T. G., YIP, Y. K., and VIL~EK, J., Virology 98, 351-363 (1979). 8. HAVELL, E. A., and VILCEK, J., Antimicrob. Agents Chemother. 2, 476-484 (1972). 9. VIL~EK, J., YAMAZAKI, S., and HAVELL, E. A., Infect. Zmmun. 18,863-867 (1977). 10. SCHECTER, Y., SCHLESSINGER, J., JACOBS, S., CHANG, K.-J., and CUATRECASAS, P., Proc. Nat. Acad. Sci. USA 75, 2135-2139 (1978). 11. OLSNES, S., and PIHL, A., Biochemistry 12,31213126 (1973). 12. LAEMMLI, U. K., Nature (London) 227,680-685 (1970). 13. STEWART, W. E., and DESMYTER, J., Virology 67, 68-78 (1975). 14. HOUSTON, L. L., J. Biol. Chem. 257, 1532-1539 (1982). 15. RAY, B., and WU, H. C., Mol. Cell. Biol. 1, 544551 (1981). 16. ANDERSON, P., TYCKO, B., MAXFIELD, F., and VIL~EK, J., Virology 117, 510-515 (1982). 17. DICKSON, R. B., WILLINGHAM, M. C., and PASTAN, I., J. Biol. Chem. 256,3454-3459 (1981).
(1982)
Synthesis and Biological Characterization of a Covalent Conjugate Interferon and Ricin Toxin 6 Chain
PAUL ANDERSON’ Department
AND
between
JAN VIL~EK
of Microbiology, New York University School of Medicine, 550 First Avenue, New York, New York 10016 Received June 22, 1982; accepted August 9, 1982
Human interferon (IFN)-d has been covalently conjugated to ricin toxin B chain (RTB) using the heterobifunctional crosslinking reagent, N-succinimidyl-3(2-pyridyldithio)propionate (SPDP). Conjugate molecules (RTB:IFN) were purified by preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and assayed for antiviral activity on human GM-258 fibroblasts in the presence or absence of competing concentrations of galactose, the specific sugar employed by RTB for cell surface binding. While the antiviral activity of native IFN-a was unchanged in the presence of galactose, the antiviral activity of RTB:IFN was markedly diminished in the presence of galactose at concentrations ranging from 1 to 100 n&f. These studies demonstrate that IFN-(Y can exert an antiviral effect on human fibroblasts after binding to the receptor of another ligand molecule (i.e., RTB).
ways be necessary for the expression of biological activity. In this paper, we describe the construction of an intermolecular conjugate between RTB and IFN (RTB:IFN) which possesses IFN activity mediated through the receptor for RTB. Our data indicate that in conjugated form IFN can exert its antiviral effect after initially binding to the cell surface receptor of another ligand. This may be useful as a method to target IFN to specific tissues or to increase the effective number of IFN molecules bound to cells. Procedures used for the propagation of human fibroblast cell strains (FS-4 and GM258) have been described (7). IFN titers were determined by a semimicro method in human FS-4 cells using mouse encephalomyocarditis virus as previously described (8). Titers of IFN-(U are expressed in international units using human IFN research standard G-023-901-527 provided by the National Institute of Allergy and Infectious Diseases. Partially purified human IFN-a was obtained from Key Interferon; the preparation contained approximately 1 X lo6 units
The binding of IFN to specific membrane receptors elicits a characteristic biological response in susceptible cells (1-3). It is not clear, however, whether receptor binding is in itself sufficient, or even necessary for the induction of the antiviral state. In the case of some polypeptide toxins (e.g., ricin and diptheria toxin), it has recently been shown that their toxic action can be expressed without binding of the toxin to its usual cell surface receptor. Thus, intermolecular conjugates between the ricin toxin A chain and either epiderma1 growth factor (4) or human chorionic gonadotropin (5) have been shown to mediate A chain toxicity via the receptor for the conjugate ligand. More recently, a conjugate between the ricin toxin B chain (RTB) and insulin has been shown to mediate insulin biological activity via the ritin receptor (6). Experiments such as these suggest that conjugate molecules may alter the receptor specificity of their biologically active components. Furthermore, they suggest that specific receptor occupancy may not ali To whom reprint
requests should be addressed.
457
0042-6822/82/160457-04$02.00/O
Copyright 0 1982by AcademicPress,Inc. All riehta of reproductionin any form reserved.
458
SHORT
COMMUNICATIONS
and 1 mg protein/ml. Antiserum to IFN(Ywas prepared as previously described (9). The covalent coupling of IFN and RTB was effected with the aid of the heterobifunctional crosslinking reagent, Nsuccinimidyl- 3(2 - pyridyldithio)propionate (SPDP). Human IFN-a was treated with 20 mM recrystallized iodoacetamide (Pierce Chem. Co.) for 18 hr at 22” to block free sulfhydryls. Amino-terminal nitrogens were then blocked by reaction with 10 mM citraconic anhydride (Sigma Chem. Co.) for 30 min at 22” at pH 6.9 (6, 10). IFN was then reacted with a twofold molar excess of SPDP (Pierce Chem. Co.) for 30 min at 22’. This solution was made 0.2 M in glycine (Miles Lab., Inc.), incubated for 15 min, dialyzed against 10 mM HCl for 6 hr at 22’, then further dialyzed against phosphatebuffered saline (PBS), pH 7.4 for 18 hr at 4”. The resulting IFN derivative (IFN-PDP) was stored at -70” for up to 30 days without loss of activity. Ricin toxin (RCA-60) was obtained from E-Y Laboratories. Toxin subunits were separated by treatment with 5% p-mercaptoethanol and 0.5 M galactose overnight at 22”. Following adsorption to DEAE-cellulose, toxin subunits were eluted with a linear NaCl gradient (O-O.15 M) as described by Olsnes and Pihl (12). Gradient elution produced two protein peaks, one containing RTB, the other RCA-60 which apparently reformed during the elution procedure. Pooled fractions from the RTB peak contained a single Coomassie blue-stainable band migrating at approximately 30,000 daltons on SDS-PAGE. Purified RTB (1 mg/ml in PBS, pH 7.4) was treated with 50 m&f dithiothreitol (Calbiochem-Behring Corp.) for 4 hr at 22”. Reduced RTB was separated from dithiothreitol by chromatography on Sephadex G-25 (fine) equilibrated with deoxygenated PBS containing 1 miV ethylenediaminetetraacetic acid. Fractions eluting in the void volume were pooled and immediately reacted with IFN-PDP at a weight ratio of 1.0 for 72 hr at 4” or 12 hr at 22”. Reaction mixtures containing RTB:IFN were purified by preparative SDS-PAGE according to the method of Laemmli (12).
Gel slices (2 mm) were eluted into MEM containing human serum albumin (1 mg/ ml) overnight at room temperature. Eluates were assayed for IFN activity (Fig. 1). Native IFN-a is resolved into two major components which migrate at approximately 16,000 and 20,000 daltons (slices 35-42, Fig. 1) as has been reported previously (13). In addition, there is a peak of IFN activity which migrates at approximately 50,000 daltons (slices 18-20, Fig. 1). This is precisely the position at which RTB:IFN would be expected to migrate based on the molecular weights of its component parts. Gel slices obtained from regions containing unreacted RTB (M, = 30,000, slices 26-28), the presence of which was established by the inclusion of actracer lz51-RTB, showed no antiviral tivity in this assay. RTB binds to galactose residues present on the surface of toxin-susceptible cells (14). We have verified that the binding of lz51-RTB to human GM-258 fibroblasts is greatly reduced in the presence of competing concentrations of galactose (Fig.
10
ib Gel Slice
30
40
FIG. 1. SDS-PAGE of RTB:IFN. RTB:IFN, prepared as described in the text, was adjusted to contain 0.1% SDS, 6% glycerol, and 0.01% bromphenol blue. After 15-30 min at room temperature, samples were applied to a 3-mm SDS-polyacrylamide slab gel containing 5% acrylamide in the stacking gel and a gradient of lo-16% acrylamide in the separating gel. Electrophoresis was for 3-4 hr at 60 mA. Following electrophoresis, gel slices (2 mm) were eluted into MEM containing HSA (1 mg/ml) and assayed for antiviral activity (0) as described in the text. The positions of molecular weight markers (0) relative to the gel slices were determined by Coomassie blue staining. The migratory positions of purified RCA-60 and RTB are indicated by arrows.
SHORT
[Goloctose]
459
COMMUNICATIONS
, mM
FIG. 2 (A). Effect of various concentrations of galactose on ‘*sI-RTB binding. Purified RTB was iodinated with the aid of chloramine-T under the following reaction conditions: 80 ~1 RTB (1 mg/ml in PBS), 10 pl i*‘I (1 mCi) in 0.5 A4 phosphate buffer, pH 7.5, 12 ~1 Me&SO, 20 ~1 chloramine-T (2.5 mg/ml in 0.5 M phosphate buffer, pH 7.5). After 25 set at room temperature, 20 pl sodium metahisulfite (5 mg/ ml in 0.5 it4 phosphate buffer) was added to stop the reaction. Following the removal of free ‘%I on Sephadex G-25, iZ51-RTB (4.5 pg/ml; 1.2 X lo6 cpm) was added to confluent monolayers of GM-258 fibroblasts for 60 min at 37” in the presence of increasing concentrations of galactose in MEM. Cells were washed four times, solubilized in 1% SDS, and counted for iz51 cpm in a Beckman gamma counter. Specific binding averaged 95% of total binding which in the absence of free galactose was 7.5 X lo5 cpm per monolayer. (B) Effect of galactose on RTB:IFN biological activity. RTB:IFN (approx. 100 units/ml in MEMHSA, from fractions 18-20, Fig. 1) or native IFN (approx. 50 units/ml in MEM-HSA, from fractions 37-42, Fig. 1) were applied to confluent monolayers of GM-258 fibroblasts for 60 min at 37” in the presence of increasing concentrations of galactose. Cells were then washed three times with MEM, overlaid with MEM containing FBS (lo%, v/v) and incubated for 16 hr before challenge with vesicular stomatitis virus (VSV) at an m.o.i. of 2-3. Virus was absorbed for 1 hr, the cells were washed extensively, and the culture fluid was harvested after one cycle of replication (9 hr). Virus titers were determined by a plaque assay in Vero cells. The graph indicates reduction in VSV yield produced by native IFN-(Y (0) or by RTB:IFN (0) in the presence of various concentrations of free galactose. The VSV yield in control cultures not treated with IFN was 8.5 X 10s PFU/ml.
2A). Similarly, the antiviral activity of both native IFN (eluted from gel slices 3542, Fig. 1) and RTB:IFN (eluted from gel slices 18-20, Fig. 1) were measured in the absence or presence of competing galac-
tose. As shown in Fig. 2B, both native IFN and RTB:IFN exert antiviral activity on human GM-258 fibroblasts. In the presence of galactose, however, the antiviral effect of RTB:IFN is markedly diminished, whereas that of native IFN is unaltered. It is interesting that when the activity of RTB:IFN is maximally inhibited by galactose, a significant amount of antiviral activity remains (0.5 log reduction at lOO150 m&f galactose). This may result from the dissociation of RTB:IFN in the presence of cells to liberate free IFN which then binds to its native receptor. Alternatively, some of the RTB:IFN may directly bind to the IFN receptor without first binding to cell surface galactose residues. The ability of antiserum to IFN-LX to neutralize the antiviral activity of free IFN and RTB:IFN is shown in Table 1. The fact that activity of RTB:IFN is abolished after treatment with this antiserum excludes the possibility that a residual toxic effect of RTB:IFN is responsible for the inhibition of virus replication. Although it is clear that RTB:IFN possesses an altered receptor specificity for IFN biological activity, the molecular mechanism by which this occurs is uncertain. Both RCA-60 (15) and RTB (14) are known to be internalized after binding to the cell surface. While the fate of the internalized ligand has not been established, TABLE
1
NEUTRALIZATION OF RTB:IFN ANTISERUM TO IFN-d IFN preparation IFN-(U (fraction RTB:IFN
39)
(fraction
19)
BY
Antiserum
IFN titer
None Anti-IFN-cY
128 <4
None Anti-IFN-cu
64 <4
a Samples eluted from the indicated gel slice (see Fig. 1) were incubated in the presence or absence of antiserum to IFN-a for 60 min at 37’. Antiserum to IFN-o, prepared in rabbits as described previously (9), was used at a final dilution of 1:lOO. IFN titers were determined as described in the text, using FS4 fibroblasts and vesicular stomatitis virus.
460
SHORT
COMMUNICATIONS
proteolytic degradation of internalized RTB could not be detected after a 3-br incubation with mouse leukemia cells (14). IFN bound to the receptor of its conjugate ligand (RTB) may be directly internalized whereupon it may interact with intracellular components to induce an antiviral state. We have previously shown that certain primary amines known to inhibit the internalization of some receptor-binding ligands also inhibit the antiviral activity of native IFNs (16). Circumstantial support for the idea that surface-bound native IFN is internalized also comes from studies employing radiolabeled IFN-a (2) or IFN--, (3) in which the maximum binding obtained at 37” is significantly greater than that obtained at 4”. These results are compatible with a mechanism involving the internalization of surface-bound IFN with the subsequent recycling of internalized receptors back to the plasma membrane. We cannot rule out the possibility that after the initial binding of RTB:IFN to a cell surface galactose residue, the IFN portion of the conjugate must also interact with the IFN receptor in order to induce the antiviral state. If RTB:IFN is internalized via the RTB receptor, it is possible that upon sequestration within an endocytic vesicle, the IFN portion of the conjugate binds to its proper receptor which could conceivably be present in the same vesicle. Alternatively, RTB:IFN may first bind to a cell surface galactose residue via its RTB component after which it may also bind to the IFN receptor while still on the cell surface. Any of these proposed mechanisms would allow the targeting of IFN to cells possessing a high number of receptors for RTB. The targeting of IFN to specific cell types would require the use of different conjugate ligands depending on the selected target tissue. We have also constructed an intermolecular conjugate between IFN-a and the serum glycoprotein, az-macroglobulin (Tycko, Anderson, Vilcek, and Maxfield, manuscript in preparation). It too was shown to exert IFN bio-
logical activity which was mediated through the receptor for the conjugate ligand (LYEmacroglobulin). The interaction of CQmacroglobulin with its cell surface receptor has been well characterized (27). A comparison of the biological activities of RTB:IFN and a2-macroglobulin:IFN may help to differentiate between the putative mechanisms listed above. ACKNOWLEDGMENTS We thank Benjamin Tycko and Frederick Maxfield for help in the preparation of interferon conjugates, and Michele Cassano for typing the manuscript. This work was supported by USPHS Grants AI-07057 and AI-12948. REFERENCES 1. AGUET, M., Nature (London) 284,459-4610980). 2. BRANCA, A. A., and BAGLIONI, C., Nature (Lendon), 294, 768-770 (1981). 3. ANDERSON, P., YIP, Y. K., and VILCEK, J., J. Biol. Chem., in press. 4. CAWLEY. D. B.. HERSCHMAN, H. R., GILLILAND, D. G.; and COLLIN, R. J.; Cell 22, 563-570 (1980). 5. OELTMANN, T. N., and HEATH, E. C., J. Biol. &em. 254, 1028-1032 (1979). 6. ROTH, R. A., MADDUX, B. A., WONG, K. Y., IWAMOTO, Y., and GOLDFINE, I. D., J. Biol. Chm. 256, 5350-5354 (1981). 7. HAYES, T. G., YIP, Y. K., and VIL~EK, J., Virology 98, 351-363 (1979). 8. HAVELL, E. A., and VILCEK, J., Antimicrob. Agents Chemother. 2, 476-484 (1972). 9. VIL~EK, J., YAMAZAKI, S., and HAVELL, E. A., Infect. Zmmun. 18,863-867 (1977). 10. SCHECTER, Y., SCHLESSINGER, J., JACOBS, S., CHANG, K.-J., and CUATRECASAS, P., Proc. Nat. Acad. Sci. USA 75, 2135-2139 (1978). 11. OLSNES, S., and PIHL, A., Biochemistry 12,31213126 (1973). 12. LAEMMLI, U. K., Nature (London) 227,680-685 (1970). 13. STEWART, W. E., and DESMYTER, J., Virology 67, 68-78 (1975). 14. HOUSTON, L. L., J. Biol. Chem. 257, 1532-1539 (1982). 15. RAY, B., and WU, H. C., Mol. Cell. Biol. 1, 544551 (1981). 16. ANDERSON, P., TYCKO, B., MAXFIELD, F., and VIL~EK, J., Virology 117, 510-515 (1982). 17. DICKSON, R. B., WILLINGHAM, M. C., and PASTAN, I., J. Biol. Chem. 256,3454-3459 (1981).