VIROLOGY
64, 349-357 (1975)
Immunological DAN1 P. BOLOGNESI, Departments
Properties
of Avian Oncornavirus
Polypeptides’
RYOTARO ISHIZAKI, GUDRUN HOPER, THOMAS C. VANAMAN, AND RALPH E. SMITH
of Surgery and Microbiology
and Immunology, Duke University North Carolina 27710
Accepted
November
Medical
Center, Durham,
13, 1974
The major polypeptides and glycoproteins of avian myeloblastosis virus and of the Prague strain of Rous sarcoma virus were isolated by gel filtration in guanidine hydrochloride (GuHCl). Hyperimmune sera prepared in rabbits against homogeneous preparations of each material were used to study the nature of the antigenic specificities on these molecules. The results indicated that (1) each component contained unique antigenic determinants; (2) each of four polypeptides (27,000, 19,000, 15,000, 12,000 daltons) contained group-specific (gs) reactivity; and (3) subgroup specific reactivity was found in the 19,000dalton polypeptide. INTRODUCTION
Two basic classes of antigenic specificities have been described for agents belonging to the chicken leukosis sarcoma virus (ChiLSV) complex. The subgroup specific class determines three distinct biological properties of ChiLSV; the host range, the interference pattern, and the specificity of neutralizing antibody (Vogt and Ishizaki, 1965; 1966; Ishizaki and Vogt, 1966). These antigens were isolated in soluble form (Tozawa et al., 1970) and are glycoproteins (Duesberg et al., 1970; Bolognesi and Bauer, 1970) located on the virion surface (Rifkin and Compans, 1971; Bolognesi et al., 1972a). The group-specific (gs) class of antigenic determinants cross-reacts among all ChiLSV and appears to be localized in the interior of the particle (Huebner et al., 1964; Bauer and Schafer, 1966; Kelloff and Vogt, 1966; Bauer and Bolognesi, 1970; Fleissner, 1971; Bolognesi et al., 1972b). The gs component was originally thought to be a single antigen but was later shown to consist of several serologically ’ These studies were supported by USPHS I-ROlCA 1571, American Cancer Society Grant VC 161, NC1 Contract NOl-CP-33308 of the Virus Cancer Program and USPHS 2-ROl-CA 12323, and USPHS Health Sciences Advancement Grant 5 SO4 RR06148. 349 Copyright 0 1975 by Academic Press, Inc. All rights of reproduction in any form reserved
distinct polypeptides (Duesberg et al., 1968; Bolognesi and Bauer, 1970; Bauer and Bolognesi, 1970; Fleissner, 1971). However, it could not be demonstrated unequivocally that each polypeptide contained gs determinants, since none of these studies were carried out with antisera prepared against purified polypeptides. Much of the evidence for the presence of gs determinants came from studies employing sera from RSV tumor-bearing hamsters (Duesberg et al., 1968; Armstrong, 1969; Fleissner, 1970). Although selected sera indeed reacted with the four major internal virion polypeptides (Bauer and Bolognesi, 1970; Fleissner, 1971), the extent of cross reactivity between polypeptides of the virion and those responsible for the induction of the hamster antibodies could not be assesseddirectly. In the present study, we have isolated the individual polypeptides by gel filtration in guanidine hydrochloride and prepared antisera against each purified component. These antisera, which show strong reactivity with their homologous proteins were used to determine the serological interrelationships among the structural components of viruses originating from several distinct ChiLSV subgroups.
350
BOLOGNESI MATERIALS
AND
ET AL.
METHODS
Virus Purification
Avian myeloblastosis virus (AMV) was obtained from the blood plasma of C/A leukemic chickens and was purified by both velocity and equilibrium sedimentation in sucrose density gradients (Bolognesi and Bauer, 1970). Over 95% of the preparation was virus of subgroup B (unpublished). Three subgroups of the Prague strain of Rous sarcoma virus were used in this study, and are designated PR-RSV-A, PR-RSV-B, and PR-RSV-C. Chicken embryo fibroblasts (CEF) were infected with the progeny of single foci, and cells were transferred until 10’ transformed cells were seeded into roller culture bottles (Smith and Bernstein, 1973). Portions of PRFIG. 1. Isolated AMV polypeptides on SDS gel RSV-C were obtained through the aid of a electrophoresis. From left to right: AMV, ~27, p19, contract to University Laboratories, Inc., ~15, and ~12. Isolation purification and analysis of Highland Park, NJ. Virus was purified polypeptide p10 will be the subject of a separate from supernatant fluids of confluent roller communication. culture bottles in a manner similar to that the antisera was designed to insure highused for AMV. titered serums. However, the amount of Purification of Major Polypeptides of AMV protein injected also enhanced the probaand PR-RSV-C bility of raising antibody to minor conPurified virus (in pellet form) containing taminating polypeptides. In fact, with the 50 mg of protein was disrupted by stirring exception of anti-p27, the remaining antifor 4 hr at 5- o in the presence of 8 M sera were found to contain some reactiviGuHCl containing 1.5 M mercaptoethanol ties against other polypeptides (see below). When this was realized, all booster injecand 0.05 M EDTA at pH 8.0. Disrupted virus was then layered on a 1.8 x 150-cm tions were made with polypeptides which had been passed through the GuHCl colcolumn of Sepharose 6B and the individual components eluted with 6 M GuHCl, 0.02 umn a second time. In addition, the dose M mercaptoethanol, and 0.05 M sodium was reduced to 100-200 pg. Purity of the acetate at pH 5.0 (Green and Bolognesi, latter polypeptides was determined by ra1974). Proteins were renatured by dialysis diolabeling with “‘1 and examination on against lo-* M mercaptoethanol for 48 hr. polyacrylamide gels (not shown). Only Protein concentration was determined by preparations showing no detectable conthe method of Lowry (Lowry et al., 1951). tamination with other polypeptides were used in the serological studies described Polypeptides Used as Antigens below. The isolation, purification, and identification of the polypeptides used in this Antisera study have been described in detail elseAntisera were prepared in rabbits to where (Green and Bolognesi, 1974). Al- each purified viral polypeptide. Immunizthough these proteins could be purified ing doses contained 0.5 mg of protein in 0.3 extensively as shown in Fig. 1, minor ml water and 0.3 ml Freund’s complete contaminants were not eliminated (see be- adjuvant (Herbert, 1973) and were injected low). The utilization of relatively large into the hind toepads of each rabbit and quantitites of protein (0.5-l mg) to prepare into several intramuscular sites in the hind
IMMUNOLOGY
OF AVIAN ONCORNAVIRUS
legs. The first two boosting doses, administered 4 and 8 wk later, contained 250 pg protein in 0.3 ml water and 0.3 ml incomplete adjuvant. Rabbits were bled 7 and 10 days after each boosting dose. Subsequently boosting doses containing 100 pg protein in water were simultaneously administered intravenously (without adjuvant) and intramuscularly (with incomplete Freund’s adjuvant) at intervals of 6-8 wk. Antisera against each polypeptide were raised in at least two animals. Normal
Sera
Sera from three normal rabbits were pooled and served as the source of normal sera. Preimmune sera were also taken from the animals to be injected. Complement
Fixation
The microcomplement-fixation (CF) test has been described (Ishizaki et al., 1973). Radioimmunoassay
The double-antibody radioimmunoassay procedure used was that described by Strand and August (1973). Polypeptides were labeled with “‘1 according to the method described by Greenwood et al. (1963). In cases where the polypeptides showed a tendency to aggregate (especially p15), improved labeling was obtained when 0.5% Triton X-100 was added to the protein solution. The specific activity of the labeled polypeptides ranged from 5 x lo3 to 2 x 10’ cpm per nanogram of protein. Between 1 and 5 nanograms of lz51labeled polypeptide in a lo-p1 volume were used in the assay. In direct radioimmunoassays, normal rabbit serum (30 ~1) and immune rabbit serum (10 ~1) were added and incubated at 37” for 3 hr and at 4” overnight. All dilutions were made in radioimmunoassay buffer (20 mA4 Tris HCl, 100 mM NaCl, pH 7.6, 1 mM EDTA containing 2 mg/ml bovine serum albumin). Goat antirabbit serum (30 ~1) (Cappel Laboratories, Downingtown, PA) was then added, incubated for 1 hr at 37” and 3 hr at 4”. Immunoprecipitates were collected by centrifugation (8000 g, 2 min) washed twice with radioimmunoassay buffer (without BSA), and counted in a
POLYPEPTIDES
351
Packard Autogamma Counter (Packard Instrument Co., Inc., Downers Grove, IL). In the competition assay, increasing amounts of unlabeled antigen (in 10 ~1) were incubated with the limiting antibody dilution (10 ~1) and normal rabbit serum (30 ~1) for 2 hr at 37”. The limiting antibody dilution precipitated 50% of the respective labeled polypeptide. A standard amount of labeled antigen (l-5 ng in 10 ~1) was then added and incubated for an additional hour at 37”. After overnight incubation at 4”, the samples were processed as described above. Preparation of Sepharose 4B Antigen finity Resins
Af-
Antigens twice purified by gel filtration on Sepharose 6B in the presence of GuHCl were attached to CNBr-activated Sepharose 4B essentially as described by Cuatrecasas (1970). Sepharose 4B (lo-ml packed bed) was exhaustively washed with deionized water on a sintered glass funnel and resuspended in 10 ml of deionized water. Two grams of cyanogen bromide (Pierce Chemical Co.) were added and the reaction mixture adjusted to pH 11 with 2 M NaOH and maintained at that pH during the reaction. The temperature was held at 30” by frequent addition of ice. The reaction was continued until base uptake had ceased. The slurry was transferred to a glass-sintered funnel, rapidly washed with a large volume of ice-cold water, resuspended in 20 ml of cold 0.2 M sodium citrate buffer, pH 6.5, and transferred to a reaction vessel. The suspension was adjusted to pH 6.5 with 1 N hydrochloric acid and 3-5 mg of purified antigen dissolved in 1.0 ml of 0.2 M sodium citrate, pH 6.5, was added. The resulting reaction mixture was stirred for 12 hr at 4’. After coupling, the resin was washed with 10 vol of cold 0.2 M sodium citrate, pH 6.5, followed by 5 vol of 0.2 M ethanolamine hydrochloride, pH 8.5. The resin was suspended in 20 ml of 0.2 M ethanolamine hydrochloride, pH 8.5, and incubated at 4” to block any remaining activated groups. After 2 hr, the material was washed with 10 vol of phosphate-buffered saline containing 0.001 M sodium azide (PBS-azide) and stored in that
352
ET AL.
BOLOGNESI
buffer at 4” until used. Resins prepared as described above have been used for periods of 6 mo with no apparent loss of affinity.
RESULTS
Characterization
of Antisera
Antibody titers were obtained for standard amounts of the respective polypeptides by complement fixation and radioimOptimal binding was obtained when the munoassay . As shown previously, each resins were gently stirred with the respec- polypeptide isolated in this fashion preciptive antigens for 1 hr at 37” then overnight itates in immunodiffusion with the respecat 4”. The resin was then sedimented at tive antiserum (Fleissner, 1971). As can be 400 g for 5 min and the supernatant re- seen in Table lA, all of the antisera reacted moved with a Pasteur pipet. Residual strongly with homologous polypeptides in material was removed by washing the resin both tests, but in some cases, minor reactwice with cold PBS-azide. Material bind- tivities were detected with heterologous Although, as indicated ing to the resin was eluted by adding 8 M polypeptides. GuHCl and incubating at room tempera- above, this probably resulted from antiture for 10 min. The resin was then washed body formation to minor contaminants in extensively and stored at 4’ in PBS-azide. the original material injected, it was also The renaturation procedure for antigens conceivable that cross-reacting determiwas essentially that used in the original nants were present on some of the virion isolation from the virus (Green and Bolog- polypeptides. The latter possibility was investigated nesi, 1974). In the case of antibody, the preparation revealed a flocculent precipi- by using radioimmunoassay to test the tate after dialysis of GuHCl which was nature of the cross reactivity between p15 easily dispersed by pipetting and mild and p27 (Table 1A). Direct radioimsonication. The antigen and antibody munoassay curves for these reactions (Fig. preparations were then employed in the 2A) showed that anti-p15 reacts strongly with ~15 but also with ~27, whereas antiserological assays.
Binding of Antigens Affinity Resins
and Antibodies
to
TABLE
1
PURIFICATION OF ANTISERA WITH ANTIGEN COLUMNS Antiserum
Resin”
Antigens
P27 CF’ b
RIA’
A Reciprocal p27 p19 p15 p12
-
512 8-32 128
64, 349-357 (1975)
Immunological DAN1 P. BOLOGNESI, Departments
Properties
of Avian Oncornavirus
Polypeptides’
RYOTARO ISHIZAKI, GUDRUN HOPER, THOMAS C. VANAMAN, AND RALPH E. SMITH
of Surgery and Microbiology
and Immunology, Duke University North Carolina 27710
Accepted
November
Medical
Center, Durham,
13, 1974
The major polypeptides and glycoproteins of avian myeloblastosis virus and of the Prague strain of Rous sarcoma virus were isolated by gel filtration in guanidine hydrochloride (GuHCl). Hyperimmune sera prepared in rabbits against homogeneous preparations of each material were used to study the nature of the antigenic specificities on these molecules. The results indicated that (1) each component contained unique antigenic determinants; (2) each of four polypeptides (27,000, 19,000, 15,000, 12,000 daltons) contained group-specific (gs) reactivity; and (3) subgroup specific reactivity was found in the 19,000dalton polypeptide. INTRODUCTION
Two basic classes of antigenic specificities have been described for agents belonging to the chicken leukosis sarcoma virus (ChiLSV) complex. The subgroup specific class determines three distinct biological properties of ChiLSV; the host range, the interference pattern, and the specificity of neutralizing antibody (Vogt and Ishizaki, 1965; 1966; Ishizaki and Vogt, 1966). These antigens were isolated in soluble form (Tozawa et al., 1970) and are glycoproteins (Duesberg et al., 1970; Bolognesi and Bauer, 1970) located on the virion surface (Rifkin and Compans, 1971; Bolognesi et al., 1972a). The group-specific (gs) class of antigenic determinants cross-reacts among all ChiLSV and appears to be localized in the interior of the particle (Huebner et al., 1964; Bauer and Schafer, 1966; Kelloff and Vogt, 1966; Bauer and Bolognesi, 1970; Fleissner, 1971; Bolognesi et al., 1972b). The gs component was originally thought to be a single antigen but was later shown to consist of several serologically ’ These studies were supported by USPHS I-ROlCA 1571, American Cancer Society Grant VC 161, NC1 Contract NOl-CP-33308 of the Virus Cancer Program and USPHS 2-ROl-CA 12323, and USPHS Health Sciences Advancement Grant 5 SO4 RR06148. 349 Copyright 0 1975 by Academic Press, Inc. All rights of reproduction in any form reserved
distinct polypeptides (Duesberg et al., 1968; Bolognesi and Bauer, 1970; Bauer and Bolognesi, 1970; Fleissner, 1971). However, it could not be demonstrated unequivocally that each polypeptide contained gs determinants, since none of these studies were carried out with antisera prepared against purified polypeptides. Much of the evidence for the presence of gs determinants came from studies employing sera from RSV tumor-bearing hamsters (Duesberg et al., 1968; Armstrong, 1969; Fleissner, 1970). Although selected sera indeed reacted with the four major internal virion polypeptides (Bauer and Bolognesi, 1970; Fleissner, 1971), the extent of cross reactivity between polypeptides of the virion and those responsible for the induction of the hamster antibodies could not be assesseddirectly. In the present study, we have isolated the individual polypeptides by gel filtration in guanidine hydrochloride and prepared antisera against each purified component. These antisera, which show strong reactivity with their homologous proteins were used to determine the serological interrelationships among the structural components of viruses originating from several distinct ChiLSV subgroups.
350
BOLOGNESI MATERIALS
AND
ET AL.
METHODS
Virus Purification
Avian myeloblastosis virus (AMV) was obtained from the blood plasma of C/A leukemic chickens and was purified by both velocity and equilibrium sedimentation in sucrose density gradients (Bolognesi and Bauer, 1970). Over 95% of the preparation was virus of subgroup B (unpublished). Three subgroups of the Prague strain of Rous sarcoma virus were used in this study, and are designated PR-RSV-A, PR-RSV-B, and PR-RSV-C. Chicken embryo fibroblasts (CEF) were infected with the progeny of single foci, and cells were transferred until 10’ transformed cells were seeded into roller culture bottles (Smith and Bernstein, 1973). Portions of PRFIG. 1. Isolated AMV polypeptides on SDS gel RSV-C were obtained through the aid of a electrophoresis. From left to right: AMV, ~27, p19, contract to University Laboratories, Inc., ~15, and ~12. Isolation purification and analysis of Highland Park, NJ. Virus was purified polypeptide p10 will be the subject of a separate from supernatant fluids of confluent roller communication. culture bottles in a manner similar to that the antisera was designed to insure highused for AMV. titered serums. However, the amount of Purification of Major Polypeptides of AMV protein injected also enhanced the probaand PR-RSV-C bility of raising antibody to minor conPurified virus (in pellet form) containing taminating polypeptides. In fact, with the 50 mg of protein was disrupted by stirring exception of anti-p27, the remaining antifor 4 hr at 5- o in the presence of 8 M sera were found to contain some reactiviGuHCl containing 1.5 M mercaptoethanol ties against other polypeptides (see below). When this was realized, all booster injecand 0.05 M EDTA at pH 8.0. Disrupted virus was then layered on a 1.8 x 150-cm tions were made with polypeptides which had been passed through the GuHCl colcolumn of Sepharose 6B and the individual components eluted with 6 M GuHCl, 0.02 umn a second time. In addition, the dose M mercaptoethanol, and 0.05 M sodium was reduced to 100-200 pg. Purity of the acetate at pH 5.0 (Green and Bolognesi, latter polypeptides was determined by ra1974). Proteins were renatured by dialysis diolabeling with “‘1 and examination on against lo-* M mercaptoethanol for 48 hr. polyacrylamide gels (not shown). Only Protein concentration was determined by preparations showing no detectable conthe method of Lowry (Lowry et al., 1951). tamination with other polypeptides were used in the serological studies described Polypeptides Used as Antigens below. The isolation, purification, and identification of the polypeptides used in this Antisera study have been described in detail elseAntisera were prepared in rabbits to where (Green and Bolognesi, 1974). Al- each purified viral polypeptide. Immunizthough these proteins could be purified ing doses contained 0.5 mg of protein in 0.3 extensively as shown in Fig. 1, minor ml water and 0.3 ml Freund’s complete contaminants were not eliminated (see be- adjuvant (Herbert, 1973) and were injected low). The utilization of relatively large into the hind toepads of each rabbit and quantitites of protein (0.5-l mg) to prepare into several intramuscular sites in the hind
IMMUNOLOGY
OF AVIAN ONCORNAVIRUS
legs. The first two boosting doses, administered 4 and 8 wk later, contained 250 pg protein in 0.3 ml water and 0.3 ml incomplete adjuvant. Rabbits were bled 7 and 10 days after each boosting dose. Subsequently boosting doses containing 100 pg protein in water were simultaneously administered intravenously (without adjuvant) and intramuscularly (with incomplete Freund’s adjuvant) at intervals of 6-8 wk. Antisera against each polypeptide were raised in at least two animals. Normal
Sera
Sera from three normal rabbits were pooled and served as the source of normal sera. Preimmune sera were also taken from the animals to be injected. Complement
Fixation
The microcomplement-fixation (CF) test has been described (Ishizaki et al., 1973). Radioimmunoassay
The double-antibody radioimmunoassay procedure used was that described by Strand and August (1973). Polypeptides were labeled with “‘1 according to the method described by Greenwood et al. (1963). In cases where the polypeptides showed a tendency to aggregate (especially p15), improved labeling was obtained when 0.5% Triton X-100 was added to the protein solution. The specific activity of the labeled polypeptides ranged from 5 x lo3 to 2 x 10’ cpm per nanogram of protein. Between 1 and 5 nanograms of lz51labeled polypeptide in a lo-p1 volume were used in the assay. In direct radioimmunoassays, normal rabbit serum (30 ~1) and immune rabbit serum (10 ~1) were added and incubated at 37” for 3 hr and at 4” overnight. All dilutions were made in radioimmunoassay buffer (20 mA4 Tris HCl, 100 mM NaCl, pH 7.6, 1 mM EDTA containing 2 mg/ml bovine serum albumin). Goat antirabbit serum (30 ~1) (Cappel Laboratories, Downingtown, PA) was then added, incubated for 1 hr at 37” and 3 hr at 4”. Immunoprecipitates were collected by centrifugation (8000 g, 2 min) washed twice with radioimmunoassay buffer (without BSA), and counted in a
POLYPEPTIDES
351
Packard Autogamma Counter (Packard Instrument Co., Inc., Downers Grove, IL). In the competition assay, increasing amounts of unlabeled antigen (in 10 ~1) were incubated with the limiting antibody dilution (10 ~1) and normal rabbit serum (30 ~1) for 2 hr at 37”. The limiting antibody dilution precipitated 50% of the respective labeled polypeptide. A standard amount of labeled antigen (l-5 ng in 10 ~1) was then added and incubated for an additional hour at 37”. After overnight incubation at 4”, the samples were processed as described above. Preparation of Sepharose 4B Antigen finity Resins
Af-
Antigens twice purified by gel filtration on Sepharose 6B in the presence of GuHCl were attached to CNBr-activated Sepharose 4B essentially as described by Cuatrecasas (1970). Sepharose 4B (lo-ml packed bed) was exhaustively washed with deionized water on a sintered glass funnel and resuspended in 10 ml of deionized water. Two grams of cyanogen bromide (Pierce Chemical Co.) were added and the reaction mixture adjusted to pH 11 with 2 M NaOH and maintained at that pH during the reaction. The temperature was held at 30” by frequent addition of ice. The reaction was continued until base uptake had ceased. The slurry was transferred to a glass-sintered funnel, rapidly washed with a large volume of ice-cold water, resuspended in 20 ml of cold 0.2 M sodium citrate buffer, pH 6.5, and transferred to a reaction vessel. The suspension was adjusted to pH 6.5 with 1 N hydrochloric acid and 3-5 mg of purified antigen dissolved in 1.0 ml of 0.2 M sodium citrate, pH 6.5, was added. The resulting reaction mixture was stirred for 12 hr at 4’. After coupling, the resin was washed with 10 vol of cold 0.2 M sodium citrate, pH 6.5, followed by 5 vol of 0.2 M ethanolamine hydrochloride, pH 8.5. The resin was suspended in 20 ml of 0.2 M ethanolamine hydrochloride, pH 8.5, and incubated at 4” to block any remaining activated groups. After 2 hr, the material was washed with 10 vol of phosphate-buffered saline containing 0.001 M sodium azide (PBS-azide) and stored in that
352
ET AL.
BOLOGNESI
buffer at 4” until used. Resins prepared as described above have been used for periods of 6 mo with no apparent loss of affinity.
RESULTS
Characterization
of Antisera
Antibody titers were obtained for standard amounts of the respective polypeptides by complement fixation and radioimOptimal binding was obtained when the munoassay . As shown previously, each resins were gently stirred with the respec- polypeptide isolated in this fashion preciptive antigens for 1 hr at 37” then overnight itates in immunodiffusion with the respecat 4”. The resin was then sedimented at tive antiserum (Fleissner, 1971). As can be 400 g for 5 min and the supernatant re- seen in Table lA, all of the antisera reacted moved with a Pasteur pipet. Residual strongly with homologous polypeptides in material was removed by washing the resin both tests, but in some cases, minor reactwice with cold PBS-azide. Material bind- tivities were detected with heterologous Although, as indicated ing to the resin was eluted by adding 8 M polypeptides. GuHCl and incubating at room tempera- above, this probably resulted from antiture for 10 min. The resin was then washed body formation to minor contaminants in extensively and stored at 4’ in PBS-azide. the original material injected, it was also The renaturation procedure for antigens conceivable that cross-reacting determiwas essentially that used in the original nants were present on some of the virion isolation from the virus (Green and Bolog- polypeptides. The latter possibility was investigated nesi, 1974). In the case of antibody, the preparation revealed a flocculent precipi- by using radioimmunoassay to test the tate after dialysis of GuHCl which was nature of the cross reactivity between p15 easily dispersed by pipetting and mild and p27 (Table 1A). Direct radioimsonication. The antigen and antibody munoassay curves for these reactions (Fig. preparations were then employed in the 2A) showed that anti-p15 reacts strongly with ~15 but also with ~27, whereas antiserological assays.
Binding of Antigens Affinity Resins
and Antibodies
to
TABLE
1
PURIFICATION OF ANTISERA WITH ANTIGEN COLUMNS Antiserum
Resin”
Antigens
P27 CF’ b
RIA’
A Reciprocal p27 p19 p15 p12
-
512 8-32 128
