Immunological properties of avian oncornavirus polypeptides

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 <2 B Reciprocal

PI9 PI5 P12

(~27) (~27) (p19).

Antibody

C Reciprocal P15

(P27)

CF

Antibody 3000 200 100
<2 <2 NT 64

Pl9


RIA

CF

P12 RIA

CF

RIA

Titer of Unpurified

Antisera

<2 128 <2 64

<2 <2 256 <2


<2 <2 <2 128


to Affinity NT 128 NT

Resins NT 2000 NT

NT NT 128

NT NT 750

NT

NT


Titer After Absorption 64 NT <2

Titer of ~15 Antibody NT

P15

<4

1500 NT
Bound to ~27 Resin NT

<4

NT

0 Antigens coupled to Sepharose 6B as indicated in Methods. NT = Not tested. b Titer represents antibody dilution at endpoint of complement fixation (CF). c Titer represents antibody dilution precipitating 50% of the labeled polypeptides (RIA).

in radioimmunoassay

IMMUNOLOGY

OF AVIAN

ONCORNAVIRUS

Reciprocal Anl~serumD~lulton

POLYPEITIDES

353

Nonogroms Cold p27 or pi5 Added

FIG. 2. Radioimmunoassay with p15 and ~27. (A) Precipitation of labeled (T) p15 and p27 by homologous or heterologous antisera; (B) Competition reactions at limiting antibody dilution (vertical dotted lines in A) with unlabeled polypeptides ~27, ~15. For example, the precipitation of labeled p27 (*) with anti-p27 was competitively inhibited by the addition of cold p27(p27); this reaction is written p27*antip27(p27).

p27 reacts essentially only with ~27. If the reaction of anti-p15 with p27 were due to a shared determinant, both polypeptides should compete for the antibody. On the other hand, if the cross reaction were due to contamination of p15 antiserum with antibodies to ~27, only p27 should compete. The vertical dotted lines (Fig. 2A) indicate the limiting dilutions chosen for the competition assay. Figure 2B shows that complete competition of the homologous reaction was obtained with 10 ng of either unlabeled p15 or ~27. However, competition of the heterologous reaction (p27-anti~15) was obtained with unlabeled p27 but not with ~15, even at the 300-ng level. Since the antigens used in the assay are greater than 99% pure, this result indicates that the ~15 antiserum was contaminated by antibodies to ~27. Similar situations existed with other cross reactions observed, but it is not clear why the contamination is not always with the adjoining peaks. From the above data, we conclude that there appears to be no significant serological relatedness among the four major virion polypeptides of ChiLSV. Purification Resins

of Antisera

Using

Affinity

Having identified unwanted reactivities in the various antisera, we attempted to remove them by absorption with the appropriate purified antigens. Purified antigen was coupled to CNBr-activated Sepharose as described in Methods and mixed with

the appropriate antisera. After incubation, the resin binding the contaminating antibody was removed by centrifugation and the residual serum was tested for reactivity. The results (Table 1B) showed that absorption completely removed the heterologous antibodies in p19, ~15, and p12 antisera with little or no loss of antibody titer to the primary antigen. Antibody bound to the resin could be eluted completely with GuHCl with a minimum loss of antibody activity (Table 1C). Elution with GuHCl was found to be superior to use of either low pH or 2 M potassium iodide, which are standard procedures for disruption of antigen-antibody complexes (Edgington, 1971). As shown in Table 1, employment of GuHCl did not release any p27 bound to the affinity resin. This latter procedure employing specific elution of antibody from the resin is a direct approach to obtain purified antibody. However, when the contaminants are known and can be purified, the method described initially, where the unwanted reactivities are selectively removed with the appropriate resin, is more rapid and does not involve denaturation and renaturation of the antibody. Antigenic Specificity Polypeptides

of the Major ChiLSV

The present analysis using AMV polypeptides was extended to polypeptides derived from a cloned avian sarcoma virus in order to determine the extent of the group

354

BOLOGNESI

specificity of these polypeptides. As indicated previously (Green and Bolognesi, 1974), the chromatograms of AMV and PR-RSV-C polypeptides by gel filtration in 6 M GuHCl were very similar and the molecular weights of the corresponding polypeptides were indistinguishable. Purified PR-RSV-C polypeptides were also injected into rabbits, and their antisera investigated in assays similar to those described above for AMV. Analyses of polypeptides ~27, ~15, and p12 from AMV and PR-RSV-C by competition radioimmunoassay are shown in Fig. 3. The results indicate that these components are serologically similar since the Prague polypeptides competed as effectively as the AMV polypeptides for the respective AMV antisera. This was not the case with p19 polypeptide since PR-RSV-C p19 was not as effective as AMV p19 in competition for the AMV p19 antiserum (Fig. 4A). The reciprocal competition yielded similar results indicating that the AMV p19 competed to a lesser extent for PR-RSV-C p19 antiserum than the homologous PR-RSV-C p19 polypeptide (Fig. 4B). These results suggest that the p19 polypeptides of AMV and IM)

PI2

80 60 40 20 i./

01

I

IO

Nonogroms Cold Anttgen Added

FIG. 3. Group specificity of ChiLSV polypeptides ~12, ~15, p27 from AMV were labeled with Y. Competition with AMV (04) and PR-RSV-C (04) polypeptides were carried out at limiting antibody dilutions determined as in Fig. 2A for the AMV polypeptides.

ET AL.

Nurogroms Cald Antigen Added

FIG. 4. Antigenic specificities of p19. Polypeptides p19 of AMV and PR-RSV-C were labeled with “‘I. The reaction in (A) employed AMV p19 lzOI and AMV p19 antiserum while that in (B) used PR-RSV-C p19 ‘*‘I and PR-RSV-C p19 antiserum. Competitition in (A) and (B) was with unlabeled p19 polypeptides of AMV and PR-RSV-C, respectively.

PR-RSV-C are not serologically identical. The presence of partial competition indicates that regions of similarity exist which may constitute group-specific determinants. That the competition is significantly less effective with the heterologous polypeptide indicates the presence of dissimilar and possibly subgroup-specific properties. To eliminate that the difference in p19 reactivity which existed between AMV and PR-RSV-C was a consequence of the fact that AMV was derived from myeloblasts and PR-RSV-C was from fibroblasts, three subgroups of Prague RSV (PR-RSV-A, PRRSV-B, and PR-RSV-C) grown in tissue culture were compared with respect to their p19 reactivity. Polypeptide p19 of PR-RSV-C was labeled with lz51 and reacted with the homologous monospecific antiserum (Fig. 5). Competition at limiting antibody dilution was achieved by utilizing the different viruses disrupted with Triton X-100. If one takes into account that p19 represents about 10% of the total virus protein, the degree of competition with the disrupted viruses indicates that the detergent released most of the p19 in serologically active form (compare to curve with isolated p19, Fig. 5). Competition with the same relative concentrations of AMV, PR-RSV-A, PR-RSVB, and PR-RSV-C gave the results shown in Fig. 5. As with the isolated polypep-

IMMUNOLOGY

OF AVIAN ONCORNAVIRUS

tides, the strongest competition was obtained with the homologous disrupted virus (PR-RSV-C). It is of interest that the B subgroup viruses (PR-RSV-B and AMV) are quite similar to one another and compete more weakly for the serum that the Prague agents of subgroup A and C. Although viruses of other subgroups need to be tested, these observations strengthen the suggestion of the presence of subgroup-specific reactivity in the p19 polypeptide. DISCUSSION

The serological analyses described above were carried out in an effort to gain a more complete understanding of the virion structural components. Homogeneous antigens and their antisera were used to examine the antigenic specificities of ChiLSV structural proteins. The results confirmed previous beliefs that the major internal virus proteins possessed group-specific determinants, but it was also found that typespecific reactivity until now attributable to the surface glycoproteins was associated with an internal polypeptide (p19). The number of kinds of antigenic determinants which constitute the group and type specificities illustrated above is not known. One must consider that the antisera used were prepared against polypeptides which may not have been fully renatured and that antibodies to several distinct regions of the molecule were probably made. Since the competitions were carried out at limiting antibody dilutions, presumably only the antibody population with high titers and strong affinities are being measured. It is impossible to determine what proportion of the molecule these may be reacting with. The antibodies present at these dilutions may be very selective and could be directed principally to only a few determinants. For example, it is conceivable that two proteins which share only one of 10 antigenic determinants against which antibody was made compete completely for one another if the antibody to the shared determinant is of high affinity and is the only one registering a titer at the 50% endpoint. In this context, the terms “group-specific reactivity” and “type-

POLYPEPTIDES

355

FIG. 5. Subgroup specificity of p19. Polypeptide p19 of PR-RSV-C was labeled with lz51 and the limiting antibody dilution of the homologous antiserum was established as in Fig. 2A. Competition was with purified polypeptide, or measured amounts of the indicated viruses disrupted with 0.2% Triton x-100.

specific reactivity” are indicative of the similarity or dissimilarity, respectively, of those antigenic determinants detectable by the antisera at limiting dilution by this procedure. Nevertheless, this is still a measure of the relatedness of these proteins and quite useful because of the sensitivity of the assay. The possible type-specific antigenicity described for p19 requires further analysis. It is of interest that this component does not appear to have a defined structural role in the virus (Bolognesi et al., 1973). In a separate study, it was shown that p19 is the most variable component in terms of quantity and electrophoretic mobility on the basis of charge (Bolognesi et al., 1974), further suggesting type or subgroup specificity. It is of interest to note that an antigenic specificity similar to that described here for p19 has been found in two internal polypeptides of the murine RNA tumor viruses. Type-specificity was demonstrated for both p12 and p30 isolated from several murine strains of virus (Tronick et al., 1973; Green et al., 1973; Strand and August, 1974; Stephenson et al., 1974). The studies described above reveal that the affinity columns used to purify the antisera may be powerful tools of rather broad applicability. A major advantage of antigen-bound resins is that they can be reused after elution of the antibody with

356

BOLOGNESI

GuHCl. For example, the resin binding ~27 has been recycled six times without appreciable loss of activity for anti-p27 antibodies. Using the same coupling procedures, it has also been possible to prepare columns coupled to antibody which bind antigen in an analogous manner. Elution of bound polypeptides can also be accomplished with GuHCl and the antibody columns can be regenerated several times. These antigen and antibody resins can be used effectively in the isolation of a number of important materials, such as selected polypeptides or polypeptide fragments as well as antibodies to specific antigen determinants. There are many important questions still to be answered regarding the nature and origin of the structural polypeptides and glycoproteins of ChiLSV. For example, it is not entirely clear which of these are bonafide virus gene products. Because antisera raised in mammals bearing RSV-induced tumors react with ~12, ~15, p19, and ~27, we favor the interpretation that these are virus-coded gene products. However, the identity of p10 remains to be established and until now has been hampered by the poor serological activity of the molecule. Unequivocal demonstration of viral or cellular origin of these components could be shown by in vitro protein synthesis with purified virus RNA. Participation of the host cell in synthesis of certain virus macromolecules may occur particularly in the glycosylation of the virus glycoproteins (gp85 and gp37) (Lai and Duesberg, 1973; Bauer et al., 1973). Furthermore, the functional roles of these components in the processes of virus infection and cell transformation is yet to be determined. Using the purified proteins, highly specific antisera and methodology described in this study, many of these questions are now open to investigation. ACKNOWLEDGMENTS We thank Dr. A. J. Langlois for the large quantities of AMV and Dr. R. Green for supplying the purified polypeptides used in these studies. REFERENCES 1, ARMSTRONG, D. (1969). Multiple

group-specific

ET AL. antigen components of avian tumor viruses detected with chicken and hamster sera. J. Viral. 3, 1333139. 2. BAUER, H., and BOLOGNESI,D. P. (1970). Polypeptides of avian RNA tumor viruses. II. Serological characterization. Virology 42, 1113-1126. 3. BAUER, H., GELDERBLOM, H., BOLOGNESI, D. P., and KURTH, R. (1973). RNA tumour virusdirected glycoproteins and their significance for the virion and the cell. In “Membrane Mediated Information” (P. W. Kent, ed.), Vol. 1. England. 4. BAUER, H., and SCHKFER, W. (1966). Origin of group specific antigen of chicken leukosis viruses. Virology 29, 494-496. 5. BOLOGNESI,D. P., and BAUER, H. (1970). Polypeptides of avian RNA tumor viruses. I. Isolation and physical and chemical analysis. Virology 42, 1097-1112. 6. BOLOGNESI, D. P., BAUER, H., GELDERBLOM, H., and HOPER, G. (1972). Polypeptides of avian RNA tumor viruses. IV. Components of the viral envelope. Virology 47, 551-566. 7. BOLOGNESI, D. P., GELDERBLOM, H., BAUER, H., MOLLING, K., and H~~PER,G. (1972). Polypeptides of avian RNA tumor viruses. V. Analysis of the virus core. Virology 47, X7-578. 8. BOLOGNESI, D. P., HOPER, G., GREEN, R. W., and GRAF, T. (1974). Biochemical properties of oncornavirus polypeptides. BBA Rev. Cancer, in press. 9. BOLOGNESI, D. P., LUFTIG, R., and SHAPER, J. H. (1973). Localization of RNA tumor virus polypeptides. I. Isolation of further virus substructures. Virology 56, 549-564. 10. CUATRECASAS,P. (1970). Protein purification by affinity chromatography: Derivatizations of agarose and polyacrylamide beads. J. Biochem. 245,3059-3065. 11. DUESBERG,P. H., ROBINSON, H. L., ROBINSON, W. S., HUEBNER, R. J., and TURNER, H. C. (1968). Proteins of Rous sarcoma virus. Virology 36, 73-86. 12. DUESBERG,P. H., MARTIN, G. S., and VOGT, P. K. (1970). Glycoprotein components of avian and murine RNA tumor viruses. Virology 41, 631-646. 13. ELXXNGTON,T. S. (1971). Dissociation of antibody from erythrocyte surfaces by chaotropic ions. J. Immunol. 106,673~680. 14. FLEISSNER, E. (1970). Virus-specific antigens in hamster cells transformed by Rous sarcoma virus. J. Viral. 5, 14-21. 15. FLEISSNER, E. (1971). Chromatographic separation and antigenic analysis of proteins of the oncornaviruses. I. Avian leukemia-sarcoma viruses. J. Viral. 8, 778-785. 16. GREEN, R. W., BOLOGNESI, D. P., SCHAFER, W.,

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33. TRONICK,S. R., STEPHENSON, J. R., and AARONSON, S. A. (1973). Immunological characterization of a low molecular weight polypeptide of murine leukemia virus. Virology 54, 199-206. 34. VOGT, P. K., and ISHIZAKI,R. (1965). Reciprocal patterns of genetic resistance to avian tumor viruses in two lines of chickens. Virology 26, 664-672. 35. VOGT,P. K., and ISHIZAKI,R. (1966). Patterns of viral interference in the avian leukosis and sarcoma complex. Virology 30, 368-374.