Isolation and characterization of the envelope glycoprotein of an avian osteopetrosis virus: Effect of host cell on antigenic reactivity

VIROLOGY

g&80-89

(1978)

Isolation and Characterization of the Envelope Glycoprotein of an Avian Osteopetrosis Virus: Effect of Host Cell on Antigenic Reactivity LINDA Department

J. VAN

of Microbiology

ELDIK’!”

AND

RALPH

and Immunology, Duke University North Carolina 27710 Accepted

June

E. SMITH” Medical

Center,

Durham,

9, 1978

The physical and antigenic properties of the surface glycoprotein (gp85) of an avian myeloblastosis-associated virus [MAV-2(O)] were compared with those of avian myeloblastosis virus (AMV) gp85. Purified MAV-2(O) contained 3- to 4-fold higher levels of gp85 than AMV, as determined by polyacrylamide gel electrophoresis and guanidine hydrochloride column chromatography. The antigenic reactivity of MAV-2(O) gp85 was examined in competition radioimmunoassays using rabbit antisera prepared against purified AMV and B77 gp85. The antigenic reactivity of MAV-2(O) as measured in competition radioimmunoassay using antiserum prepared against AMV gp85 depended on the source of the virus; MAV-2(O) isolated from chicken embryo fibroblast cultures showed considerably less antigenic reactivity than did MAV-2(O) isolated from the serum of osteopetrotic chickens. In contrast, MAV-2(O) from serum or grown in chicken embryo fibroblasts showed similar antigenic reactivities in competition radioimmunoassays using antiserum prepared against gp85 from B77. A comparison of the antigenic reactivities of the purified glycoproteins from MAV-2(O) (grown in fibroblasts) and AMV (isolated from plasma) demonstrated that the two glycoproteins showed equal reactivity using anti-B77 gp85 serum, but that MAV-2(O) gp85 showed at least 106fold less antigenic reactivity than AMV gp85 in competition radioimmunoassay using anti-AMV gp85 serum. These results together with those previously published (Van Eldik et al., (1978). Virology 86, 193-204.) suggest that MAV-2(O) and AMV gp85 show similar host-dependent glycosylation patterns in z&o, which differ from the pattern observed when the viruses are grown in chicken embryo fibroblasts.

preparations contain more than one associated virus (Moscovici and Vogt, 1968) and several myeloblastosis-associated viruses have been isolated from AMV stocks (Moscovici, 1975). One myeloblastosis-associated virus [MAV-2(O))], a subgroup B virus, induces a high incidence of osteopetrosis (Smith and Moscovici, 1969). Although MAV-2(O) also causes nephroblastoma and lymphoid leukosis in a small percentage of infected chickens, myeloblastosis has never been observed (Smith et al., 1976; Banes and Smith, 1977). The oncogenic spectrum of MAV-2(O), therefore, differs considerably from AMV. The molecular basis for the differences in oncogenic spectrum between MAV-2(O) and AMV has not been determined. Observations that the virus surface glycoproteins are involved in determining the host range,

INTRODUCTION

Avian myeloblastosis virus (AMW4 has been shown to cause at least four types of neoplasms in the chicken: myeloblastosis, nephroblastoma, lymphoid leukosis, and osteopetrosis (Beard, 1963). Standard AMV ’ This work is part of a dissertation submitted by L. J. V. E. to the Department of Microbiology and Immunology, Duke University Medical Center for the degree of Doctor of Philosophy. ’ Present address: The Rockefeller University, 1230 York Ave., New York, N. Y. 10021. ’ To whom all correspondence should be addressed. * Abbreviations used: AMV, avian myeloblastosis virus; CEF, chicken embryo fibroblasta; GuHCl, guanidine hydrochloride; MAV-2(O), myeloblastosis-aasociated virus of subgroup B inducing osteopetrosis; MEM, Eagle’s Minimal Essential Medium; PAGE, polyacrylamide gel electrophoresis; RIA, radioimmunoassay; SDS, sodium dodecyl sulfate. 0042.6822/78/0901-0080$02.00/0 Copyright 0 1978 by Academic Press. Inc. All righti of reproduction in any form reserved.

80

HOST

CELL

AND

MAV-2(O)

interference, and neutralization properties of avian RNA tumor viruses (Hanafusa, 1965; Ishizaki and Vogt, 1966; Vogt and Ishizaki, 1965, 1966) suggest that these glycoproteins play an essential role in virus penetration and the infectious process. Although the major surface glycoprotein (gp85) of AMV has been extensively studied (Tozawa et al., 1970: Bolognesi et al., 1972; Porter and Winzler, 1975), little is known about the analogous glycoprotein of MAV30). MAV-2(O) gp85 was purified from virus grown in chicken embryo fibroblasts (CEF) and its .physical and antigenic properties compared with those of AMV gp85. AMV was chosen for this comparison because a) it is the parental virus of MAV-2(O) (Smith and Moscovici, 1969), b) the strain of AMV used for most of the studies was of a subgroup B host range (Ishizaki et al., 1975) and, therefore, the nondefective associated virus presumably possessed a gp85 similar to MAV-2 (0)) and c) antisera against gp85’s were available which were chosen for their carbohydrate-dependent and carbohydrate-independent nature (Van Eldik et al., 1978). We report here that MAV-2(O) has 3- to 4-fold higher levels of gp85 than does AMV, as determined by polyacrylamide gel electrophoresis (PAGE) and guanidine hydrochloride (GuHCl) column chromatography. In addition, the antigenic reactivity of MAV-2(O) gp85 was studied by competition radioimmunoassay (RIA). We report that MAV-2(O) preparations show differences in antigenic reactivity with anti-AMV gp85 serum depending on whether MAV2(O) was isolated from CEF supernatant fluids or from the serum of osteopetrotic chickens. In addition, purified MAV-2(O) gp85 showed at least lOO-fold less antigenic reactivity than AMV gp85 in competition RIA using anti-AMV gp85 serum. These results suggest that host-dependent glycosylation of gp85 may influence antigenic reactivity of the glycoprotein. MATERIALS

AND

METHODS

Viruses and cell culture. CEF were prepared from 11-day Spafas (Norwich, Corm.) embryos as previously described (Smith and Bernstein, 1973). Secondary CEF were

13385 ANTIGENICITY

81

infected with B77, AMV, or plaque-purified MAV-2(O) (Smith et al., 1976) at a multiplicity of 0.01. Virus was isolated from infected CEF cultures by centrifuging the supernatant fluid at 1500 r-pm (8OOg)for 15 min to remove cells, centrifuging at 7000 rpm (8OOg) for 20 min for further clarification, then pelleting the virus at 24,000 rpm (80,OOOg)for 35 min. Virus was purified by equilibrium sedimentation in 15-60% (w/v) sucrose gradients (Smith and Bernstein, 1973). Supernatant fluids from cultures of AMV-infected chicken myeloblasts were kindly provided by Dr. A. Langlois, Duke University. AMV was isolated from the supernatant fluids as described above. AMV was also isolated from the plasma of leukemic chickens. Serum MAV-2(O) was isolated directly from the serum or plasma of osteopetrotic chickens. Virus strains used in this study are shown in Table 1. Polyacrylamide gel electrophoresis. Virus polypeptides were analyzed by electrophoresis in 10% acrylamide, 0.1% sodium dodecyl sulfate (SDS) gels as described by Bolognesi and Bauer (1970). Gels were stained in a solution of 0.25% (w/v) Coomassie blue in methanol:water:acetic acid (5:5:1) and destained in 7.5% (v/v) acetic acid, 5% (v/v) methanol. Gels were scanned with a Quik scan (Helena Laboratories) interfaced with a Digital PDP ll/lO computer. The peaks obtained in the scans were integrated relative to the total Coomassie blue-staining material and compared to the amount of protein loaded on the gels (determined by the procedure of Lowry et al. (1951)). Radioactive labeling of virus protein. Virus protein was labeled with [‘4C]glucosamine in Eagle’s Minimal Essential Medium (MEM) containing 25% of the normal glucose concentration. This low glucose medium was kindly provided by Dr. R. Ishizaki, Duke University. MAV-2(O) infected CEF in roller bottle cultures were starved for 2 hr in low glucose MEM, then labeled in 50 ml media containing 25 PCi of [‘“C] glucosamine (57 mCi/mmol, New England Nuclear). Virus-containing supernatant fluids were harvested every 24 hr followed by the addition of fresh unlabeled low glu-

82

VAN

ELDIK

AND

TABLE VIRUS Virus AMV

History

grown

in myelo-

blasts

AMV

from

Serum

MAV-2(O)

AMV grown [(MAV-B(TG)] MAV-2(O) CEF Serum grown AMV-A

plasma

in

CEF

grown

in

MAV-2(O) in CEF

Pr-RSV-C

-

1

STRAINS --

AMV injected into chickens, myeloblasts harvested and cultured in uitro. AMV injected into chickens, virus isolated from the plasma of leukemic animals MAV-2(O) injected into 12. day embryos, virus harvested from serum of OSteopetrotic animals at 3-4 weeks of age. AMV plaque-purified on CEF, nontransforming virus isolated. MAV-2(O) plaque-purified 3 times in CEF. Serum MAV-2(O) as above, grown in CEF for 1 week. AMV selected for subgroup A host range. Pr-RSV-C focus-purified three times on CEF. -

case MEM. The labeled virus was pelleted as described above, and the polypeptides were analyzed by SDS-PAGE. Preparation of gp85 and anti-gp85 sera. The virus surface glycoprotein gp85 was purified by GuHCl column chromatography and DEAE-ion exchange chromatography as previously described (Green and Bolognesi, 1974; Rohrschneider et al., 1975). Purified AMV gp85 and B77 gp85 were gifts of Drs. R. Green, and A. Hizi, Duke University, respectively. Rabbit antiAMV gp85 sera and anti-B77 gp85 serum were prepared and characterized as previously described (Van Eldik et al., 1978), and were kindly provided by Dr. D. P. Bolognesi, Duke University, and Dr. J. Li, Duke University, respectively. Radioimmunoassays. Purified gp85 was labeled with ““I by the method of Greenwood et al. (1973). Radioimmunoassays were performed with modifications of the method of Strand and August (1973), as previously described (Van Eldik et al., 1978). The specific antisera were used at the concentration predetermined by direct

SMITH

USED

Source -___ c Supplied directly by Dr. A, J. Langlois, Duke University. , Supplied directly by Dr. A. ( J. Langlois, Duke University.

c

Reference -__~ Beaudreau et al., 1958

Eckert

’ Our laboratory. , 1

Smith and 19fi9.

I 1 Originally supplied Thomas Graf, grown , our laboratory ! Our laboratory

-

et al.,

by in

, Our laboratory 1 ( Supplied directly by Dr. A. J. Langlois. Originally supplied by Dr. P. K. Vogt, grown in our laboratory

1951

Moscovici,

Smith

et al., 1976

Smith

et al., 1976

This Ishizaki

work et al.,

Smith and 1973

1975

Bernstein,

radioimmunoassay to give 50% precipitation of the “? counts of the homologous ““I-labeled antigen. RESULTS

Analysis of MAV-Z(0) Electrophoresis

Polypeptides

by Gel

The electrophoretic mobility of MAV2(O) polypeptides in 10% acrylamide, 0.1% SDS gels was examined, and Fig. 1 shows a typical electropherogram. It can be seen that MAV-2(O) contained bands that comigrated with the four major polypeptides characteristic of avian RNA tumor viruses: ~27, p19, ~12, and p15/10. It is also evident from Fig. 1 that MAV-2(O) contained more Coomassie blue-staining material migrating in the position of gp85 than did AMV or PrRSV-C. The amount of material migrating in the position of gp85 was quantified by determining the area of the gp85 peak as a percentage of the total Coomassie bluesbaining material. Analyses of scans of 23 gels showed that the material in the gp85 position in the MAV-Z(0) gels contained

HOST CELL AND MAV-2(O)

lo

Fro. 1. SDS-PAGE analysis of avian RNA tumor polypeptides. Virus was isolated and analyzed by electropboresis in 10% acrylamide, 0.1% SDS gels as described in Materials and Methods. Electrophoresis was from top to bottom. Virus preparations contained 30-40 pg protein and each molecular weight standard contained 5-10 pg protein. Samples are from left to right: MAV-2(O) from CEF, Serum MAV-2(O) grown in CEF for 1 week, AMV; Pr-RSV-C. Molecular weight standards [top to bottom: bovine serum albumin (66,906 MW), ovalbumin (43,999 MW), chymotrypsinogen (25,700 MW), myoglobin (17,200 MW), cytochrome c (11,700 MW)]. Arrows indicate the positions of the major virus polypeptides, from top to bottom: gp65, ~27, p19, ~12, p15/10. virus

7.14% of the total Coomassie blue stain, whereas AMV gp85 was 2.14% of the stain, and Pr-RSV-C gp85 was 2.23% of the stain. Thus, by the criterion of gel scans, MAV2(O) contained 3.33% more gp85 than AMV and 3.20% more gp85 than Pr-RSV-C. To confirm that the material in this band was glycoprotein, MAV-2(O) was labeled with [‘*C]glucosamine, the virus was purified, disrupted, and analyzed by SDSPAGE. A major peak of radioactivity migrated with the same mobility as the gp85 detected by Coomassie blue staining (Fig. 2). A minor [‘*C]glucosamine peak co-migrated with Coomassie blue bands in the gp37 region of the gels (Fig. 2). These results indicate that the Coomassie bluestaining material in the gp85 region of the gels contained carbohydrate. Purification of MAV-2(O) Polypeptides by GuHCl Column Chromatography MAV-2(O) polypeptides were purified by GuHCl column chromatography as described in Materials and Methods. A typical column profile with the accompanying

83

gp65 ANTIGENICITY

30

50

70

SD

FIG. 2. SDS-PAGE analysis of [‘%]glucosaminelabeled MAVd(0). [‘4C]glucosamine-labeled MAV2(O) (36,999 cpm) was analyzed by electrophoresis as described in Materials and Methods. Radioactivity was determined by slicing the gel into l-mm sections, incubating each segment in 0.5 ml of protosol/ toluene/water (910~1) at 37” overnight, then counting the segments in a PPO-POPOP scintillation cocktail (4 g PPO; 0.1 g POPOP; 1 liter toluene). Unlabeled MAV-2(O) (30 pg) was electrophoresed on a parallel gel and stained with Coomassie blue as described in Materials and Methods. Electrophoresis was from left to right.

SDS-polyacrylamide gels of each polypeptide peak is shown in Fig. 3. It is evident that the individual peaks were well resolved and that the polypeptides were relatively homogeneous as assayed by SDS-PAGE. The MAV-2(O) gp85 fraction from the GuHCl column was further purified by DEAE-cellulose ion-exchange chromatography inasmuch as this purification step eliminated minor bands seen by SDSPAGE of the gp85 fraction from GuHCl chromatography. All of the studies reported here used gp85 purified by both GuHCl chromatography and DEAE-cellulose chromatography. A quantitation of the gp85s from MAV2(O), AMV and Pr-RSV-C was made by calculating the areas under the gp85 peaks of GuHCl-chromatographed MAV-2(O), AMV and Pr-RSV-C. The peak areas for AMV and Pr-RSV-C polypeptides were determined from published GuHCl column profiles (Green and Bolognesi, 1974). The ratios of the gp85 peak area to p27 peak area were calculated to provide an internal normal for differences in the amount of protein loaded on the GuHCl columns. By this procedure, the gp85/p27 peak ratios

a4

VAN

ELDIK

AND

SMITH

were 0.83 for MAV-2( 0)) 0.24 for AMV, and 0.21 for Pr-RSV-C. Thus, using the criterion of peak area ratios from GuHCl columns, MAV-2(O) contained 3.46 times more gp85 than AMV and 3.95 times more gp85 than Pr-RSV-C. These results are consistent with the comparison of the gp85 levels obtained by integration of Coomassie blue-staining bands by gel scans, described above. Influence of Host Cell on Antigenic tivity of MA V-2(0) gp85

I

c ., w

Reac-

It has been reported (Duesberg et al., 1970; Tozawa et al., 1970; Bolognesi et al., 1972) that the surface glycoprotein (gp85) of avian RNA tumor viruses demonstrates subgroup specificity in competition RIA, in that viruses of the same subgroup show similar competition patterns, whereas viruses of other subgroups compete less efficiently. When viruses of three subgroups were compared by competition RIA using anti-AMV gp85 serum it was found that MAV-2(O), a subgroup B virus, did not compete as efficiently as AMV-B, a virus of primarily subgroup B (Fig. 4). AMV-A and Pr-RSV-C, viruses of subgroups A and C, respectively, also showed no competition (Fig. 4). Neither purified nor unpurified MAV2(O) competed as efficiently as AMV (data not shown). This result excluded the possibility that the gp85 of MAV-2(O) was stripped off during sucrose gradient purification of the virus and was, therefore, unavailable for competition. The expected subgroup-specific antigenicity was obtained by isolating MAV-2(O) directly from the serum of osteopetrotic chickens and analyzing the virus by competition RIA. Serum MAV-2(O) competed as efficiently as AMV for the anti-AMV gp85 serum (Fig. 5). This observation suggested that growth of MAV-2(O) in the animal resulted in an antigenic change in gp85, which resulted in efficient competition with AMV gp85. This suggestion was supported by the following experiment. CEF were infected with MAV-2(O) isolated from the serum of osteopetrotic chickens and the virus was allowed to replicate in culture for 1 week. MAV-2(O) was then

0

20

40

60

80

100

120

140

Fractton Number

FIG.

3. GuHCl column profile of MAV-2(O) polyMAV-2(O) (30 mg) was disrupted and the polypeptides were isolated by GuHCl column chromatography as described in Materials and Methods. The large, eluting peak (fractions 1-12) contained nonpolypeptide virus components. Peaks were pooled, and aliquots of each peak were analyzed by SDSPAGE. The six peaks representing the viral polypeptides are shown with their corresponding SDS-polyacrylamide gels. The left well contained MAV-2(O) virus (30 pg) and was included for comparison. 1, gp85; 2,p27; 3, p19; 4, ~15; 5, ~12; 6, ~10.

peptides.

,\

I 01

,

IO 100 Compehg Prorefn hg)

1000

10000

FIG. 4. Analysis of viruses of different subgroups in competition RIA. ““I-labeled AMV gp85 (25,000 cpm) and a limiting antibody dilution (1:lOOO) of antiAMV gp85 serum were used in the reactions. Competing antigens were homologous AMV gp85 (0- - -0) or viruses of subgroups A [AMV-A ([1--o)], subgroup B [MAV-2(O) (M), AMV-B (H), MAV2(TG) (A--A)], and subgroup C [Pr-RSV-C

(A--A)].

CELL

HOST

AND

MAVS(0)

isolated from the supernatant fluid of the virus-infected CEF culture and examined by competition RIA. As shown in Fig. 5, the serum virus lost the capacity to compete for anti-AMV gp85 serum after tissue culture passage. To further define the effect of tissue culture passage on the immunological specificity of gp85, AMV was grown in tissue culture and examined by RIA. When AMV was grown in myeloblasts, it retained the ability to compete for anti-AMV gp85 serum (Fig. 5). However, when AMV was grown in CEF [MAV-2(TG)], it did not compete for antibody (Fig. 4). In order to determine whether the differences observed in RIA between AMV gp85 and MAV-2(O) gp85 were due to differences in the glycoproteins themselves, or in the conformation or arrangement of the glycoproteins on the virus particle, the gp85 from MAV-2(O) was purified by GuHCl chromatography and DEAE chromatography. The purified MAV-2 (0) gp85 was examined by competition RIA using anti-AMV gp85 serum. Fig. 6 shows that three preparations of MAV-2(O) gp85 demonstrated at least lOO- to lOOO-fold less competition than AMV gp85 for anti-AMV gp85 serum. In

Compettng

Protein

gp85

ANTIGENICITY

85

fact, the isolated glycoprotein was no more reactive than intact MAV-2(O) and less reactive than intact AMV particles (Fig. 6). The RIA data using anti-AMV gp85 serum was summarized by calculating the amount of protein required for 50% competition in the various gp85 RIA measurements, and the tabulations are shown in Table 2. AMV grown in myeloblasts or isolated from plasma required the same amount of protein for 50% competition (47-50 ng), whereas MAV-2(O) isolated from the serum of osteopetrotic chickens required 232 ng of protein (4.6 times more protein than AMV). However, MAV-2(O) grown in CEF required at least 50 times more protein (3000 ng) for 50% competition than did AMV. Purified MAV-2(O) gp85 required 227 times more protein for 50% competition than did AMV gp85. Influence of Carbohydrate peptide on Antigenie MAV-2(O) gp85

and PolyReactivity of

Our observation that the immunological reactivity of gp85 in RIA depended on the host cell in which the virus was propagated suggested that the glycosylation pattern may be important for the antigenic specificity of gp85. This suggestion was confirmed (Van Eldik et al., 1978) when we determined that the immunological reactivity of AMV gp85 using anti-AMV gp85 sera depended upon an intact carbohydrate side chain. Previous studies (Van Eldik et al.,

ingl

5. Effect of host cell on reactivity of virus in competition RIA. ““I-labeled AMV gp85 (32,000 cpm) and a limiting antibody dilution (1:2000) of anti-AMV gp85 serum were used in the reactions. Viruses were grown in different host cells as described in Materials and Methods. Competing antigens were homologous AMV gp85 (O---O), MAV-2(O) from CEF (M), Serum MAVd(0) (O--O), Serum MAV2(O) grown in CEF for one week (A---A), AMV from plasma (t--l), and AMV grown in myeloblasts (U-U). Each point represents the mean f SD of triplicate determinations. FIG.

FIG. 6. Analysis of purified gp85 in competition RIA. ‘*‘I-labeled AMV gp85 (31,000 cpm) and a limiting antibody dilution (1:1500) of anti-AMV gp85 (O---O), three preparations of purified MAV-2(O) gp85 (O-- -4), AMV from plasma (M), and MAV-2(O) from CEF (O---4).

86 TABLE COMPETITION RADIOIMMUNOASSAY gp85 AND ANTI-AMV Competing species

VAN

AND

SMITH

2 WITH

gp85

Viruses AMV grown in myeloblasts AMV from plasma Serum MAV-2(O) AMV grown in CEF [MAWZ(TG)] MAV-2(O) from CEF Serum MAV-P(0) grown in CEF AMV-A Pr-RSV-C Glycoproteins AMV gp85 MAV-2(O) gp85

ELDIK

[T]AMV

SERUM

Nanogram protein required for 505 competition

-

47.0 50.4 232.0 >1680.0” 2995.8 >[email protected]” 1420.0 >2ooo.o”

3.8 864.5

nt

FIG. 7. Analysis of MAV-2(O) in heterologous competition RIA. ““I-labeled AMV gp85 (38,000 cpm) and a limiting antibody dilution (1:lOO) of anti-B77 gp85 serum were used in the reactions. Competing antigens were B77 gp85 (O---O), Serum MAV-2(O) (o----O), and MAV-2(O) from CEF (M).

0 The symbol (>) indicates that even at the highest concentration of protein, 50% competition was not achieved.

1978) also showed that when competition RIAs were performed using anti-B77 gp85 serum, the immunological reactivity of AMV gp85 or B77 gp85 did not depend upon an intact carbohydrate side chain. The antigenic reactivity of MAV-Z(0) gp85 was, therefore, examined in competition RIAs using anti-B77 gp85 serum. Figure 7 shows the results obtained when serum MAV-2(O) and CEF-grown MAV-2(O) were compared in a competition RIA utilizing anti-B77 gp85 serum. It can be seen that MAV-2(O) from serum and MAV-2(O) from CEF both competed for anti-B77 gp85 serum. In fact, MAV-2(O) from CEF competed with slightly more efficiency than MAV-2(O) from serum (Fig. 7). To further compare the antigenic reactivities of MAV-2(O) gp85 and AMV gp85, the competition patterns of the purified glycoproteins were examined in heterologous and homologous competition RIAs using anti-B77 gp85 serum. Figure 8 shows that in the heterologous RIA using ‘Y-AMV gp85 and anti-B77 gp85 serum, purified B77 gp85, MAV-2(O) gp85, and AMV gp85 showed similar competition for anti-B77 gp85 serum. In the homologous RIA using ‘““I-B77 gp85 and anti-B77 gp85 serum (Fig. 9), MAV-2(O) gp85 and AMV gp85 showed similar competition for anti-B77 gp85 se-

FIG. 8. Analysis of purified gp85 in heterologous competition RIA. ““I-labeled AMV gp85 (31,000 cpm) and a limiting antibody dilution (1:lOO) of anti-B77 gp85 serum were used in the reactions. Competing antigens were B77 gp85 (A-A), AMV gp85 (Ck-O), and MAV-2(O) gp85 (U).

rum, although both MAV-2(O) gp85 and AMV gp85 competed less efficiently than the homologous B77 gp85. In addition, MAV-2(O) from CEF and AMV from plasma showed similar reactivity in the competition RIA using anti-B77 gp85 serum, reaching a plateau level of approximately 25% competition at a concentration of approximately 1000 ng competing protein (Fig. 9). These results indicate that MAV-2(O) gp85 and AMV gp85 contain determinants not recognized by anti-B77 gp85 serum. DISCUSSION

These studies tigenic reactivity

demonstrate of MAV-2(O)

that the anas measured

HOST

CELL

AND

MAV-2(O)

tion RI.4. ‘251-labeled B77 gp85 (24,000 cpm) and a limiting antibody dilution (1:lOO) of anti-B77 gp85 serum were used in the reactions. Competing antigens were B77 gp85 (A-A), AMV gp85 (C--0), MAV2(O) gp85 [W), AMV from plasma (U--U), and MAV-2(O) from CEF (-1.

in competition RIA using antiserum prepared against AMV gp85 depends upon the host cell in which the virus is grown. Further, competition radioimmunoassay analysis of the purified glycoproteins from MAV-2(O) and AMV suggests that the changes in antigenic reactivity are due to alterations in the glycoprotein. A comparison of the antigenic reactivities of MAV-2(O) gp85 and AMV gp85 demonstrates that MAV-2(O) gp85 shows at least NO-fold less reactivity than AMV gp85 using anti-AMV gp85 serum. A possible explanation for this observation is that the MAV-2(O) virus particle may contain less gp85 than AMV. However, data presented here suggest that MAV-2(O) contains 3-4 times more gp85 than does AMV. The apparent increased levels of MAV-2(O) gp85 may represent an artifact of the isolation procedures, although MAV-2(O) and AMV were isolated and purified using identical conditions. Another explanation for the differences in antigenic reactivities between MAV-2(O) gp85 and AMV gp85 may be that hostdependent glycosylation of gp85 influences the antigenic reactivity of the glycoprotein. This possibility is supported by previous findings (Van Eldik et al., 1978) that the reactivity of AMV gp85 in RIA using this anti-AMV gp85 serum depends upon an intact carbohydrate side chain. Inasmuch as the ability of the anti-AMV gp85 serum to react in RIA is determined by the carbohydrate side chain structure of the gp85,

gp85

ANTIGENICITY

87

a change in host cell may result in alterations in the anti-AMV gp85 reactivity. The anti-AMV gp85 serum was prepared against gp85 purified from a serum-isolated AMV (Van Eldik et al., 1978). As shown in the study presented here, MAV-2(O) isolated from serum shows a similar competition pattern with AMV isolated from serum. However, when the host cell of the virus is a chicken embryo fibroblast, then MAV2 (0) or AMV no longer shows efficient competition. It seems unlikely that the antigenie change observed could be due to a contaminating virus. First, the same virus was grown in the animal, and then for 1 week in CEF, after which time the antigenic change was present. It seems unlikely that a variant strain of virus with the same host range could emerge in such a short time. Second, infected animals were housed in an isolation facility. Third, contaminating viruses have not been detected by several criteria, including interference assay, endpoint titrations and examination of infected and contact control chickens for the presence of Marek’s disease virus, avian leukosis virus of subgroup A, and infectious bursal agent. Analysis of the antigenic reactivity of MAV-2(O) gp85 in competition RIA using anti-B77 gp85 serum (prepared against CEF-grown B77) indicates that MAV-2(O) isolated from serum and MAV-2(O) isolated from CEF supernatant fluids show similar reactivities. In addition, the purified glycoproteins from MAV-2(O) and AMV show equal antigenic reactivity in competition RIA using anti-B77 gp85 serum. These observations are consistent with our findings (Van Eldik et al., 1978) that the reactivity of the anti-B77 gp85 serum does not depend upon an intact gp85 carbohydrate side chain, but is affected by the state of the polypeptide portion of the glycoprotein. Results presented here also suggest that changes in the host cell of the virus do not significantly alter the structure of the polypeptide chain of MAV-2(O) gp85, inasmuch as serum MAV-2(O) and CEFgrown MAV-2(O) show similar competition patterns using anti-B77 gp85 serum. Thus, these results suggest that the differences in antigenic reactivity between

88

VAN

ELDIK

MAV-2(O) gp85 and AMV gp85 are due to host-dependent glycosylation of the glycoproteins. This study does not address the nature of the host cell for replication of MAV-2(O) in the animal. However, the study does suggest that changes in glycosylation occur when the virus is grown in uiuo and in vitro, and that these changes influence antigenic reactivity. Chemical comparison of the carbohydrate side chain structure of the two glycoproteins will be required to directly demonstrate differences in glycosylation. These structural studies must await the isolation of enough MAV-2(O) gp85 to perform direct chemical analyses of the molecule, such as determination of amino acid and carbohydrate compositions and tryptic peptide maps. The functional significance of the carbohydrate in the viral glycoproteins is unclear. Treatment of Pr-RSV-C with tunicamycin, an antibiotic which inhibits glycosylation, results in a decrease in infectivity and a loss of detectable gp85 in polyacrylamide gel electrophoresis (Schwarz et al, 1976). There have been conflicting reports that there is a correlation between glycopeptide size and host range of avian tumor viruses (Lai and Duesberg, 1972; Galehouse and Duesberg, 1978). However, mutants of Rous sarcoma virus have been isolated which appear to have alterations in the glycoprotein gp85 and which are defective in specification of virus host range (Mason and Yeater, 1977; Zarling et al., 1977). The nature of the defects in the structure of the glycoprotein in these reports has not been determined. The above reports and the studies presented here suggest that alterations in gp85 can affect, biological and immunological properties of the virus. It is possible that changes in the carbohydrate side chain structure of MAV-2(O) gp85 and increased levels of gp85 may have functional significance in the specific interactions of MAV2(O) with target cell receptors that determine its oncogenic spectrum. Further studies are necessary to define the structural components of gp85 that are involved in these processes. ACKNOWLEDGMENTS We thank

Drs.

R. Green

and A. Hizi

for purified

AND

SMITH

gp85, Drs. D. P. Bolognesi and J. Li for anti-gp85 sera, and Dr. A. Langlois for AMV-containing supernatant fluids. We also gratefully acknowledge Drs. J. C. Paulson and D. M. Watterson for many helpful discussions and advice. This research was supported by National Cancer Institute Research Grants HOI-CA-12323 and POI-CA-14236 L. J. V. E. was the recipient of National Science Foundation Graduate Fellowship 338-0019. REFERENCES BANES, A. J., and SMITH, R. E. (1977). Biological characterization of avian osteopetrosis. Infect. Zmmune. 16,876-884. BEADREALJ, G. S., BECKER, C., SHARP, D. G., PAINTER, J. C., and BEARD. J. W. (1958). Virus of avian myeloblastosis. XI. Release of the virus by myeloblasts in tissue culture. J. Nat. Cancer Inst. 20, 351-581. BEARD, J. W. (1963). Avian virus growths and their etiological agents. Adc. Cancer Rex 7, l-127. BOLOGNF,SI, D. P., and BAUEK, H. (1970). Polypeptides of avian RNA tumor viruses. I. Isolation and physical and chemical analysis. Virology 42, 1097-l 112. BOLOGNESI, D. P., BAUER, H., GELDERBLOM, H., and HUPER. G. (1972). Polypeptides of avian RNA tumor viruses. IV. Components of the viral envelope. Virology 47, 551-566. DUESBERC,, P. H., MARTIN, G. S., and VOGT, I’. K. (1970). Glycoprotein components of avian and murine RNA tumor viruses. Virolog-v 41, 631-646. ECKEHT, E. A., BEARD, D., and BEARD, J. W. (1951). Dose response relations in experimental transmission of avian erythromyeloblastic leukosis. I. Host response to the virus. J. Nat. Cancer Inst. 12, 447-463. GAI.EHOUSE, D. M., and DUESHERG, P. H. (1978). Glycoproteins of avian tumor virus recombinants: evidence of intragenic crossing-over. J. Vzrol. 25, 86-96. GREEN, R. W., and BOI,O~NESI, D. P. (19i4). Isolation of proteins by gel filtration in 6 M guanidinium chloride: application to RNA tumor viruses. Anal. Biochem. 57, 108-I 17. GREEKWOOD, F. C., HUNTER, W. M., and GLOBER, J. W. (1973). The preparation of ‘,“I-labelled human growth hormone of high specific radioactivity. Biothem. J. 89 114-123. HANAFUSA, H: (1965). Analysis of the defectiveness of Rous sarcoma virus. III. Determining influence of a new helper virus on the host range and susceptibility to interference of RSV. Vwology 25, 248-255. ISHIZAKI, R., LANGLOIS, A. J., and BOLOGNESI, D. P. (1975). Isolation of two subgroup-specific leukemogenie viruses from standard avian myeloblastosis virus. J. Viral. 15, 906-912. ISHIZAKI, R., and VOC,T, P. K. (1966). Immunological relationships among envelope antigens of avian tumor viruses. Virology 30, 375-387. LAI, M., and DUESBERG, P. H. (1972). Differences

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CELL

AND

MAV-2(O)

between the envelope glycoproteins and glycopeptides of avian tumor viruses released from transformed and from nontransformed cells. Virology 50, 359-372.

LOWRY, 0. H., ROSEBROUGH, N. J., FARR, A. L., and RANDALL, R. (1951). Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193, 265-275. MASON, W. S., and YEATER, C. (1977). A mutant of Rous sarcoma virus with a conditional defect in the determinant(s) of viral host range. Virology 77, 443-455. MOSCOVICI, C. (1975). Leukemic transformation with avian myeloblastosis virus: present status. CUFF. Top. Microbial.

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Moscov~cr, C., and VOGT, P. K. (1968). Effects of genetic cellular resistance on cell transformation and virus replication in chicken hematopoietic cell cultures infected with avian myeloblastosis virus (BAI-A). Virology 35,487-497. PORTER, W. H., and WINZLER, R. J. (1975). Puriflcation and chemical characterization of the major glycoprotein of avian myeloblastosis virus. Arch. Biochem.

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ROHRSCHNEIDER, L., BAUER, H., and BOLOGNESI, D. P. (1975). Group-specific antigenic determinants of the large envelope glycoprotein of avian oncornaviruses. Virology 67, 234-241. SCHWARZ, R. T., ROHRSCHNEIDER, J. M., and SCHMIDT, M. F. G. (1976). Suppression of glycoprotein formation of Semliki forest, influenza, and avian sarcoma virus by tunicamycin. J. Virol. 19,782-791. SMITH, R. E., and BERNSTEIN, E. H. (1973). Production and purification of large amounts of Rous sar-

gp85

ANTIGENICITY

89

coma virus. Appl. Microbial. 25,346-353. R. E., and MOSCOVICI, C. (1969). The oncogenie effects of non-transforming viruses from avian myeloblastosis virus. Cancer Res. 29,1356-1366. SMITH, R. E., DAVIDS, L. J., and NEIMAN, P. E. (1976). Comparison of an avian osteopetrosis virus with an avian lymphomatosis virus by RNA-DNA hybridization. J. Virol. 17, 160-167. STRAND, M., and AUGUST, J. T. (1973). Structural components of oncogenic RNA viruses: Znterspec ZZ, a new interspecies antigen. J. Biol. Chem. 248, 5627-5633. TOZAWA, H., BAUER, H., GRAF, T., and GELDERBLOM, H. (1970). Strain-specific antigen of the avian leukosis sarcoma virus group. I. Isolation and immunological characterization. Virology 40,530-539. VAN ELDIK, L. J., PAULSON, J. C., GREEN, R. W., and SMITH, R. E. (1978). The influence of carbohydrate on the antigenicity of the envelope glycoprotein of avian myeloblastosis virus and B77 avian sarcoma virus. Virology 86, 193-204. VOGT, P. K., and ISHIZAKI, R. (1965). Reciprocal patterns of genetic resistance to two avian tumor viruses in two lines of chickens. Virology 26,664-672. VOGT, P. K., and ISHIZAKI, R. (1966). Patterns of viral interference in the avian leukosis and sarcoma complex. Virology 30, 368-374. ZARLING, D. A., MOSSER, A. G., and TEMIN, H. M. (1977). Spontaneous mutations affecting the host range of the B77 strain of avian sarcoma virus involve type-specific changes in the virion envelope antigen. J. Viro.!. 21, 105-112. SMITH,