Vol. 26, No. 1

JOURNAL OF VIROLOGY, Apr. 1978, p. 159-164

0022-538X/78/0026-0159$02.00/0 Copyright X) 1978 American Society for Microbiology

Printed in U.S.A.

Bovine and Ovine Leukemia Viruses I. Characterization of Viral Antigens WOLFGANG ROHDE,' GEORG PAULI,V JAN PAULSEN,2 ETTI HARMS,t* AND HEINZ BAUER1 Institut ficr Virologie, Fachbereich Humanmedizin' and Institut fi;r Hygiene und Infektionskrankheiten der Tiere,' Justus Liebig- Universitat Giessen, 63 Giessen, West Germany Received for publication 28 October 1977

A comparative study on several virus-specific antigens of bovine and ovine leukemia viruses is presented. In addition to the major immunologically reactive, nonglycosylated antigen p21, which has a molecular weight of 21,000, both viruses share two glycoproteins of apparent molecular weights of 60,000 (gp6O) and 32,000 (gp32), respectively. Immunological cross-reaction suggests that ovine and bovine leukemia viruses are genetically highly related.

Evidence has accumulated for the viral etiol- Cells were grown in Dulbecco minimal essential meogy of enzootic leukosis in adult cattle and sheep dium supplemented by 5 to 10% heat-inactivated calf (21). Short-term lymphocyte cultures from leu- serum; the medium was collected every 24 h and frozen kemic animals produce C-type particles which at -200C. For virus purification, cell debris and denatured share several distinct characteristics (release of protein removed centrifugation, by low-speed Agem visharep in an was concentrated clarified medium genome, and the were virus by a budiag process,7ity RNA reverse transcriptase actiVity) of aVian and Amicon DC-2 concentrator equipped with a hollow mammalian leukosis viruses. For bovine leuke- fiber cartridge, type HlP100. Virus was pelleted from mia virus (BLV), molecular hybridization has the concentrate in an SW27 rotor at 25,000 rpm at 4°C revealed that the virus is exogenous to the bo- for 2 h through a cushion of 30% (wt/vol) sucrose in vine species (2, 9) and that its genetic infornma- TE buffer (10 mM Tris-hydrochloride, pH 7.4, and 1 tion can be acquired by the host cell genome in mM EDTA). The pellet was resuspended in TE buffer, the form of integrated proviral DNA as a result and virus was purified by equilibrium density centrifugation in linear 20 to 70% (wt/vol) sucrose gradients of horizontal transmission. This latter observation on the mechanism of containing TE buffer. Virus was located by determining reverse transcriptase activity using polyadenylic the acid-decathymidylic viral spread hass spurred speculations that acid as the exogenous template/ tatthe leukosis viruses associated with leukemic cattle primer. Growth and purification of labeled virus. For and sheep are related, if not identical, and that interspecies transmission might have taken labeling of viral proteins, cell cultures were grown in place in nature. Indeed, immunological cross- roller bottles, and growth medium was replaced by reaction was demonstrated for ovine leukemia modified Dulbecco medium containing 20 uCi of Lvirus (OLV) and BLV with sera from leukemic [3S]methionine (590 Ci/mmol) per ml or 10 ,uCi of Danimals by various techniques (20). One major [6-3H]glucosamine hydrochloride (19 Ci/mmol) per h, and virus was collected afterand16purified antigen (p21) was found to be indigenous to both ml. Medium was centrifugation by isopycfurther viruses elucidconcentrated .. above. For polyacrylamide oTo (18). elucidate this relation- nic banding asby described viruses furtner ship, we have extended our studies on the bio- gel electrophoresis (PAGE) and immunoprecipitation, chemical characterization of virus-specific pro- virus was pelleted from appropriate fractions of the teins. The data obtained suggest that OLV and gradient. In vitro labeling of viral proteins. Purified OLV BLV are genetically highly related viruses, and that horizontal transmission from one species to or BLV was disrupted in 0.1 M borate buffer (pH 8.1) containing 0.5% Nonidet P-40 and dialyzed exhausanother might have occurred in nature. tively against 0.1 M borate buffer (pH 8.1). Viral proteins (20 Ig) were labeled with 1 mCi of "I (carrier MATERIALS AND METHODS by the chloramine T method, reisolated by gel Virus propagation and purification. Fetal lamb free) filtration on Sephadex G-25 (medium) in sterile water, adpucingBLV weretanly kidnercells prop kidney cells continously producing BLV were kndly and lyophilized. Sera. Antisera for immunodiffusion tests and raprovided by M. J. Van der Maaten. OLV was isolated from cell lines obtained by cocultivation of fetal lamb dioimmunoprecipitation were obtained from leukemic

veukosirspruea aspurrated spthleulation

continously

skin cells with lymphocytes from leukemic sheep (19). t Present address: Department of Microbiology, University of Illinois, Urbana, IL 61801.

cattle and sheep. In instances, immunoglobulin G fractions were isolated by gel filtration on Sephadex G-200 columns and used for preparation of antisera against

159

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bovine and ovine immunoglobulin G by boosting rabbits three times with 10 mg of particular immunoglobulin G (16). Serological procedures. (i) Immunodiffusion and complement fixation. Both techniques for the detection of antigenic material using homologous as well as heterologous antisera have been described in detail (12, 20). For analysis of precipitin lines, 125Ilabeled viral proteins were mixed with unlabeled material as a carrier, and the precipitin lines formed were cut out and washed exhaustively with 0.0625 M Trishydrochloride (pH 6.8) to remove unspecifically bound, labeled protein prior to PAGE. (ii) Indirect immunoprecipitation. Radioimmunoprecipitation was performed according to published procedures (16). Radioisotope-labeled cells or purified virus were disrupted in lysis buffer (0.02 M Tris-hydrochloride, pH 7.4; 0.05 M NaCl; 0.5% Nonidet P-40) containing 1 mM phenylmethanesulfonyl fluoride, 3 mM L-1-tosylamide-2-phenylmethyl chloromethyl ketone, and 3 mM N-a-p-tosyl-L-lysine chloromethyl ketone to inactivate proteases. Residual membrane complexes were removed by centrifugation, and the supernatant was used for immunoprecipitation with the individual serum. Rabbit anti-immunoglobulin G serum sufficient to provide complete precipitaton of antigen-antibody complexes was added. The precipitate was collected by low-speed centrifugation, washed twice with ice-cold lysis buffer and once with ice-cold distilled water, and pelleted again for PAGE analysis. For competition experiments, the serum was preadsorbed with unlabeled antigen preparations followed by indirect immunoprecipitation as outlined above. PAGE. Separation of proteins under denaturing conditions was achieved in N,N'-methylenebisacrylamide-cross-linked polyacrylamide (ratio of acrylamide to N,N'-methylenebisacrylamide, 37.5:1) cylindrical or slab gels using the Laemmli buffer system (10). Protein samples were heated in sample buffer (0.0625 M Tris-hydrochloride, pH 6.8; 2% sodium dodecyl sulfate [SDS]; 4% 2-mercaptoethanol; 0.01% bromophenol blue) at 100°C for 5 min before electrophoretic separation. Proteins were visualized by staining with Coomassie blue and destaining with 7.5% acetic acid in 5% methanol at 60°C or, after fixing the gel overnight, with 3.5% perchloric acid and direct staining with Coomassie G250 (23). Cylindrical gels containing labeled proteins were cut into 1-mm segments, and radioactivity was determined after solubilization with Soluene 350 in a liquid scintillation counter (3H, 14C, 35S) or without manipulation in a gamma counter

(1251).Materials. Reagents for PAGE were obtained from

Bio-Rad Laboratories. The protease inhibitors were products of Serva. The Radiochemical Centre, Amersham, supplied the radioisotope-labeled compounds; 125I was a product of New England Nuclear.

RESULTS RESULTS PAGE of viral proteins. A comparative analysis of pmteins from purified ovine and bovine leukemia viruses showed that major differences of protein composition are not obvious (Fig. la). Furthermore, it is clear, in view of the

J. VIROL.

limited genome size, that not all polypeptides copurifying with virus particles can be encoded by the viral genome. In connection with immuInconnetion twh i bythgiraldatagee. (see below; 18), the existence of nologrcal three virus-specific proteins can be ascertained. Two of them are glycoproteins; when [3H]glucosamine-labeled virus was coelectrophoresed with unlabeled virus preparations on a cylindrical gel, two dominant glycoprotein peaks appeared (Fig. lb). According to their apparent molecular weights, they have been designated and gp32, although considerable deviation gp6O is fnd dpe2n ongh polyaramide systs found depending on the polyacrylamide sys tem used for analysis (gp60 was found in the range of 53,000 to 60,000 daltons; for gp32, values of 26,000 to 35,000 daltons were obtained). The previously identified viral antigen p21 (18), which is indigenous to both viruses, was not labeled with glucosamine and did not bind to concanavalin A-Sepharose columns, whereas gp6O and gp32 did (data not shown). Thus, p21 is not a glycoprotein. Irrespective of the poly. st , system, this antigen exhibits an apacrylamlde parent molecular weight of 21,000. The low-molecular-weight region is not well resolved (Fig. la and b) and probably contains the recently characterized p15 (8) or p16 (4). Our assignment of viral antigens is in good agreement with the data of Deshayes and colleagues (4), who by complement fixation identified four virus-specificpolypeptides (gp6O, p35, p24, and p16) in BLVp BL . Antigens involved in the precipitin formation The serological analysis of viral antigens from either purified OLV and BLV or concentrated supernatants from virus-producing tissue culture by the double immunodiffusion test gave rise to the formation of one or two precipitin lines, depending on the particular antigen or antiserum preparation. Precipitin lines of identity were formed with homologous and heterolandhetens o sera T (Fig. 22a). ogous sera fig. a). To identify the antigens involved, i-labeled proteins from purified OLV andd BLV, respectively, were included in the double immunodiffusion assay. The precipitin lines I and II were cut out and washed with 0.0625 M Tris-hydrochloride (pH 6.8) to remove unspecifically bound labeled protein. The agar was made 0.1% in SDS and 4% in mercaptoethanol heated at 100'C for 5 mi, and layered on top of cylindrical 15% polyacrylamide gels. For BLV, such an analysis is shown in Fig. 2b. Precipitin line I contained one antigen, which from its molecular weight (21,000) is identical to p21 previously identified by radioimmunoprecipitation of [35S]methionine-labeled virus preparations (18). Line II, which was formed by the "ether-sensitive" antigen (14, 17), consisted of two polypeptides. In all probability, they correogou

VOL. 26,1978

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(rnm) FIG. 1. Polyacrylamide gel electrophoresis ofpurified BLV and OLV. (a) Preparative analysis ofpurified virus by SDS-PAGE on 10% polyacrylamide slab gels; electrophoresis was carried out at 4°C and 30 mA for 28 h, the gel was fixed with 3.5% perchloric acid, and proteins were stained directly with Coomassie G250. Marker proteins: BSA (bovine serum albumin, 68,000 daltons), OVA (ovalbumin, 45,000 daltons), CHY (chymotrypsin, 24,500 daltons), CYT (cytochrome c, 12,400 daltons). (b) Analytical SDS-PAGE of BLV. Coelectrophoresis of BL V with rH]glucosamine-labeled BLV was carried out on a cylindrical 9% polyacrylamide gel at 4°C and 4 mA per gel. The gel was stained with Coomassie brilliant blue and, after scanning, was sliced into 1-mm segments to determine radioactivity.

spond to the glycoproteins gp6O and gp32, although in this particular analysis a lowered apparent molecular weight was observed (54,000 and 26,000, respectively). Under nonreducing conditions (omission of mercaptoethanol), both protein peaks were reduced drastically, and most of the counts were found in the upper portion of the gel (Fig. 2b). This finding might suggest that gp32 appears in precipitin line II, because it is covalently linked to gp6O by disulfide bonds. Glycoproteins reacting in the indirect radioimmunoprecipitation. With [3S]methionine-labeled antigen preparations, we have previously demonstrated that one major antigen, p21, is precipitated by homologous and heterologous sera from both OLV and BLV (18). Immunoprecipitation of [3H]glucosamine-labeled viral proteins of ovine and bovine origin, fol-

lowed by SDS-PAGE analysis, revealed that two glycoproteins with apparent molecular weights of 60,000 and 32,000 participate in this reaction (Fig. 3a). These proteins were not precipitated by normal control sera (Fig. 3c). Preadsorption of sera with proteins from purified OLV prevented the immunoprecipitation of the BLVspecific glycoproteins (Fig. 3b). These data confirn conclusions, based on the inhibition of syncytia formation (13), that OLV and BLV share

cross-reacting glycoproteins.

DISCUSSION OLV and BLV are C-type RNA tumor viruses that share several cross-reacting antigens (20) and that cannot be distinguished morphologically (13). In this communication we further

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J. VIROL.

existence of one major immunologically reactive, nonglycosylated protein with an apparent molecular weight in the range of 21,000 to 25,000 (p21 to p25), several groups have reported on the presence of viral glycoproteins with molecOO ^>-ular weights ranging from 45,000 to 69,000 (4, 5, 8, 15). Our data on the PAGE analysis of [3H]glucosamine-labeled, purified OLV and BLV, of indirect radioimmunoprecipitates of viral glycoproteins, and of the precipitation bands formed in the double immunodiffusion of "I25-labeled proteins strongly suggest that OLV and BLV encode for two glycoproteins with apparent molecular weights of 60,000 (gp6O) and 32,000 (gp32), respectively. These molecular weights were determined from internal markers (e.g., from coelectrophoresis of [3H]glucosaminelabeled proteins with "4C-amino acid-labeled Semliki Forest virus proteins; Fig. 3), but deviations from these values occur with other polyacrylamide systems and antigen preparations. Similar observations have been reported for the glycoprotein gp34 of murine mammary tumor virus (7). Thus, the high-molecular-weight glycoprotein gp6O very likely corresponds to the gp6O of Deshayes et al. (4) and to the gp5l described by Devare and Stephenson (5) for BLV. In addition we describe a second immunologically reactive glycoprotein, gp32. Both glycoproteins constitute the precipitin line II in the double immunodiffusion test which is formed by the "ether-sensitive" antigen(s). Thus, these glycoproteins may be considered to be constituents of the viral envelope and involved in syncytia formation with appropriate indicator cells (6, 13). The finding that gp6O and gp32 are linked via disulfide bonds may reflect their actual interaction on intact virus particles, although we cannot exclude that covalent linkage has occurred after solubilization of the proteins. In ;°. ...-J Rous sarcoma virus, the two membrane-associated viral glycoproteins, gp85 and gp35, are held FIG. 2. Double immunodiffusion of BLV-specific together by S-S bridges (11) and form the knobantigens. (a) Precipitin lines formed with serum from like projections on the outer viral envelope (1). a leukemic sheep (0) and with sera from two leukemic Under mild reducing conditions, gp85 is seleccattle (BI, B2) and one healthy cow (B3). (b) Analysis tively removed, while gp35 remains associated of '25I-labeled BLV antigens trapped in precipitin with the intact virion (G. Pauli, W. Rohde, and lines I and II by SDS-PAGE on cylindrical 15% E. Harms, Arch. Virol., in press). A similar model polyacrylamide gels at 3 mA per gel was as described could be imagined for the interaction of gp6O in the text. and gp32 of OLV and BLV. Together with the nonglycosylated protein substantiate the high relatedness of both viruses p21, which constitutes precipitin line I, three by biochemical studies on the polypeptides spec- cross-reacting virus-specific antigens have been ified by the viral genome. In view of the confus- ascertained for OLV and BLV. The recently ing reports on various virus-specific proteins, we described BLV antigen p15 (8) or p16 (4) is not have found it necessary to characterize the an- prominent in the double immunodiffusion test tigens involved in immunological cross-reac- of total virus preparations. Also, reverse trantions. While there is general agreement on the scriptase has not yet been identified biochemi-

PIN,

BOVINE AND OVINE LEUKEMIA VIRUSES

VOL. 26, 1978

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FIG. 3. Indirect radioimmunoprecipitation of 3H]glucosamine-labeled BL Vproteins. (a) SDS-PAGE analysis of BL V glycoproteins precipitated with serum from a leukemic sheep. (b) Same as (a), except serum was preadsorbed with 5 ug of purified OLV proteins. (c) SDS-PAGE analysis of BL V glycoproteins precipitated with a negative control serum. Arrows indicate position of "C-amino acid-labeled proteins from Semliki Forest virus; numbers are apparent molecular weights (xl,000).

cally, although antibodies directed against the enzyme from BLV are present in sera of leukemic cattle (24). The degree of genetic relatedness of OLV and BLV has yet to be established. Immunological

byvariousr.,ologic observed byvaxiousserologicross-reactions, as observed.

cal procedures including inhibition of radioimmunoprecipitation by preadsorption of sera, demonstrate that the same antigenic determinants are recognized on the viral proteins from both viruses. Furthermore, the three antigens, gp6O, gp32, and p21, are indigenous to both OLV and BLV. Although the existence of a distinct 60-70S viral RNA genome has been described

This study was supported by the Deutsche Forschungsge-

meinschaft, SFB 47. LITERATURE

CID

iTERATURECITr eldebo D. P., H. Bauer, H. Gelderblom, and G. Bolognesi,1972. 1.L.Blge Polypeptides H

Huiper. IV. Components

H

adG

of avian RNA tumor viruses. of the viral envelope. Virology

47:551-566. 2. Callahan, R., M. M. Lieber, G. J. Todaro, D. C.

Graves, and J. F. Ferrer. 1976. Bovine leukemia virus

genes in the DNA of leukemic cattle. Science

192:1005-1007.

3. Crawford, L. V., and R. R. Gesteland. 1973. Synthesis of polyoma proteins in vitro. J. Mol. Biol. 74:627-634.

L., D. Levy, A. L. Parodi, and J. P. Levy. 4. Deshayes, 1977. Proteins of bovine leukemia virus. I. Characterization and reactions with natural antibodies. J. Virol.

21:1056-1060. for BLV (2, 9, 22), we have not yet been able to 5. Devare, S. G., and J. RI Stephenson. 1977. Biochemical isolate labeled RNA in amounts sufficient for and immunological characterization of the major envebiochemical characterization. Thus, we cannot glycoprotein of bovine leukemia virus. J. Virol. the geneticlope anything relatedness (as genetic on the 23:443-447. say anything 6. Diglio, C. A., and J. F. Ferrer. 1976. Induction of would be possible from hybridization experisyncytia by the bovine C-type leukemia virus. Cancer ments with complementary DNA) or similarities Res.A.36:1056-1067. in nucleotide sequence by T, oligonucleotide S., C. J. Williams, and A. A. Pomenti. 1977. Dion, antigens of vlral viral antlgens fingerprinti7Characterization The major structural proteins of mammary tumor virus: fingerprinting. Characterlzatlon of is techniques for isolation. Anal. Biochem. 82:18-28. by tryptic peptide mapping in progress to 8. Kaaden, 0. R., B. Frenzel, B. Dietzschold, F. Weiland, establish the genetic relationship of OLV and and M. Mussgay. 1977. Isolation of a p15 polypeptide BLV, as long as direct studies on the viral gefrom bovine leukemia virus and detection of specific on say

not feasible. feasible. nome are not nome are

ACKNOWLEDGMENTS We thank U. Schneider and H. Reincke for technical assistance.

antibodies in leukemic cattle. Virology 77:501-509.

Y. 9. Kettmann, R., D. Portetelle, M. Mammerickx, Cleuter, D. Deckegel, M. Galoux, J. Ghysdael, A. Burny, and H. Chantrenne. 1976. Bovine leukemia virus: an exogenous RNA oncogenic virus. Proc. Natl.

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Acad. Sci. U.S.A. 73:1014-1018. 10. Laemmli, U. K. 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature (London) 227:680-685. 11. Leamnson, R. N., and M. S. Halpern. 1976. Subunit structure of the glycoprotein complex of avian tumor virus. J. Virol. 18:956-968. 12. Miller, J. M., and M. J. Van der Maaten. 1974. A complement-fixation for the bovine leukemia (C-type) virus. J. Natl. Cancer Inst. 53:1699-1702. 13. Ogura, H., J. Paulsen, and H. Bauer. 1977. Crossneutralization of ovine and bovine C-type leukemia virus-induced syncytia formation. Cancer Res. 37:1486-1489. 14. Onuma, M., C. Olson, L. E. Baumgaertner, and L. D. Pearson. 1975. An ether-sensitive antigen associated with bovine leukemia virus infection. J. Natl. Cancer Inst. 55:1155-1158. 15. Onuma, M., C. Olson, and D. M. Driscoll. 1976. Properties of two isolated antigens associated with bovine leukemia virus infection. J. Natl. Cancer Inst. 57:571-578. 16. Pauli, G., and H. Ludwig. 1977. Immunoprecipitation of Herpes simplex virus type 1 antigens with differeat antisera and human cerebrospinal fluids. Arch. Virol. 53:139-155. 17. Pauli, G., W. Rohde, H. Ogura, E. Harms, H. Bauer, and J. Paulsen. 1976. Comparative immunological studies on bovine and ovine C-type particles, p. 45-48. In E. Burmy (ed.), Bovine leukosis: various methods of molecular virology. Commission of the European Communities. Coordination of Agricultural Research, Brus-

sels.

J. VIROL. 18. Paulsen, J., W. Rohde, G. Pauli, E. Harms, and H. Bauer. 1975. Comparative studies on ovine and bovine C-type particles. Comparative leukemia research. Bibl. Haematol. Basel 43:190-192. 19. Paulsen, J., R. Rudolph, R. Hoffmann, E. Weiss, and T. Schliesser. 1972. C-type virus particles in phytohemagglutinin-stimulated lymphocyte cultures with reference to enzootic lymphatic leukosis in sheep. Med. Microbiol. Immunol. 158:105-112. 20. Paulsen, J., R. Rudolph, and J. Miller. 1974. Antibodies to common ovine and bovine C-type virus specific antigen in serum from sheep with spontaneous leukosis and from inoculated animals. Med. Microbiol. Immunol.

159:105-114. 21. Piper, C. E., D. A. Abt, J. F. Ferrer, and R. R. Marshak. 1975. Seroepidemiological evidence for horizontal transmission of bovine C-type virus. Cancer Res. 35:2714-2716. 22. Portetelle, D., R. Kettmann, M. Mammerickx, Y. Cleuter, J. Galoux, A. Burny, and H. Chantrenne. 1975. Biochemical characterization of bovine leukemia virus. Comparative leukemia research. Bibl. Haematol. Basel 43:363-365. 23. Reisner, A. H., P. Nemes, and C. Buchholtz. 1975. The use of Coomassie brilliant blue G250 perchloric acid solution for staining in electrophoresis and isoelectric focusing on polyacrylamide gels. Anal. Biochem. 64:509-516. 24. Wuu, K. D., D. C. Graves, and J. F. Ferrer. 1977. Inhibition of the reverse transcriptase of bovine leukemia virus by antibody in sera from leukemic cattle and immunological characterization of the enzyme. Cancer Res. 37:1438-1442.