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Purification and Serological Characterization of the Major JOURNAL OF VIROLOGY, Mar. 1976, p. 727-736 Vol. 17, No. 3 copyright © 1976 American Society for Microbiology Printed in U.S.A. Purification and Serological Characterization of the Major Envelope Glycoprotein from AKR Murine Leukemia Virus and Its Reactivity with Autogenous Immune Sera from Mice JAMES N. IHLE, TIMOTHY P. DENNY, AND DANI P. BOLOGNESI* Basic Cancer Research Program, NCI Frederick Cancer Research Center, Frederick, Maryland 21701, and Duke University Medical Center, Durham, North Carolina 27710* Received for publication 7 October 1975 The major envelope glycoprotein (gp7l) from AKR murine leukemia virus (MuLV) was purified and its serological reactivity with heterologous and autogenous immune mouse sera was examined. Homologous and interspecies competition radioimmunoassays using antisera to Rauscher-MuLV gp69/71 or Friend-MuLV gp7l or antisera to feline leukemia virus to precipitate '25I-labeled gp7l from various MuLV showed that distinct differences exist between Rauscher- or Friend-MuLV and AKR-MuLV glycoproteins. Characteristically, the AKR-MuLV gp7l, in contrast to FLV or RLV gp7l, does not compete fully in homologous or interspecies radioimmunoassays with iodinated Friend or Rauscher glycoproteins. Purified 125I-labeled AKR-MuLV gp7l, in contrast to the Rauscher- or Friend-MuLV glycoproteins, reacts with normal (autogenous im- mune) mouse sera in direct radioimmune precipitation assays. Competition experiments further demonstrate that this is a predominant immunological reactivity of normal mouse sera which had previously been detected by radioimmune precipitation assay against intact virions. The major glycoprotein constituents of both Friend- and Rauscher-MuLV infectivity Rauscher and Friend murine leukemia viruses (10, 19). A lesser but distinct neutralization was (MuLV) have been purified (16, 20). Two glyco- also demonstrated for Gross virus, indicating peptides which have not been separated are the participation ofgroup-specific determinants present in the Rauscher-MuLV preparations in the neutralization reaction. Furthermore, and have an approximate molecular weight of Hunsmann et al. (10) have demonstrated inter- 69,000 and 71,000 (gp69/71), respectively. The ference of Friend- and Gross-MuLV infectivity Friend-MuLV gp7l, however, consists of a sin- with purified Friend-MuLV gp7l. These studies gle glycoprotein with a molecular weight of support the notion that this and analogous approximately 71,000. Using heterologous oncornavirus glycoproteins represent the major monospecific antisera to these glycoproteins, virion envelope components which mediate the several studies have demonstrated cross-reac- attachment ofthe virus to the cell during infec- tive components in a variety of type-C viruses, tion (1, 10, 24). suggesting that similar glycoproteins are basic Not only is gp7l localized on the virion structural elements of this class of viruses (21). surface, but it also appears to be consistently Distinct differences also exist among the glyco- expressed on the surface of virus-infected proteins of the various agents, demonstrating and/or transformed cells at sites not usually the presence of type-, group- and interspecies- associated with budding virions (J.N. Ihle, J.C. specific determinants comparable to those ini- Lee, J. Longstreth, and M. G. Hanna, Jr., in tially described for the virion p30 (3, 5). In the press; H. Schwarz, M. Claviez, G. Hunsmann, case of Rauscher-MuLV gp69/71, the predomi- V. Moennig, and W. Schafer, Virology, in nant determinants are type- and group-specific press). A similar situation was described for with only a small fraction of the molecule the analogous avian virus glycoprotein, gp85 bearing interspecies cross-reactive sites (21). (4). These sites may be significant in the cyto- Both the Rauscher- and Friend-MuLV glyco- toxic reactivity of monospecific antisera to gp7l proteins have been shown to have a number of against such cells (6). However, all of the anti- biologically significant properties. Monospecific genic determinants expressed on the virion heterologous antisera to Rauscher-MuLV surface may not be expressed on the cell sur- gp69/71 and Friend gp7l strongly neutralize face (Ihle et al., in press). It has been suggested 727 728 IHLE, DENNY, AND BOLOGNESI J. VIROL. that the expression of the MuLV glycoprotein were at 4 C. The virus was pelleted from the media gp69/71 occurs on the surface of not only virus- through 5 ml of 25% sucrose in PNE (0.05 M sodium replicating cells but also cells of several histo- phosphate, pH 7.0, 0.1 M NaCl, 0.001 M EDTA) in a linked to the Spinco SW25.2 rotor at 25,000 rpm for 1 h. The logical types and has been GIx virus-containing pellets were resuspended in a mini- differentiation marker (2, 22). The conse- mal volume of PNE and the virus was further purified quences of this expression, however, are un- by centrifugation on a linear 15 to 50% isopycnic clear. sucrose gradient in PNE overnight at 25,000 rpm in a The involvement of envelope glycoprotein in Spinco SW25.1 rotor. The virus band at 1.16 g/cc was the autogenous immune response in mice to collected and diluted with PNE; and the virus was their endogenous type-C viruses has been dem- concentrated by pelleting at 25,000 rpm for 2 h in a onstrated (Ihle and Hanna, Comtemp. Top. Spinco SW25.1 rotor resuspended in PNE and stored Immunobiol., in press; Ihle et al., in press; at -170 C. 11, 12). These studies demonstrated the exis- Rauscher-MuLV was either purified, as above, from the JLS V-5 cell line or was obtained from the tence of natural antibodies specific for type-C Office of Program Resources and Logistics, National viruses in a variety of mouse strains. Analysis Cancer Institute, Bethesda, Md. Friend-MuLV was of the immune complexes formed between obtained from the Eveline cell line which is derived these normal sera and disrupted labeled virus from the STU mouse strain (18a) and was purified as has suggested that gp7l, as well as gp43, and a previously described (7). FeLV (Rickard strain) was virus envelope-associated polypeptide (pl5[E]) a generous gift of F. de Noronha, Cornell University. are naturally antigenic to mice. Additional Purification of Rauscher-MuLV gp7l and p30. experiments have suggested that the predomi- Rauscher-MuLV was dialyzed against 0.01 M sodium nant immune response, however, is directed phosphate, pH 7.0, 0.01 M NaCl overnight at 4 C. and Hanna, Contemp. Top. Virion particles were subsequently pelleted by centrif- against gp7l (Ihle ugation at 25,000 rpm for 1 h at 4 C (Spinco SW25.1 Immunobiol., in press). rotor). For purification of the Rauscher-MuLV glyco- Perhaps a more relevant question regarding protein, the supernatant fraction was made 80% the above studies is the relationship of the ammonium sulfate and left at 4 C overnight. The various glycoproteins to one another. The precipitate was subsequently collected by centrifuga- Friend- and Rauscher-MuLV used in the major- tion (10,000 rpm, 20 min) and redissolved in a mini- ity of the above studies may not be representa- mal volume of 0.01 M sodium phosphate buffer, pH tive of the endogenous type-C viruses of mice 7.0, 0.01 M NaCl, and insoluble material was re- moved by centrifugation. The supernatant was then which include the Gross or AKR viruses (9) or chromatographed on a G-150 Sephadex column the recently described xenotropic viruses (13). equilibrated with 0.01 M sodium phosphate, pH 7.0, In fact, using disrupted viruses it could be 0.01 M NaCl. Generally, the glycoprotein elutes shown by competition radioimmunoassays that from this column as a homogeneous peak slightly distinct serological differences exist between behind the void volume. The purified glycoprotein Gross and Friend or Rauscher glycoproteins (21) was then concentrated by precipitation with am- consistent with the cross-neutralization studies. monium sulfate as above, dialyzed against the above Clearly serological assays using the endogenous buffer, and stored at -20 C. This material revealed these relation- only one band on sodium dodecyl sulfate (SDS) virus glycoproteins would clarify acrylamide gels and not the two components re- ships and also greatly aid the ability to measure ported by Strand and August (20). To distinguish it viral activities in the natural host. Because of from the latter, it will be designated as Rauscher- these considerations, we have purified the MuLV gp71. AKR-MuLV gp7l and compared its serological To purify the Rauscher-MuLV p30 the virus pellet properties to Rauscher- and Friend-MuLV after dialysis (see above) was resuspended in a gp7l, using both heterologous antisera mono- minimal volume ofdistilled water and made 0.1 M in specific for these glycoproteins as well as au- lithium di-iodosalicylate (LDS). After incubation at various mouse 4 C for 30 min, the virus was centrifuged at 25,000 togenous immune sera from rpm for 1 h as described above. The supernatant was strains. subsequently dialyzed against distilled water over- night and then against 0.01 M sodium phosphate, MATERIALS AND METHODS pH 7.0, 0.01 M NaCl for an additional 12 h. Insoluble Viruses. AKR-MuLV was purified from an AKR material was removed by centrifugation and the embryonic fibroblast cell line which spontaneously supernatant was applied to a G-150 column in the initiated replication of virus. The original cell line was same buffer. The protein peak eluting at a position obtained from W. P. Rowe (National Cancer Insti- of V1/V1 = 0.5 was subsequently precipitated with tute, Bethesda, Md.). The cells were maintained on ammonium sulfate (80% saturation), resuspended in Eagle minimal essential medium supplemented with a minimal volume, and dialyzed against 0.01 M 2 mM glutamine and 10% fetal calf serum. Media sodium phosphate, pH 7.0. Final purification of the were collected daily and clarified by centrifugation at p30 was achieved on a DEAE-Sephadex column 5,000 rpm for 20 min.
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