Purification and Serological Characterization of the Major

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 immune) 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 removed 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 overnight 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. All subsequent manipulations equilibrated with 0.01 M sodium phosphate, pH 7.0. VOL. 17, 1976 AKR VIRUS GLYCOPROTEIN 729 The p30 was subsequently eluted as a homogeneous overnight. All dilutions were made in radioimmunoas- peak with 0.01 M sodium phosphate, pH 7.0, 0.1 M say buffer (20 mM Tris-hydrochloride, pH 7.6, 100 NaCl, concentrated as above with ammonium sul- mM NaCl, 1 mM EDTA containing 2 mg of bovine fate, dialyzed, and stored at -20 C. serum albumin [BSAI). Antiserum to the appro- Antisera. Antiserum to Rauscher-MuLV gp7l was priate immunoglobulins (30 ,ul) (mouse or rabbit; prepared by immunizing rabbits intramuscularly Cappel Laboratories, Downington, Pa.) was then with 150 1Ag of glycoprotein in an equal volume of added and incubated for 1 h at 37 C and 3 h at 4 C. Freund complete adjuvant. An additional 150 ,ug in Immunoprecipitates were collected by centrifuga- saline was given intravenously 10 days later. Rabbits tion (8,000 x g, 2 min), washed twice with radio- were bled from the ear vein 2 weeks later and immunoassay buffer (without BSA), and counted in subsequently at weekly intervals. In contrast to the a Packard Autogamma Counter (Packard Instru- two anti-gp7l sera described below, this serum re- ment Co., Inc., Downers Grove, Ill.). acted primarily in a type-specific fashion. A goat In the competition assay, increasing amounts of antiserum to Rauscher-MuLV gp69/71 was also ob- unlabeled antigen (or virus) were incubated with a tained from T. August, Albert Einstein (20). Rabbit limiting antibody dilution (10 Ml) and the appropriate antiserum to Friend-MuLV gp7l was prepared as normal serum (30 Ml) for 2 h at 37 C. The limiting described (9). The amount of Friend-MuLV glyco- antibody dilution, determined by direct double-anti- protein injected was about five times greater than body radioimmunoassay, precipitated 50% of the for Rauscher-MuLV gp7l. An antiserum to purified respective labeled antigen. A standard amount of FeLV was prepared in goats (7) and was used for labeled antigen (1 to 5 ng in 10 Ml) was then added and interspecies assays. This serum was absorbed ex- incubated for an additional hour at 37 C. After haustively with normal cat cells, normal cat overnight incubation at 4 C, the samples were proc- plasma, fetal calf serum, and sheep erythrocytes essed as described above. (7). Radioimmunoprecipitation assay. The radioim- Purification of AKR-MuLV gp7l. Approximately munoprecipitation (RIP) assay against intact radioac- 50 mg of AKR-MuLV in 5 ml of PNE was made 0.003 tively labeled AKR virus has been described in detail M in LDS and incubated at 4 C for 30 min. Intact elsewhere (12). In brief, 0.1 ml of the test serum was virions were subsequently removed by centrifugation serially diluted twofold in PNE buffer, 0.1 ml (6,000 at 25,000 rpm for 1 h in a Spinco SW50.1 rotor. The counts/min) of labeled virus was added, and the supernatant was then dialyzed against 0.01 M sodium mixture was incubated 1 h at 37 C to allow the phosphate, pH 7.0, 0.01 M NaCl overnight at 4 C. formation of immune complexes. Subsequently, a Insoluble material was removed by centrifugation and volume of 0.1 ml of anti-mouse gamma-globulin the supernatant was made 80% saturated in ammo- (Cappel) diluted 1:2 in PNE was added; the mixture nium sulfate. The precipitated protein was collected was incubated again at 37 C for 1 h and then at 4 C for by centrifugation, redissolved in 0.5 ml of 0.01 M 2 h. The precipitates were collected by centrifugation sodium phosphate, 0.01 M NaCl, pH 7.0, and then at 1,200 x g for 15 min, and the supernatant was immediately applied to a Sephadex G-150 column (1 removed for determination of radioactivity. The pre- by 60 cm). The gp71 elutes as a homogeneous peak cipitates were washed three times with PNE, resus- from this column slightly behind the void volume. pended in 0.4 ml of PNE, and prepared for counting. The material was concentrated by ammonium sulfate All samples were counted in 10 ml of Aquasol (New precipitation as described above, dialyzed against England Nuclear, Boston, Mass.) in a Packard Tri- 0.01 M sodium phosphate, pH 7.0, 0.01 M NaCl, and Carb scintillation counter. The degree of precipi- stored at -170 C. The recovery was approximately 0.7 tation is expressed as the percentage of counts in the mg from the initial 50 mg of virus. precipitate relative to the combined counts in the Preparation of Friend-MuLV gp7l and p30. precipitate and the first supernatant. Friend-MuLV gp71 was prepared according to the Other methods. SDS polyacrylamide gel electro- method described by Moennig et al. (16) which uses phoresis was performed according to the procedure of osmotic shock for release of the antigen, followed by Weber and Osborn (23). Protein concentrations were density gradient centrifugation, and affinity chroma- determined by the method of Lowry et al. (14). tography on concanavalin A-agarose columns. Friend- MuLV p30 was purified by gel filtration in guanidine hydrochloride (7). RESULTS Radioimmunoassay. The double-antibody radi- Purification of AKR-MuLV gp7l. Attempts oimmunoassay procedure used was that described by to purify the AKR-MuLV gp7l using the proce- Strand and August (20). Polypeptides or glyco- dures described for Rauscher-MuLV gp69/71 proteins were labeled with 1251 according to the (20) or Friend-MuLV gp7l (16) have been method described by Greenwood et al. (8). The unsuccessful, specific activity of the labeled components ranged primarily due to the inability to from 5 x 103 to 2 x 104 counts/min per ng of mate- solubilize the glycoprotein from the virion (Ihle, rial. unpublished observations). Therefore, the effect Between 1 and 5 ng of '25I-labeled p30 or gp7l in a of LDS on the virus was investigated since this 10-Al volume was used. In direct radioimmunoassays, compound has been routinely used to solubilize normal serum (30 Al) and immune serum (10 Ml) were glycoproteins from erythrocytes (15). The effect added and incubated at 37 C for 3 h and 4 C of various concentrations of LDS on the release 730 IHLE, DENNY, AND BOLOGNESI J. VIROL. of virion components is shown in Fig. 1. With quently purified by ammonium sulfate pre- increasing concentrations of LDS, progressively cipitation and Sephadex G-150 chromatography more solubilization is observed until 80% of the as described. The resultant preparation was protein has been solubilized at the highest LDS homogeneous by SDS-polyacrylamide gel elec- concentrations. Since we were primarily inter- trophoresis (Fig. 2) and was used in subsequent ested in the virion envelope components, we studies. Recoveries were normally 1 to 2% of examined the solubilized proteins after treat- the initial total protein which constitutes ap- ment of the virus with 0.003 M LDS. As seen in proximately 50% of the available glycoprotein Fig. 2, this treatment releases three proteins in the virus. with molecular weights of approximately Serological characterization of AKR- 71,000, 30,000, and 12,000 on SDS-polyacryla- MuLV gp7l. The reaction of 125I-labeled AKR- mide gel electrophoresis. The 71,000-molecular- MuLV gp7l with various antisera is shown in weight component stained with periodic acid- Fig. 3. Approximately 90% of the labeled pro- Schiff which is indicative of a glycoprotein tein is precipitated by monospecific antisera to (data not shown). The 30,000-molecular-weight Rauscher-MuLV gp69/71 (from T. August) and protein reacts with monospecific antisera to Friend-MuLV gp7l. For comparison, the titra- Rauscher-MuLV p30. The identity of the tion curves obtained with 125I-labeled Friend- 12,000-molecular-weight component is un- MuLV gp7l are also shown. The comparable known, but has several properties associated with Friend-MuLV p12 (unpublished data). The 71,000-molecular-weight protein was subse-

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0 6 12 24 48 96 LDS CONCENTRATION (mM) FIG. 1. Effect of LDS on disruption ofAKR-MuLV. [Illeucine-labeled AKR-MuLV (150,000 counts/ min, 6 tg) in PNE (0.25 ml) was incubated with the FIG. 2. SDS-polyacrylamide gel electrophoresis of indicated concentrations of LDS for 30 min at 4 C. virion proteins. All samples were incubated 12 h at 37 The samples were diluted to 2.0 ml with distilled C in 1% SDS, 1% mercaptoethanol, and 0.01 M water and centrifuged at 25,000 rpm for 60 min at 4 C sodium phosphate, pH 7.0, before electrophoresis on in a SW50.1 Spinco rotor. The percent soluble is 10% polyacrylamide gels for 6 h at 8 mA per tube. Left that proportion of the radioactivity remaining in the to right, 20 ,g of LDS solubilized virion proteins, 20 supernatant after centrifugation. ug ofpurified AKR-MuL Vgp71. 731 VOL. 17, 1976 AKR VIRUS GLYCOPROTEIN titers of these antisera with both glycoproteins The direct precipitation assays demonstrate indicate the presence of common antigenic cross-reactivity but leave unresolved both the determinants. extent and nature (e.g., type, group, or interspecies) of the shared antigenic sites. To answer these questions, it was necessary to examine the capacity of AKR-MuLV gp71 to compete in homologous assays with the Rauscher- or 100 - Friend-MuLV glycoproteins. Figure 4A shows a I- 0 that Friend-MuLV gp7l competes effectively I-. for the precipitation of 125I-Friend-MuLV gp7l K by its homologous antiserum. Similarly, cJ DI. Rauscher-MuLV gp7l is able to compete fully z 50 - in this reaction, although about fivefold more DI Rauscher-MuLV gp7l is necessary to achieve the same degree of competition. The similarity of the slope and the complete competition by the two glycoproteins indicate a high degree of

0 -0 o 0-0o o relatedness. In marked contrast, the maximum 10 100o 1000 1o,00 100,000 competition achieved with the AKR-MuLV I/SERUM DILUTION gp7l was 60% and occurred at protein concentrations 4,000-fold higher than were required for FIG. 3. Precipitation of Friend- and AKR-MuLV the Friend-MuLV glycoprotein to yield compa- glycoproteins by various antisera. An antiserum rable competition. Furthermore, the slope of the prepared against Friend-MuLV gp7l was titered competition curve is significantly less pro- against iodinated Friend-MuLV gp7l (4) or AKR- MuLV gp7l (U). Similarly, a rabbit antiserum to nounced than those of the Friend- or Rauscher- Rauscher-MuLV gp7l was titered against iodinated MuLV glycoproteins. Taken together these Friend-MuLV gp7l (0) or AKR-MuLV gp7l (A). observations demonstrate that, although sero- The precipitation of Friend- or AKR-MuLV glyco- logically cross-reactive, the AKR-MuLV gp7l protein by normal rabbit serum is also shown (0). lacks many antigenic sites common to the

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01 0.1 10 100 10 00 01 10 100 1000 10,000 NANOGRAMS gp?1 NANOGRAMS DISRUPTED vIRus FIG. 4. Homologous double-antibody radioimmunoassay. An antiserum to Friend-MuLV gp7l was used for the precipitation of iodinated Friend-MuLV gp7l in the competition assays in A and B. Competing antigens included the purified glycoproteins from Friend (0), Rauscher (A) and AKR (0) in A and disrupted virions ofFriend (0), Gross (B), AKR (-), and FELV (A) in B. In C and D a rabbit antiserum to Rauscher- MuLV gp7l (Materials and Methods) was used for the precipitation of iodinated Rauscher-MuLV gp7l. Competing antigens included Rauscher-MuLV gp7l (0) or AKR-MuLV gp7l (0) in C or disrupted Rauscher-MuLV (0) or AKR-MuLV (0) in D. 732 IHLE, DENNY, AND BOLOGNESI J. VIROL. Rauscher- or Friend-MuLV glycoproteins. This portion of Friend-MuLV gp7l is distinctly dif- is not attributable to loss of antigenicity of the ferent from the Rauscher or Friend glycopro- glycoprotein during purification as can be seen teins or the native viruses. In fact, the curves in Fig. 4B, which shows the results of competi- obtained reflect differences as striking as those tions with the native viruses. Clearly, AKR as seen in the homologous assays (Fig. 4). These well as Gross viruses compete very poorly for observations suggest that not all of the "inter- 125I-Friend-MuLV gp7l in comparison to in- species" antigenic determinants of Rauscher tact Friend virus. Furthermore, the competition and Friend virus glycoproteins which are curves obtained with the viruses were analogous shared with FeLV are present on AKR or Gross to those obtained with the purified glyco- MuLV gp7l molecules. proteins in the percent competition, displace- Reactivity of AKR-MuLV gp7l with auto- ment, and slope. genous immune sera. Previous experiments This difference is further emphasized in simi- have demonstrated the natural occurrence of lar studies using a rabbit antiserum to Rausch- antibodies specific for endogenous type-C vi- er-MuLV gp7l which appears to be more type ruses in a variety of mouse strains (12). Im- specific than that to Friend-MuLV gp7l. Com- mune precipitations of labeled disrupted virion petition assays using this antiserum are shown preparations have suggested natural immune in Fig. 4 (C and D). Neither AKR-MuLV gp7l reactivity to the virion surface constituents nor AKR virus competed for precipitation of gp7l, gp43, and p15(E) (12). We, therefore, '251-Rauscher-MuLV gp7l by this antiserum. examined the reactivity of autogenous immune To further define the serological properties of sera with '2II-labeled AKR-MuLV gp7l (Fig. 6). the AKR-MuLV gp7l, we investigated its abil- Pooled sera from 1-year (B6C3)F1 [(C57BL/6 x ity to compete in an interspecies assay. For C3H/Anf)F2] mice and 3-month C3H/Anf mice these studies a potent antiserum to purified had titers of 1:1,280 and 1:320 in an RIP assay FeLV prepared in goats which effectively pre- using ['H leucine-labeled intact AKR virions cipitates Friend-MuLV gp71 (7) was used. As (Fig. 6A). These same sera had titers of 1:320 shown in Fig. 5 (A and B), the relative capac- and 1:80 in a direct double-antibody radio- ity of AKR-MuLV gp7l or AKR virus to com- immunoassay against 125I-labeled AKR-MuLV pete with antibody recognizing the interspecies gp7l (Fig. 6B) precipitating 85% ofthe iodinated AKR gp7l. The specificity of this precipitation was further examined by SDS-polyacrylamide A l0 - gel electrophoresis of the immune precipitates. As seen in Fig. 7, the profiles contained a single radioactive peak at approximately the position corresponding to a molecular weight of 71,000, 50u identical to the peak of radioactivity precipitated by antisera to Friend-MuLV gp7l. Fur- z thermore, a pool of sera from 2-month (B6C3)F, t: mice which had no detectable 0ru antibodies against a1 intact virions failed to react with 1251-labeled B AKR-MuLV gp7l. Lastly, when the 1-year 100 - (B6C3)F, sera were absorbed with AKR virus and then reacted with either 125I-labeled AKR- z MuLV gp71 or [3Hlleucine-labeled virions, no 0-- .0;- precipitating antibodies were detectable by either assay. These results provide a direct /.7. demonstration of the presence of antibodies directed against AKR-MuLV gp7l in sera from normal mice and suggest the correlation between these antibodies and precipitating anti- 0.01 01 1 10 100 1000 10.000 100,000 bodies against intact virions. NANOGRAMS gp71 The reaction of 1-year (B6C3)F, sera with FIG. 5. Interspecies double-antibody radioim- 126I-labeled Rauscher-MuLV is munoassay. An antiserum prepared in goats to FeLV gp7l also shown was used to precipitate iodinated Friend-MuLV in Fig. 6. In contrast to the results obtained with gp7l. Competing antigens included the purified AKR-MuLV gp7l, these sera had only weak glycoproteins from Friend (0), Rauscher (A), or titers (<1:10) against this glycoprotein. These AKR (O) MuLV in A and disrupted Friend (0), results emphasize the serological differences Rauscher (A), AKR (O) or Gross (A) MuLV in B. between Rauscher- and AKR-MuLV gp7l and VOL. 17, 1976 AKR VIRUS GLYCOPROTEIN 733

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10 20 40 80 160 32'0 640 128025605120 1/SERUM DILUTION 2/ \.... \ FIG. 6. Titration of various mouse sera. Sera from '. ; . , 1-year (B6C3)Fl mice (0), 2-month (B6C3)F, mice (0), 3-month C3HIAnf mice (0), and serum from 1-year (B6C3)F, mice which had been incubated for 5 10 15 20 25 30 35 40 45 45 min at 37 C and 45 min at 4 C with 1 mg of AKR-MuLV before removal of the virus by centrifu- SECTION NUMBER gation (25,000 rpm, 60 min, 4 C) (A) were titered FIG. 7. SDS-polyacrylamide gel electrophoresis of against [3HJleucine-labeled intact AKR-MuLV by immune precipitates. Sera from (B6C3)F, mice (A) or RIP (A) or in double-antibody radioimmunoassay a rabbit antiserum to Rauscher-MuLVgp7l (B) were against 12"I-labeled AKR-MuLV gp7l (B). In addi- reacted with iodinated AKR-MuLV gp7l (30,000 tion, sera from 1-year (B6C3)F, mice were titered in counts/min, 5 to 6 ng) at dilutions of 1:10 and 1:100, double-antibody radioimmunoassay against 125I-la- respectively, for 3 h at 37 C and 18 h at 4 C. Immune beled Rauscher-MuLVgp71(U, B). complexes were precipitated by the addition of 0.1 ml of the appropriate anti-gamma-globulin antiserum as described in Materials and Methods. The immune precipitates were collected, washed, and electro- suggest that the autogenous immune response phoresed as described in Fig. 2. in mice to the virion glycoprotein has type specificity for AKR-MuLV gp7l. This, however, may not be true for other naturally antigenic precipitation of leucine-labeled AKR virions at virion components (J. Ihle, J. Domotor, and limiting dilutions of (B6C3)F, sera. The results K. Bengali, manuscript in preparation). are shown in Fig. 8. Approximately 1 utg of Previous experiments have indicated that the AKR-MuLV gp7l was required to give 50% predominant immune response in (B6C3)F, inhibition of the precipitation of approximately mice is directed against gp7l, although some 1 ,g of virus. This inhibition was specific in that activity is directed at gp43 and p15(E). To Rauscher-MuLV gp7l did not compete in the further examine this question we evaluated the reaction. Although complete competition was ability of AKR-MuLV gp71 to compete for not achieved, the results are compatible with 734 IHLE, DENNY, AND BOLOGNESI J. VIROL. I 00 homologous assay using Friend-MuLV gp7l and its antiserum showed that AKR-MuLV gp7l and AKR virus can be easily distinguished from the Rauscher- or Friend-MuLV glycoproteins I- or the respective viruses. These results using purified AKR-MuLV gp7l substantiate the 2 0. 50 studies by Strand and August (21) which indi- 7// cated type-specific differences among murine z w type-C glycoproteins. However, these authors a: did not find differences in the interspecies reac- w tivities of various MuLV. In this study, a similar analysis using AKR- and Gross-MuLV, as well as the purified AKR-MuLV gp7l, showed distinct differences relative to the correspond- 1 0 1 00 1 000 1 0, 000 ing Rauscher or Friend components in the NANOGRAMS gp7l ability to compete for anti-FeLV antiserum in an interspecies assay. This lack of correlation Fit;. 8. Competition for precipitation of intact could be due to the serological purity of the virions by (B6C3)F, serum with AKR-MuLV gp7l. virus strains, or possibly the nature of the One-year (B6C3)F, serum (1:320) was reacted with heterologous anti-FeLV antiserum used. In [3H]leucine-labeled AKR-MuLV under conditions fact, preliminary evidence supports the latter identical to those used in the RIP assays but in the and suggests that it may be due to the fact presence of increasing amounts of AKR-MuLV gp7l that the anti-FeLV antiserum employed in this or Rauscher-MuLV gp7l (0). (0) study was absorbed with normal cat constituents which may have removed activity that the predominant immune against normal cross-reactive mouse antigens. the hypothesis These questions are presently under investiga- response against the virus in these mice is the virion envelope glycopro- tion. Nevertheless, our studies show that anti- directed against genic differences can exist even in the "inter- tein gp71. species" regions of MuLV glycoproteins and DISCUSSION suggest that such assays should be used cau- tiously for both quantitative and qualitative Techniques have been developed for purifica- measures of virus-specific expression. tion of the gp7l from AKR virus and are The reaction of AKR-MuLV gp7l with sera described above. Attempts to use procedures from normal (i.e., nonimmunized) mice is ex- previously described for Rauscher-MuLV tremely significant. RIP assays with intact or gp69/71 (20) and Friend-MuLV gp7l (16) were disrupted virus have previously suggested such found not to be applicable with AKR-MuLV. an immune response (11), and the results pre- This, in part, reflects the more tenacious associ- sented here provide a direct substantiation of ation of the AKR-MuLV glycoprotein with the these studies. The titers of these normal sera are virion. Nevertheless, the results demonstrate low relative to heterologous rabbit antisera to that LDS at appropriate concentrations can be gp7l. Interestingly, however, the natural titers used to preferentially solubilize the virion glyco- that exist are comparable to those obtained protein. Furthermore, these conditions appear against BSA in mice immunized against BSA to be nondenaturing for the serological reactiv- under optimal conditions. Furthermore, some of ity ofthis glycoprotein with autogenous immune the "natural" titers against AKR-MuLV gp7l sera. The general utility of this reagent, in terms are 2- to 10-fold higher than those which can be of solubilization and purification of other virion induced in mice by active immunization with components, now needs to be examined. In this Friend-MuLV gp7I (Ihle et al., manuscript in regard, we have routinely used higher concen- preparation). trations (0.1 M) to solubilize virus for the In contrast, normal mouse sera do not react subsequent purification of p30 from both with Rauscher-MuLV gp7l. This underlines the Rauscher- and AKR-MuLV (see Materials and serological and presumably structural differ- Methods). ences between the Rauscher or Friend glyco- AKR-MuLV gp7l is serologically related to proteins and the AKR-MuLV gp7l. It also Rauscher- or Friend-MuLV gp7l as demon- demonstrates that autogenous immune sera strated by the ability of antisera specific for the react predominately with the type-specific de- latter components to precipitate the purified terminants of AKR-MuLV or AKR-MuLV AKR glycoprotein. However, competition in a gp7l. The antibodies found in normal mouse VOL. 17, 1976 AKR VIRUS GLYCOPROTEIN 735 sera are clearly the products of an immune Inc., and grant no. NO1-CP-33308 (Virus Cancer Program) response to the expression of viral antigens. In from the National Cancer Institute. this regard, a number of strains of mice We are indebted to Werner Schafer for providing the FLV in gp7l glycoprotein and its antiserum for this study. We are addition to those presented here were examined also grateful to A. Swenson for excellent technical assistance. and a good correlation between the expression of ecotropic viruses and reactivity against AKR- LITERATURE CITED MuLV gp7l was consistently detected, although 1. Bolognesi, D. P., H. Baluer, H. Gelderblom, and G. no significant reactivity was observed against Htkper. 1972. Polypeptides of avian RNA tumor viruses. IV. Components f the viral envelope. Virology Rauscher- or Friend-MuLV gp7l. These data 47:551-556. serve to emphasize that the natural immune 2. Del Villano, B. C., B; Nave, B. P. Croaker, R. A. response whi h has been measured thus far is Lerner, and F. J. Dixon. 1974. Tlte oncornavirus probably directed against endogenous mouse glycoprotein gp69/71: a constituent of the surface of normal and malignant thymocytes. J. Exp. Med. viruses which are serologically related to AKR. 141:198-205. It is interesting to note, however, that when 3. Geering, G., T. Aoki, and L. J. Old. 1970. Shared viral several mouse strains were actively immunized antigen of mammalian leukemia viruses. Nature (Lon- with Friend-MuLV gp7l a response was ob- don) 266:265-266. tained was 4. Gelderblom, H., H. Bauer, and T. Graf. 1972. Cell-sur- which type specific for the Rauscher- face antigens induced by avian RNA tumor viruses: or Friend-MuLV glycoproteins and did not detection by immunoferritin technique. Virology react with AKR-MuLV gp7l (Ihle et al., manu- 47:416-425. script in preparation). Clearly, the type-specific 5. Gilden, R. V., S. Oroszlan, and R. J. Huebner. 1971. response in mice to virion Coexistence of interspecies specific antigenic determi- glycoproteins suggests nants on the major structural polypeptide of mamma- that natural antibodies could exist against other lian C-type viruses. Nature (London) New Biol. endogenous or horizontally transmitted agents 231:107-108. with yet undefined specificity. 6. Grant, J. P., D. D. Bigner, P. J. Fischinger, and D. P. The reasons for Bolognesi. 1974. Expression of murine leukemia virus the narrow specificity of the structural antigens on the surface of chemically in- mouse antibodies for these glycoproteins remain duced murine sarcomas. Proc. Natl. Acad. Sci. U.S.A. poorly understood. Studies by Del Villano et al. 71:5037-5041 (2), which may have bearing on this point, have 7. Green, R. W., D. P. Bolognesi, W. Schafer, L. Pister, shown G. Hunsmann, and F. de Noronha. 1973. Polypep- that large quantities of glycoprotein tides of mammalian oncornaviruses. I. Isolation and molecules which share antigenic specificities serological analysis of polypeptides from murine and with Rauscher-MuLV gp69/71 are present in a feline C-type viruses. Virology 56:565-579. number of mouse tissues in the absence of virus 8. Greenwood, F. C., W. M. Hunter, and J. W. Glover. production. 1963. The preparation of "'I-labeled human growth Detection of these "normal" glyco- hormone of high specific radioactivity. Biochem. J. proteins was made with radioimmunoassays 89:114-123. specific for the interspecies determinant of 9. Gross, L. 1951. Pathogenic properties and vertical trans- Rauscher-MuLV gp69/71. That natural anti- mission of the mouse leukemia agent. Proc. Soc. Exp. bodies can be Med. 78:342-348. found in free form in the presence 10. Hunsmann, G., V. Moennig, L. Pister, E. Seifert, and of such glycoproteins remains a challenging W. Schafer. 1974. Properties of mouse leukemia vi- question. The fact that the mouse seems to ruses. VIII. The major viral glycoprotein of Friend recognize only subdeterminants of the viron leukemia virus. Seroimmunological, interfering and hemagglutinating capacities. Virology 62:307-318. glycoproteins suggests at least two explana- 11. Ihle, J. N., M. G. Hanna, Jr., L. E. Roberson, and F. tions: (i) the mouse is "tolerant" to the group T. Kenney. 1974. Autogenous immunity to endogenous and interspecies determinants of virion glyco- RNA tumor virus: identification of antibody reactivity proteins; or (ii) the mouse can respond to all of to select viral antigens. J. Exp. Med. 136:1568-1581. the virion 12. Ihle, J. N., M. Yurconic, Jr., and M. G. Hanna, Jr. gp7l determinants. However, the 1973. Autogenous immunity to endogenous RNA tumor "normal" glycoproteins which appear to share virus: radioimmune precipitation assay of mouse serum group and interspecies sites but not those which antibody levels. J. Exp. Med. 138:194-208. are type specific with the endogenous virion 13. Levy, J. A. 1973. Xenotropic viruses: murine leukemia glycoproteins could viruses associated with NIH Swiss, NZB and other form complexes with such mouse strains. Science 182:1151-1153. antibodies leaving in free form only those that 14. Lowry, 0. H., N. J. Rosebrough, A. L. Farr, and R. are type specific. The availability of purified J. Randall. 1951. Protein measurement with the Folin AKR-MuLV gp7l whose type-specific determi- phenol reagent. J. Biol. Chem. 193:265-275. nants are clearly 15. Marchesi, V. T., and E. P. Andrews. 1971. Glycopro- recognized by a variety of tein isolation from cell membranes with Lithium Di- normal mouse sera tested should make it possi- iodosalicylate. Science 174:1247-1248. ble to resolve these questions. 16. Moennig, V., HI. Frank, G. Hunsmann, I. Schneider, and W. Schafer. 1974. Properties of mouse leukemia ACKNOWLEDGMENTS viruses. VII. The major viral glycoprotein of Friend This investigation was supported by Public Health leukemia virus. Isolation and physicochemical proper- Service Grant no. NO1-CO-25423 (with Litton Bionetics, ties. Virology 61:100-111. 736 IHLE, DENNY, AND BOLOGNESI J. VIROL.

17. Nowinski, R. C., and S. L. Kaehler. 1974. Antibody to of oncogenic RNA viruses: Interspec II, a new interspe- leukemia virus. Widespread occurrence in inbred mice. cies antigen. J. Biol. Chem. 248:5627-5633. Science 185:869-871. 21. Strand, M., and J. T. August. 1975. Structural proteins 18. Obata, Y., H. Ikeda, E. Stockert, and E. A. Boyse. ofmammalian oncogenic RNA viruses: multiple anti- 1975. Relation of G x antigen of thymocytes to envelope genic determinants of the major internal protein and glycoprotein of murine leukemia virus. J. Exp. Med. envelope glycoprotein. J. Virol. 13:171-180. 141:188-197. 22. Tung, J.-S., E. S. Vitetta, E. Fleissner, and E. A. 18a. Selfert, E., M. Claviez, H. Frank, G. Hunsmann, H. Boyse. 1975. Biochemical evidence linking the G, Schwarz, and W. Schafer. 1975. Properties of mouse thymocyte surface antigen to the gp69/7 1 envelope leukemia viruses. XII. Production of substantial glycoprotein of murine leukemia virus. J. Exp. Med. amounts of Friend leukemia virus by a suspension 141:198-205. tissue culture line (Eveline suspension cells). (In 23. Weber, K., and M. Osborn. 1969. The reliability of German) Z. Naturforsch. 30:698-700. molecular weight determinations by dodecyl sulfate- 19. Steeves, R. A., M. Strand, and J. T. August. 1975. polyacrylamide gel electrophoresis. J. Biol. Chem. Structural proteins of mammalian oncogenic RNA 244:4406-4412. viruses: murine leukemia virus neutralization by anti- 24. Witter, R., H. Frank, V. Moennig, G. Hunsmann, J. sera prepared against purified envelope glycoprotein. J. Lange, and W. Schafer. 1973. Properties of mouse Virol. 14:187-189. leukemia viruses. IV. Hemagglutination of surface 20. Strand, M., and J. T. August. 1973. Structural proteins components. Virology 54:330-345.