Cellular changes in the thymuses of preleukemic AKR mice: correlation with changes in the expression of murine leukemia viruses.

Thymus cells of preleukemic and leukemic AKR mice express on their cell surface elevated levels of antigens associated with the murine leukemia virus (MuLV) proteins gp70 and p30. The gp70 antigenicity is contained in a 70,000 dalton polypeptide that corresponds to the viral envelope protein, while the p30 antigenicity is contained in two polypeptides of 85,000 and 95,000 daltons that correspond to glycosylated forms of the polyprotein product of the gag gene.The expression of these viral coded proteins on the cell surface of thymocytes varies both quantitatively with the age of the mouse and qualitatively with the cellular populations that express these antigens. Four discrete stages in the leukemic pathway can be identified. First, low numbers of cells from the thymuses of young (2 month old) AKR mice express p30 (<0.25%) and gp70 (2–7%) antigens. Expression of gp70 antigen is restricted to large cells in the subcapsular region of the thymus. Second, thymuses of 6 month old AKR mice show a selective depletion of cortical thymocytes with a concomitant increase in the medullary region of the thymus. Thymus cells of these mice contain elevated numbers of cells that express an increased concentration of p30 and gp70 antigens. Viral antigens are found on the surface of all large cells of the subcapsular region of the thymus, and in variable numbers (2–85%) of small cells of the cortical and medullary regions. Third, the thymuses of some 8 month old AKR mice demonstrate selective hypertrophy of a single thymic lobe. The enlarged lobe contains a population of cells that are intermediate in size between the small cortical cells and leukemic blast cells. This new cell population expresses elevated levels of p30 and gp70 viral antigens. These cells, which are not leukemic (since transfer of high numbers of these cells to syngeneic hosts does not induce transplantable disease), may represent preleukemic thymocytes. Fourth, thymuses of mice with overt leukemia contain primarily leukemic blast cells. These cells express extremely high levels of viral antigens on their cell surfaces, and upon transfer of these cells to syngeneic hosts, they rapidly induce transplantable leukemias.The increased expression of viral antigens on the surface of thymus cells is correlated with an increased production of infectious ecotropic and xenotropic MuLV in the thymus. During aging, the percentage of cells producing ecotropic MuLV increases 10-fold, while the percentage of cells producing xenotropic MuLV increases 100 fold.     

Identification of an FMR cell surface antigen associated with murine leukemia virus-infected cells.

FMR antigens are found on the surface of cells infected with Friend, Moloney, and Rauscher murine leukemia viruses (MuLV). These antigens are serologically distinct from the G cell surface antigens that are found on cells infected with endogenous MuLV (AKR and Gross virus). Cell surface antigens of both virus groups are immunogenic in mice, and immunization with appropriate virus-infected cells leads to the production of cytotoxic antisera. The cytotoxic activity of FMR antisera can be absorbed by disrupted preparations of Rauscher MuLV, but not by AKR MuLV. FMR antisera precipitate the viral envelope proteins gp70, pl5(E), and p12(E) from detergent-disrupted preparations of [3H]leucine-labeled MuLV. The reaction of these antisera with p15(E) and p12(E) proteins is directed against group-specific antigens and can be absorbed with AKR MuLV; in contrast, the reaction of these antisera with gp70 is directed against type-specific antigens and is absorbed only by viruses of the FMR group. In immune precipitation assays with detergent-disrupted 125I surface-labeled cells, FMR antisera react only with type-specific antigens of the viral envelpe protein. On the basis of these findings we conclude that the FMR cell surface antigen is a determinant on the MuLV env gene product.     

Detection of polymorphism in BALB/c leukemia viruses with mouse antisera.

Antisera produced in mice recognize primarily type-specific antigenic determinants on both the major core protein, p30, and the major envelope proteins, gp70 and p15(E), of the endogenous leukemia viruses (MuLV) of BALB/c mice. Three different mouse sera were investigated in detail. (i) Antisera prepared in C57BL/6 mice against the AKR leukemia K36 reacted with the gp70, p15(E), and p30 proteins of MuLV. Certain pools of the C57BL/6 anti-AKR K36 serum contained antibodies which serologically distinguished the p30 proteins of N-ecotropic, B-ecotropic, and xenotropic BALB/c MuLV. (ii) Antisera prepared in BALB/c mice against the BALB/c sarcoma 1315 contained antibodies that reacted with a type-specific antigen of the 1315 MuLV gp70 that is not found on other BALB/c MuLV. (iii) The normal sera of multiparous BALB/c mice contained antibodies that reacted with gp70 and p15(E) proteins of ecotropic MuLV. Sera from some of these mice contained antibodies that serologically distinguished the gp70 of N-ecotropic and B-ecotropic BALB/c viruses. These results emphasize the utility of mouse antisera in the serological typing of MuLV. Furthermore, the antigenic differences observed in the p30 and gp70 proteins should be of particular use in the future analysis of recombinant BALB/c MuLV.     

Monoclonal antibody to the amino-terminal L sequence of murine leukemia virus glycosylated gag polyproteins demonstrates their unusual orientation in the cell membrane.

To analyze cell surface murine leukemia virus gag protein expression, we have prepared monoclonal antibodies against the spontaneous AKR T lymphoma KKT-2. One of these antibodies, 43-13, detects an AKR-specific viral p12 determinant. A second monoclonal antibody, 43-17, detects a novel murine leukemia virus-related antigen found on glycosylated gag polyproteins (gp95gag, gp85gag, and gp55gag) on the surface of cells infected with and producing ecotropic endogenous viruses, but does not detect antigens within these virions. The 43-17 antibody immunoprecipitates the precursor of the cell surface gag protein whether in its glycosylated or unglycosylated state, but does not detect the cytoplasmic precursor of the virion gag proteins (Pr65gag). Based on these findings, we have localized the 43-17 determinant to the unique amino-terminal part of the glycosylated gag polyprotein (the L domain). We have determined that gp95gag contains L-p15-p12-p30-p10 determinants, whereas gp85gag lacks the carboxyterminal p10 determinant, and gp55gag lacks both p30 and p10 carboxy terminal determinants. Analysis of cell surface gag expression with the 43-17 antibody leads us to propose that the L domain plays a crucial role in (i) the insertion and orientation of murine leukemia virus gag polyproteins in the cell membrane and (ii) the relative abundance of expression of AKR leukemia virus versus Moloney murine leukemia virus glycosylated gag polyproteins in infected cells.     

Two species of type C viral core polyprotein on AKR mouse leukemia cells.

Two species of glycosylated type C viral core polyprotein were identified on the surface of AKR spontaneous leukemia cells. One of these cell surface polyproteins was shown by immunoprecipitation to have antigenic determinants of murine leukemia virus p30, p15, p12, and p10; the other had murine leukemia virus p30, p15, and p12, but not p10, determinants. Both species were also expressed on thymocytes from 6-month-old, preleukemic AKR mice.     

Autologous immune responses to the major oncornavirus polypeptides in unmanipulated AKR/J mice.

The autologous immune response of AKR/J mice to the structural proteins of murine leukemia virus (MuLV) was examined. Immunoglobulins from the renal glomeruli were chemically eluted, separated from antigens, recovered, and tested for immunological reactivity against MuLV structural proteins. Analyzing immune precipitates obtained after mixing radiolabeled Tween-disrupted MuLV preparations with eluates from AKR/J mice on sodium dodecyl sulfategel electrophoresis, we found evidence of antibodies to the major classes of MuLV structural components: gp70, gp45, p30, and one or more proteins in the 10,000- to 15,000-dalton class. Using rate zonal centrifugation we confirmed that the eluates from AKR/J glomeruli contained antibody(s) that bound specifically to p30. These results indicate that AKR/J mice spontaneously mount immune responses against the major oncornavirus polypeptide antigens.     

Structural studies of retroviruses: characterization of oligomeric complexes of murine and feline leukemia virus envelope and core components formed upon cross-linking.

To examine the protein proximity and subunit organization of type C retroviruses, preparations of AKR murine leukemia virus were treated with bifunctional cross-linking reagents and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The cross-linked components obtained were characterized by immunoprecipitation with monospecific antisera against purified viral proteins, followed by SDS-PAGE analysis both before and after cleavage of the cross-links. With these procedures, complexes of both viral envelope and core components were identified. The major envelope subunit obtained was a large (apparent molecular weight of 450,000 to 500,000), glycosylated complex, composed of four to six gp70-p15(E) subunits. This complex was detected over a 100-fold range of cross-linker concentration and thus seems to represent a particularly stable viral substructure. The cross-linked complexes of the core proteins consisted of oligomers of p30 dimers, suggesting that the p30 dimer is a basic structural unit of the viral core. When virion preparations, which had previously been disrupted with the nonionic detergent Nonidet P-40, were cross-linked, the envelope complex was still observed, indicating that this structure is stable in the presence of Nonidet P-40. A similar envelope structure was observed for feline leukemia virus, suggesting that such a complex may be a conserved feature of oncornavirus structure.     

Immune response of the mouse to the major core protein (p30) of ecotropic leukemia viruses.

Sera from normal C57BL/6 mice contained low titers of antibodies against proteins of MuLV. Sera from C57BL/6 mice that were immunized with allogeneic leukemia cells sometimes contained high-titered antibodies against the p15 protein of MuLV; these antibodies detected group-specific antigenic determinants of the p15 protein, since reactions were observed with the p15 proteins of both AKR and Moloney viruses. In contrast, antisera prepared in C57BL/6 mice against the AKR leukemia K36 reacted strongly with the p30 protein of MuLV, as well as with p15. Antibodies in the C57BL/6 anti-AKR K36 sera detected group-specific antigenic determinants of the p30 protein; reactions were observed with the C57BL/6 anti-AKR K36 serum and the p30 proteins of both AKR and Moloney viruses. It was concluded that mice do have the capacity to respond immunologically to antigenic determinants of the MuLV p30 protein, although in most circumstances this is not observed.     

Characterization of Molecular Species Carrying Gross Cell Surface Antigen

The Gross cell surface antigen (GCSA), associated with expression of endogenous Gross-type murine leukemia virus (G-MuLV) in tissues of mice, is defined by the cytotoxic reaction of a C57BL/6 antiserum, anti-AKR spontaneous leukemia K36, with cells of the Gross virus-induced C57BL/6 leukemia, Emale symbolG2. Sequential lactoperoxidase-catalyzed radioiodination of Emale symbolG2 cells, Nonidet P-40 lysis, precipitation with anti-K36 serum, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis identified molecules with properties of polyproteins encoded by the gag region of the viral genome. These cell surface species could also be labeled by in vitro culturing of Emale symbolG2 with radioactive glucosamine. The viral specificity of these molecules and their participation in the GCSA typing system were established as follows. (i) Absorption of anti-K36 serum with GCSA(+), but not GCSA(-), leukemias led to a marked decrease in precipitation of these proteins. (ii) The same Emale symbolG2 cell surface proteins were also precipitated by antisera against the MuLV virion proteins p30 and p15. (iii) Anti-K36 was shown to possess antibodies against Gross virus p30 and p15. (iv) "Clearing" the Emale symbolG2 lysate of molecules reactive with anti-p30 or anti-p15 sera removed molecules reactive with anti-K36 serum. (v) Absorption of anti-K36 serum with disrupted G-MuLV virions or with Gross p30 or p15 removed GCSA cytotoxic antibodies; partial absorption was achieved with disrupted Rauscher-MuLV (R-MuLV) or with R-MuLV p30, and no absorption was found with R-MuLV p15. These data show that Emale symbolG2 cells express, on their surfaces, MuLV core polyproteins that apparently can be glycosylated and on which the determinants of GCSA are located.     

Synthesis and glycosylation of polyprotein precursors to the internal core proteins of Friend murine leukemia virus

Synthesis and post-translational processing of murine leukemia virus proteins were analyzed in a murine cell line (Eveline) that produces large amounts of Friend lymphatic leukemia virus. Immunoprecipitation of l-[(35)S]methionine-labeled cell extracts demonstrated that several different virus-specific proteins antigenically related to the virion core (gag) proteins p12 and p30 become radioactive within 1 min of labeling and exhibit labeling kinetics characteristic of primary translation products. The most abundant of these were proteins with molecular weights of 75,000 and 65,000. There were, in addition, two large glycosylated polyproteins with apparent molecular weights of 220,000 and 230,000, which were precipitated by antisera to p30 or p12 but not by antiserum to the major envelope glycoproteins gp69/71. Several lines of evidence, including labeling with d-[(3)H]glucosamine and binding to insolubilized lectins, suggested that the 75,000-dalton internal core polyprotein is slowly processed to form a glycoprotein with an apparent molecular weight of 93,000. On the contrary, the 65,000-dalton protein appeared to be an immediate precursor to the virion core proteins. Its processing can involve intermediates containing p30 and p12 antigens with molecular weights of 50,000 and 40,000; however, the latter did not appear to be obligatory intermediates. The detection of the 40,000-dalton protein suggested that the genes for p30 and p12 are adjacent on the viral genome. These results indicated that there are several pathways of synthesis and post-translational processing of polyprotein precursors to the gag proteins and that several of these polyproteins are glycosylated. A comparison of gag precursor processing in rapidly growing, slowly growing, and stationary cells indicated that different pathways are favored under different conditions of cell growth. Our analysis of envelope glycoprotein synthesis has confirmed the existence of two rapidly labeled 90,000-dalton glycoproteins, which appear to be precursors to the envelope glycoproteins gp69/71.     

Expression of viral antigens on the membrane of normal and leukemic thymocytes of AKR mice]

Expression of antigenous determinants of structural proteins G-MuLV (p10, p12, p30, gp14, gp17) and R-MuLV (gp69/71, p15) on thymocytes of normal and leukemic AKR mice was studied by membrane immunofluorescence. From this sign sharp difference between normal and malignant thymocytes was shown. A possible role in the antitumour immunity of antigens to structural viral proteins MuLV expressed on the leukemic cell membrane is discussed.     

Presence of murine leukemia virus envelope proteins gp70 and p15(E) in a common polyprotein of infected cells.

The murine leukemia virus envelope proteins, p15(E) and gp70, exhibit a mode of processing distinct from that of virion core proteins according to three criteria. First, the incorporation of both p15(E) and gp70 into virions is more sensitive to the metabolic analogue 2-deoxy-D-glucose than the incorporation of core proteins. Second, the kinetics with which the newly synthesized envelope proteins appear in the released virions is delayed relative to the appearance of core proteins. Third, immunoprecipitation of large polypeptides from infected cells reveals the presence of gp70 and p15(E) in a common precursor distinct from the core polyprotein.     

MCF-specific murine monoclonal antibodies made against AKR-247 MCF virus recognize a unique determinant associated with the gp70-p-15(E) complex.

Hybridomas obtained from (NFS X AKR)F mice immunized with syngeneic cells infected with AKR-247 MCF virus produced antibodies specific for only AKR-247 or closely related MCF viruses which hare a previously defined MCF antigen (MCFA-3). These monoclonal antibodies recognized a new type of viral antigenic determinant which appeared to be a conformational determinant associated with the env precursor polyprotein (pr80env) or its disulfide-linked gp70-p15(E) complex (gp80) but not with free gp70 or p15(E) or any other virion or virus-induced protein.     

Anomalous reactions of mouse alloantisera with cultured tumor cells. II. Cytotoxicity is caused by antibodies to leukemia viruses.

Certain alloantisera prepared in mice against H-2 region membrane antigens were found to be unexpectedly cytotoxic for murine sarcoma and leukemia cells in culture. This anomalous cytotoxicity was shown to be the result of antibody in these alloantisera directed against the p15 and gp70 envelope proteins of Mu LV which were present on the surface of the tumor target cells. Sera from aged unimmunized mice of strains used for the preparation of alloantisera also contained antibodies against MuLV protein p15 and gp70 that were cytotoxic for sarcoma and leukemia cells, which indicates that these antibodies occurred naturally in mice. These results independently confirm earlier findings of the widespread occurrence in mouse serum of antibodies reactive with MuLV. The presence of antibody against MuLV in mouse serum which can cause cytotoxic reactions with tumor cells points to the fact that particular caution should be used during the typing of murine sarcomas or leukemias for cell surface antigens, since mouse antisera may yield cytotoxicity (or other serologic reactions) based on anti-MuLV specificities, rather than on anticipated antigens.     

Monoclonal antibodies specific for wild mouse neurotropic retrovirus: detection of comparable levels of virus replication in mouse strains susceptible and resistant to paralytic disease.

We used AKR/J mice to produce monoclonal antibodies specific for a neurotropic ecotropic (WM-E) virus initially isolated from wild mice. The rationale for this approach involved the observation that these mice were immunologically hyporesponsive to endogenous ecotropic virus (Akv) but fully responsive to type-specific determinants of WM-E. Hybridoma cell lines derived from mice immunized with both denatured and viable virus produced antibodies with specificity for three viral membrane-associated polypeptides, gp70, p15(E), and p15gag. Epitopes specific for WM-E virus were detected in each of these polypeptides. Cross-reactivity with Friend ecotropic virus (Friend murine leukemia virus) was observed with some gp70- and p15gag-specific antibodies, but no reactivity with endogenous Akv ecotropic virus was seen. The majority of these antibodies did not react with either xenotropic or mink cell focus-forming viruses. Two WM-E-specific anti-gp70 antibodies reacting with different determinants had virus-neutralizing activity in the absence of complement, suggesting that the respective epitopes may participate in receptor binding or virus penetration events. We used these monoclonal antibodies in initial studies to examine the replication of WM-E virus in neonatally inoculated AKR/J mice which are fully resistant to the paralytic disease induced by this virus. Since these mice express high levels of endogenous ecotropic virus, standard assays for ecotropic virus cannot be used to study this question. We present evidence that the resistance to disease does not involve a resistance to virus replication, since these mice expressed levels of viremia and virus replication in spleen and lumbar spinal cord comparable to susceptible NFS/N mice at a time when the latter began to manifest clinical signs of lower-motor-neuron pathology.     

A murine leukemia virus mutant with a temperature-sensitive defect in membrane glycoprotein synthesis.

Cells infected with a temperature-sensitive mutant (ts-26) of Rauscher murine leukemia virus (R-MuLV) or with wild-type virus were labeled with 35S-methionine, and cell extracts were examined for radioactive polypeptides which could be precipitated by monospecific antisera to viral proteins. When shifted from permissive (31 degrees C) to nonpermissive (39 degrees C) temperature, cells infected with ts-26 rapidly begin to accumulate gPr90enr, the glycoprotein precursor to the membrane envelope glycoprotein gp70 and to the membrane-associated protein p15E. Simultaneously, formation of these mature virion proteins ceases. In addition, lactoperoxidase-catalyzed surface labeling with 125I--iodine indicates that the plasma membrane of cells infected with ts-26 becomes depleted of gp70 antigens at 39 degrees C. Nevertheless, at 39 degrees C these cells release defective MuLVs which lack gp70 and p15E but contain an outer membrane. The released particles also contain an aberrantly processed form of the major virion core protein p30, and many of these virion cores have an unusual immature crescent shape. It has previously been reported that cells infected with the ts-26 mutant of R-MuLV process a 65,000 dalton precursor (Pr65gag) of the virion core proteins more slowly at 39 degrees C than do cells infected with wild-type virus (Stephenson, Tronick and Aaronson, 1975). Although we have confirmed these results, this effect is relatively small and it is known that various alterations of MuLV assembly can lead secondarily to inhibited processing of Pr65gag. We propose that the ts-26 mutant has a primary temperature-sensitive defect in membrane glycoprotein synthesis and that this change causes pleiotropic effects on core morphogenesis.     

Immunological properties of Fc receptor on lymphocytes. 7. Suppression of mixed lymphocyte reaction by Fc receptor-bearing splenic lymphocytes.

Two Rauscher virus (R-MuLV)-induced leukemias, RBL-5, a virus-producer, and RBL-3, a nonproducer, were compared for their expression of cell surface antigens (RCSA), by cytotoxicity, membrane immunofluorescence, R-MuLV neutralization, and complement-fixation assays using syngeneic anti-crude membrane sera as well as anti-gp69/71, anti-p30, and anti-R-MuLV sera. There was a higher concentration of gp69/71 and p30 on the RBL-3 membrane than on RBL-5. This presumably is due to a cellular block at a step before assembly of virus, resulting in accumulation of these viral structural proteins (VSP). The common antigen on RBL-3 and RBL-5 is called RCSAa and may be composed of gp69/71 (RCSAa1) and an unidentified antigen (RCSAa2) which reacts more effectively with syngeneic antisera than against anti-VSP sera. An antigen abundant on the membrane of RBL-3 but not on RBL-5 is designated RCSAb, which is probably identical with p30. An unidentified antigen designated RCSAc is present on RBL-5, but not on RBL-3. Ascites cells of both RBL-3 and RBL-5 exhibited less RCSA than did the cultured lines.     

Purification and serological characterization of the major envelope glycoprotein from AKR murine leukemia virus and its reactivity with autogenous immune sera from mice.

The major envelope glycoprotein (gp71) 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 gp71 or antisera to feline leukemia virus to precipitate 125I-labeled gp71 from various MuLV showed that distinct differences exist between Rauscher- or Friend-MuLV and AKR-MuLV glycoproteins. Characteristically the AKR-MuLV gp71, in contrast to FLV or RLV gp71, does not compete fully in homologous or interspecies radioimmunoassays with iodinated Friend of Rauscher glycoproteins. Purified 125I-labeled AKR-MuLV gp71, 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.     

Genetic alterations of RNA leukemia viruses associated with the development of spontaneous thymic leukemia in AKR/J mice.

T1-oligonucleotide fingerprinting and mapping were used to study the expression of RNA leukemia viruses in leukemic and preleukemic AKR/J mice, with techniques designed to minimize the loss or inadvertent selection of viruses in vitro before biochemical analysis. In leukemic animals, complex mixtures of ecotropic and mink-tropic viruses were expressed. Unique but similar polytropic virus-like genomes were present in each tumor isolate. In preleukemic mice, viral isolates from the thymus that were grown on NIH3T3 fibroblasts contained genomes with non-Akv polytropic virus-related oligonucleotides. This phenomenon was not evident in fingerprints of viruses from the spleen and bone marrow of the same animals. Remarkably, the non-Akv oligonucleotides located in the 3' portion of the P15E gene, the U3 noncoding region, and the 5' part of the gp70 gene were often expressed independently. Our results suggest the following. (i) Recombinant viruses can be detected in the thymuses of young preleukemic AKR mice and increase in relative abundance with age. (ii) During in vivo generation of the recombinant leukemogenic viruses, the selection of polytropic virus-related sequences in the 3' part of p15E and the U3 region and the 5' portion of gp70 occurs independently. (iii) Independent biological properties encoded in the gp70 and p15E regions of env of the recombinant viruses may mediate viral selection or leukemogenicity. (iv) The leukemogenic polytropic viruses of AKR/J mice arise via genetic recombination involving at least three endogenous viral sequences.