Search

A panel of eight monoclonal antibodies recognizing individual antigenic determinan (epitopes) on the envelope proteins (gp70 and p15(E)) of murine leukemia virus (MuLV) has been used to probe the serological properties of recombinant dualtropic (MCF) viruses. Since six of these monoclonal antibodies showed specificity for epitopes that were exclusive to endogenous N-ecotropic MuLV, it was reasoned that they might provide a means to determine the relative genetic contribution of ecotropic information in the env gene of dualtropic isolates. It was anticipated that the loss of ecotropic-specific sequences in these viruses would be reflected by a loss of reactivity with the ecotropic-specific monoclonal antibodies. This expectation proved to be correct, as several of the monoclonal antibodies that reacted with endogenous ecotropic MuLV of AKR mice failed to react with isolates of recombinant dualtropic MuLV from the same strain. In addition, it was noted that the “nonreactivity” of the monoclonal antibodies with different dualtropic viruses occurred in characteristic patterns. A common feature of the patterns was coordinate loss of expression of the gp70 a , gp70 d , and gp70 e epitopes from all AKR dualtropic MuLV. In contrast to the common deletion of ecotropic-specific epitopes, the gp70 b , gp70 c , and p15(E) a epitopes were variably deleted in different recombinant viruses.

The C3H/10T1/2 cell line, established from mouse embryo fibroblasts, is highly sensitive to postconfluence inhibition of division and is susceptible to malignant transformation by chemical carcinogens in culture. Normal, chemically transformed and spontaneously transformed clones of C3H/10T1/2 cells were examined for the production of infectious murine leukemia virus (MuLV) and MuLV and mouse mammary tumor virus (MTV) antigens. Criteria for the expression of oncornaviruses included the production of: (1) MuLV and MTV gs-antigens, (2) MuLV GL and GT cell surface antigens, (3) DNA polymerase-containing particles, and (4) infectious virions. Normal C3H/10T1/2 cells were free of oncornaviruses by all four of these criteria. Transformation of these cells to malignancy by chemical carcinogens did not result in an increased production of MuLV or MTV products; hence, these cells demonstrated the same virus-free phenotype as the parental cell line. Attempts to rescue transforming viral information from transformed C3H cell lines by superinfection with leukemia viruses were unsuccessful. Repressed endogenous MuLV (but not MTV) could be induced from all cell lines by treatment with 5-iododeoxyuridine (IUdR). Induction of MuLV in normal cells occurred in three phases: I, an initial transient production of low-titered virus; II, a 3–4-week interval where virus production fell to a lower level and then gradually increased. This was accompanied by the recruitment of virus-free cells in the culture to produce MuLV gs-antigens and DNA polymerase-containing particles; and III, the constitutive production of high-titered virus. Transformed cells were more sensitive to treatment with IUdR than normal C3H/10T1/2 cells. These cells underwent a more rapid induction of MuLV, contained a larger fraction of cells that expressed MuLV functions, continued the production of MuLV throughout the second phase of induction, and had an earlier onset of the third phase of high-titered MuLV production. Oncornaviruses induced from IUdR-treated cells showed diversity in their host range and in their ability to produce XC plaques; none of these viruses, however, were capable of transforming normal C3H/10T1/2 fibroblasts in culture.

Hybrid cell lines that produce monoclonal antibodies against the proteins of murine leukemia virus (MuLV) have been prepared by the fusion of mouse myeloma cells with the lymphocytes of mice that were immunized with MuLV-producing leukemia cells. Preliminary screening of culture fluids from these hybrid cells for reactivity on a panel of viruses led to the selection of cell lines that produced antibodies against highly polymorphic antigenic sites of the viral proteins. Inoculation of the hybrid cells into syngeneic mice resulted in the formation of ascites fluids that contained high titers of monoclonal antibodies. These ascites fluids demonstrated 100,000-fold variations in reactivity with the proteins of different isolates of MuLV. Monoclonal antibodies of this type now provide new tools for the precise serological classification and detection of murine leukemia viruses.

Mice of different inbred strains varied in their immune response to endogenous leukemia viruses (MuLV). The ability of mice to produce antibodies against the gp70 proteins of xenotropic and ecotropic MuLV was to some extent dependent upon the level to which these mice naturally expressed their endogenous leukemia viruses. Three patterns of response were observed upon immunization with AKR leukemia cells: (1) Mice of low leukemic strains, which did not contain inducible ecotropic or xenotropic MuLV, produced antibodies that reacted with the gp70 proteins of both xenotropic and ecotropic MuLV; (2) mice of low leukemic strains, which contained low levels of ecotropic and inducible xenotropic MuLV, produced antibodies that reacted primarily with the gp70 proteins of ecotropic MuLV; and, (3) mice of high leukemic strains, which contained high levels of ecotropic and inducible xenotropic MuLV, failed to produce antibodies that reacted with the gp70 proteins of either xenotropic or ecotropic MuLV. Antibodies induced by immunization with leukemia cells reacted with type-specific antigenic determinants of the gp70 protein; by absorption analysis distinctive antigenic determinants could be identified on the gp70 proteins of BALB xenotropic, NZB xenotropic, and AKR ecotropic MuLV.

Group-specific viral proteins, p27, p19, p15, and p12, have been purified from different strains of avian leukemia-sarcoma viruses, and their chemical and serological properties have been compared. Functionally homologous proteins from two different viral isolates had similar amino acid compositions. The major constituent of the viral ribonucleoprotein, p12, possesses the highest content of hydrophilic amino acids (53%), while the core shell protein, p27, has the lowest (42%). In isolates from six viral subgroups the equivalent proteins were serologically indistinguishable by immunodiffusion analyses with monospecific anti-sera. By contrast, for the four different proteins from the same viral isolate, no serological cross reactions were found, nor could structural homologies be demonstrated by amino acid and peptide analyses.

Leukemia cells from mice of the AKR and C58 strains produced leukemia viruses (MuLV) in culture that failed to induce syncytia in the XC plaque assay (XC − ), were poorly infectious for mouse embryo fibroblasts, and yielded low levels of infectious progeny upon the establishment of infection. In host range analysis the majority of these viruses were N-ecotropic, although one leukemia cell line also produced xenotropic MuLV. Viral isolates from five different leukemia cell lines were oncogenic upon inoculation into newborn AKR mice. In contrast, viruses that occurred in high titer in the tissues and sera of normal AKR mice were not oncogenic in vivo and in culture they efficiently induced (with N-ecotropism) syncytia in the XC plaque assay (XC + ). These findings demonstrate that the transforming agent for spontaneous leukemogenesis differs in biological properties from the endogenous MuLV that is commonly observed in mice of high leukemic strains.

Mild Tween 80-ether treatment of mouse mammary tumor virus (MTV) results in disruption of the viral membrane and release of subviral components. Prolonged lipid extraction, however, results in virtually complete solubilization of the virion. Soluble proteins have been separated by Sephadex G-200 chromatography and used for immunization of rats These antisera identify five viral antigens (designated MTV-s1, s2, s3, s4, and s5). The s1 and s2 antigens are group-specific, and are the antigens of the viral nucleoid. MTV-s3 antigen is a structural protein of the viral membrane. Poly-acrylamide gel electrophoresis of MTV shows five major polypeptides in the virion; the molecular weights of these are p1 (90,000), p2 (70,000), p3 (52,000), p4 (33,000), and p5 (23,000). The major protein of the virus is p3; it is found in the nucleoid and carries the s1 antigen. Protein p5 is the structural protein of the membrane, and it carries the s3 antigen. Antisera prepared against isolated MTV proteins were used also in immunofluorescence tests to determine the intracellular deposition of MTV antigens. These studies show that all MTV structural proteins are synthesized cytoplasmically. Screening of a variety of neoplasms by immunofluorescence and immunodiffusion tests demonstrates MTV infection in only three types of neoplastic diseases, mammary tumors, ML+ leukemias, and Leydig cell tumors of MTV-infected mice. Mammary tumors synthesize all viral structural proteins, while the synthesis of MTV proteins in the ML+ leukemias and Leydig cell tumor is limited to the s1 and s2 antigens. Electron microscopic examination of these tissues confirms the synthesis and accumulation of viral nucleocapsids (A particles) without the production of budding virions.

Antisera prepared in C57BL/6 mice against the AKR K36 leukemia cells detect] group-specific and class-specific antigenic determinants on the major core protein (p30) of murine leukemia virus (MuLV). Sera that contain a predominance of antibodies against class-specific antigens react preferentially with the p30 proteins of ecotropic MuLV; these sera do not react with the p30 proteins of xenotropic MuLV. The use of MuLV class-specific antisera in immunological assays enables the assignment of p30 antigens in viruses and tissues to ecotropic or xenotropic origins.

Hybrid cells that produced monoclonal antibodies against the envelope proteins of murine leukemia virus (MuLV) were prepared by the polyethylene glycol-mediated fusion of a mouse myeloma cell line with lymphocytes from mice immunized with allogeneic MuLV-producing leukemia cells. Twenty-three independent cell lines were cloned and inoculated into syngeneic mice for the production of ascites fluids that contained high-titered (20–75 mg/ml) monoclonal antibodies. Six serologically distinct specificities were detected when these ascites fluids were tested on a broad panel of MuLV and non-murine retra iruses. Prototype cell lines producing monoclonal antibodies that were representative of each pattern of reaction were selected for further study. In immune precipitation assays each of the prototype antibodies reacted with viral envelope proteins; three of these identified antigenic determinants on p15(E), while three others identified antigenic determinants on gp70. The p15(E) antigenic determinants were shared by a diverse panel of MuLV. One of these p15(E) antigenic determinants was also found in feline leukemia virus. The gp70 antigenic determinants, on the other hand, had a more restricted distribution and were found in only selected isolates of MuLV.

Recently, we have shown that several isolates of recombinant, dualtropic (MCF) murine leukemia virus (MuLV) can induce amplification of MuLV gag and env gene-coded antigens on thymocytes of young AKR mice and, subsequently, can accelerate leukemia in these same animals. With respect to env gene-coded antigens, it is unclear whether antigen amplification represents expression of env gene products of the input virus on the surface of infected thymocytes or whether infection by MCF viruses induces the expression of other endogenous env gene sequences. Infection by antigenically marked viruses has allowed us to decide between these alternatives. We have found that AKR MCF isolates 69L1,13, and 247 can be distinguished serologically by specific patterns of reactivity with mouse and rat monoclonal antibodies that recognize 10 distinct epitopes (single antigenic determinants) of AKR ecotropic MuLV gp70 and p15(E) proteins. Accordingly, 1-monthold AKR mice were injected intrathymically with either MCF 69L1, MCF 13, or MCF 247 viruses and thymocytes of virus-injected mice were then assayed for amplification of specific epitopes at 32—36 days postinjection. Quantitative expression of MuLV antigens was determined by flow microfluorometry using a fluorescence-activated cell sorter. The patterns of reactivity with monoclonal antibodies which were originally determined for each virus in vitro were found to breed true on thymocytes infected in vivo . Moreover, the phenotype of virus recovered in vitro from virus accelerated leukemias was identical to that of the injected virus. Thus, the preleukemic change of MuLV antigen amplification appears to represent expression on thymocytes of env gene products encoded by the infecting MCF viral genome which continues to be expressed in the resulting leukemias without apparent changes in the recombinant env gene.

Hybrid cell lines were prepared by the fusion of mouse myeloma cells with the spleen cells of C57BL/6 mice that were immunized with the AKR leukemia K36. Approximately 10% of the hybrid cells produced immunoglobulins that reacted in antibody binding assays with AKR MuLV. By the combined use of low-density passage and cloning, seven independent cell lines were isolated. These cells produced antiviral antibodies at a level of 3–15 μg/ml of culture fluid. Inoculation of the hybrid cells into syngeneic mice resulted in the formation of tumors (hybridomas) that secreted extremely high levels of monoclonal antibodies (5–15 mg/ml) into the serum or ascites fluid. Five of the hybrid cell lines produced immunoglobulins of the IgM subclass, one produced IgG2a, and one produced IgG2b. In high-resolution two-dimensional polyacrylamide gels, these immunoglobulins showed the limited heterogeneity in heavy and light chains that would be expected for monoclonal products. Radioimmune precipitation assays demonstrated that the monoclonal antiviral antibodies reacted with the p15(E) protein of ecotropic MuLV; these antibodies did not react with the p15(E) protein of xenotropic MuLV. In contrast, rabbit antiserum prepared against purified p15(E) reacted equally well with ecotropic and xenotropic MuLV. The sera or ascites fluids from hybridoma-bearing mice had antibody titers 75- to 100-fold higher than the sera from conventionally immunized mice or rabbits. Serological analysis demonstrated that the monoclonal antibodies reacted with the cell surface of virus-producing leukemia cells, but not with normal thymocytes. Furthermore, monoclonal anti-pl5(E) antibodies of the IgG2a subclass mediated lysis of the virion of ecotropic MuLV in the presence of complement.

Monoclonal antibodies produced by hybrid cells in culture are chemically homogeneous reagents that react with constant avidity to single antigenic determinants (epitopes). As such, these antibodies are remarkably specific probes for small regions of complex antigenic proteins. We have used a panel of monoclonal antibodies in competition binding assays to investigate the arrangement of six epitopes on the envelope proteins of AKR leukemia virus. It was reasoned that if two epitopes were adjacent to each other on a single protein, the binding of antibody to one epitope would sterically hinder the binding of antibody to the other epitope. On the other hand, if the two epitopes were at distant sites on the protein, the binding of antibody to one epitope would not influence the binding of antibody to the other epitope. The results of these competition binding assays demonstrated the presence of two distinct antigen sites on both the gp70 and p15(E) envelope proteins. With the gp70 protein, one antigen site contained the gp70b and gp70c epitopes; the other antigen site on this protein contained the gp70a epitope. With p15(E), one of the antigen sites contained the p15(E)b and p15(E)c epitopes, while the other site contained the p15(E)a epitope. These findings demonstrate the utility of this type of serological analysis for the study of the tertiary structure of individual viral proteins.

Athymic nude ( nu/nu ) mice of the BALB/c and C57BL/6 strains produced lower titers of antibody to endogenous leukemia viruses (MuLV) than did comparably aged thymus-intact ( nu/+ ) littermates. The majority of these athymic mice also produced high levels of infectious MuLV in their sera, whereas none of the thymus-intact mice contained detectable infectious virus. These findings suggest that natural immunity to endogenous MuLV is involved in the regulation of expression of these viruses in mice of low-leukemic strains.

Group-specific antigens of the mammary tumor virus (MTV-s1) and of Gross (wild-type) murine leukemia virus (MuLV-s1), demonstrable with specific precipitating antisera, are present in high concentration in the milk of naturally infected mice. With two exceptions, MTV-s1 was restricted to mice of strains with a high incidence of mammary tumors; the two exceptions were individual mice of strains free of MTV by foster-nursing and may be explained by the presence of virus of the same antigenic group. MuLV-s1 has a wide distribution among mouse strains. It occurs not only in mice with a high incidence of leukemia, but also in mice of low-incidence strains. This points to the existence of naturally occurring variants with MuLV-s1 group specificity and G (Gross) type specificity, but with little or no leukemogenic activity. An analogous relationship exists between the weakly oncogenic nodule-inducing virus and MTV, which belong to the same antigenic group. In only one mouse strain were MTV-s1 and MuLV-s1 found together in the milk, an observation suggesting that viruses of the two types may to some extent exclude one another. NZB mice are G+, have MuLV-s1 in their milk and tissues, and carry virus morphologically indistinguishable from murine leukemia virus. It is an open question whether this virus is etiologically related to the autoimmune disease of NZB mice.

Mild Tween 80-ether treatment of mouse mammary tumor virus (MTV) results in disruption of the viral membrane and release of subviral components. Prolonged lipid extraction, however, results in virtually complete solubilization of the virion. Soluble proteins have been separated by Sephadex G-200 chromatography and used for immunization of rats These antisera identify five viral antigens (designated MTV-s1, s2, s3, s4, and s5). The s1 and s2 antigens are group-specific, and are the antigens of the viral nucleoid. MTV-s3 antigen is a structural protein of the viral membrane. Poly-acrylamide gel electrophoresis of MTV shows five major polypeptides in the virion; the molecular weights of these are p1 (90,000), p2 (70,000), p3 (52,000), p4 (33,000), and p5 (23,000). The major protein of the virus is p3; it is found in the nucleoid and carries the s1 antigen. Protein p5 is the structural protein of the membrane, and it carries the s3 antigen. Antisera prepared against isolated MTV proteins were used also in immunofluorescence tests to determine the intracellular deposition of MTV antigens. These studies show that all MTV structural proteins are synthesized cytoplasmically. Screening of a variety of neoplasms by immunofluorescence and immunodiffusion tests demonstrates MTV infection in only three types of neoplastic diseases, mammary tumors, ML+ leukemias, and Leydig cell tumors of MTV-infected mice. Mammary tumors synthesize all viral structural proteins, while the synthesis of MTV proteins in the ML+ leukemias and Leydig cell tumor is limited to the s1 and s2 antigens. Electron microscopic examination of these tissues confirms the synthesis and accumulation of viral nucleocapsids (A particles) without the production of budding virions.

A soluble antigen (MTV-s1), a subunit of the mammary tumor virus (MTV), is demonstrable by immunoprecipitation with rabbit antiserum prepared against MTV from infective milk of RIII mice. MTV-s1 antigen occurs in mammary tumors of MTV+ mice, milk of MTV+ mice and in ML+ leukemias. MTV-s1 antigen is released from intact MTV by treatment with ether. Sera prepared in mice against mammary tumors or against ML+ leukemias have precipitating antibody for MTV-s1, showing that the mouse is capable of an immune response to MTV. The demonstration of MTV-s1 in tumor extracts or in the milk of individual mice in immunoprecipitation provides the most rapid and direct method for detecting infection with MTV.