Monoclonal antibodies against murine leukemia viruses: identification of six antigenic determinants on the p 15(E) and gp70 envelope proteins.

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.     

Phenotypic mixing between murine leukemia viruses: characteristics of ecotropic virus infection of heterologous cells.

Ecotropic murine leukemia viruses (MuLV) were capable of infecting mink, rabbit, duck, guinea pig, human, and cat cells, but not chicken cells, when phenotypically mixed with xenotropic MuLV. Mink cell cultures inoculated with the phenotypically mixed pools showed XC plaques with one-hit dose response kinetics; these plaques represented colonies derived from outgrowth of single infected cells rather than spreading areas of infection, which showed two-hit kinetics. Clonal lines of mink and rabbit cells chronically infected with N-tropic ecotropic (AKR-L1), B-tropic ecotropic (WN1802B), and NB-tropic ecotrpic (Friend) MuLV were established. Production of virus by these xenogenic cell lines was generally much lower than by mouse cells, as was the number of cells with gs-antigen detected by immunofluorescence. However, essentially all cells registered as virus-producers on infectious center plating. Treatment with IUdR enhanced virus production by 10-fold, and immunofluorescence staining as well, in the three infected mink cell lines. The viruses retained their ecotropic and Fv-1-determined host range characteristics through more than 6 to 12 months of chronic virus production in the heterologous cells.     

Expression of cell surface Friend virus gp70 does not block reinfection by ecotropic murine leukemia viruses

The effect of cell surface gp70 expression on reinfection by murine leukemia virus was studied in Friend virus (FV)-induced erythroleukemia cell clones. A clone (2C) which released gp70-positive infectious virions and expressed large amounts of cell surface ecotropic FV gp70 was not superinfectable by ecotropic viruses, whereas another clone (7C) which expressed similar amounts of cell surface FV gp70 but released gp70-deficient virions showed no resistance to superinfection. The lack of interference to superinfection of 7C cells suggested that newly budded gp70-positive virions, rather than cell surface gp70 molecules, were responsible for the interference to superinfection seen in 2C cells which released infectious virus.     

Abelson murine leukemia virus-infected cell lines defective in transformation.

The isolation of two distinct classes of transformation-defective cell lines nonproductively infected with Abelson leukemia virus (AbLV) is described. One group was selected as spontaneous revertants of an AbLV-transformed mink cell line. Mutants of this group are shown to be defective both in transformation and in expression of gag gene proteins (p15 and p12) encoded by the amino terminal region of the AbLV genome. Superinfection of such cell clones by various helper viruses led to rescue of wild-type AbLV, arguing that the transformation defect in the morphologically reverted clones involves a defect in cellular genes influencing transformation rather than in the viral genome itself. Mutants of the second group were isolated by screening single cell clones, newly infected by AbLV pseudotype virus, for expression of p12 antigen in the absence of either transformation or virion production. Twenty such mutants were selected. All clones except one expressed high levels of p15 and p12 in the absence of detectable levels of other gag gene-coded structural proteins. One clone was also positive for envelope glycoprotein (gp70) apparently encoded by a replication-defective helper virus mutant. In the second group of mutant cell clones, attempts to isolate wild-type AbLV were unsuccessful. Moreover, transformation could be induced following superinfection with wild-type AbLV pseudotype virus but not with superinfection by helper virus alone. The possibility that these latter mutants may be viral rather than cellular in origin is discussed.     

Monoclonal antibodies to xenotropic and MCF murine leukemia viruses derived during the graft-versus-host reaction

The humoral immune response during adult graft-versus-host reaction (GVHR) was assessed by the recovery of antibody-forming host spleen cells using cell fusion techniques. Two parent → F₁ strain combinations were studied, (B6 × D2)F₁ and (NFS × AKR)F₁ injected with the respective parental spleen cells. Hybridomas derived from recipient spleens were found to produce antibody with predominant specificity for murine leukemia virus (MuLV) envelope (env) polypeptides. The recovery of anti-MuLV monoclonal antibodies was dependent on the donor strain. Thus, D2 → (B6 × D2)F₁ and AKR → (NFS × AKR) resulted in a high incidence of anti-MuLV antibody production among the primary fusion products whereas no anti-MuLV hybridomas were recovered when B6 or NFS, respectively, were used as donors. Hybridomas derived from D2 → (B6 × D2)F₁ produced anti-MuLV antibodies of two general specificities: (1) broadly reactive, detecting determinants expressed by ecotropic and xenotropic MuLV, and (2) xenotropic MuLV specific. The latter group included antibodies reacting with all xenotropic MuLV and antibodies with xenotropic MuLV strain specificity. Xenotropic MuLV-specific determinants tere expressed by gp70, p15(E), and the gp70-p15(E) complex (gp90). This is to our knowledge, the first report of monoclonal antibodies specific for xenotropic MuLV. In contrast, hybridomas derived from AKR → (NFS × AKR) produced antibodies which reacted predominantly with unique determinants of a subgroup of MCF viruses. These results suggested that the anti-MuLV antibody repertoire expressed during GVHR is influenced by the endogenous MuLV of the respective mouse strain combination.     

Topological mapping of murine leukemia virus proteins by competition-binding assays with monoclonal antibodies

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 gp70^b and gp70^c epitopes; the other antigen site on this protein contained the gp70^a 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.     

Preparation and properties of monoclonal antibodies against murine interferon-beta

Wistar rats were immunized with total crude, poly(I) poly(C)-induced mouse L cell interferon. Spleen cells of one rat producing serum antibodies against Mu IFNβ were fused with the NS-1 mouse myeloma cell line using standard procedures. Two hybridomas secreting IgG antibodies to Mu IFNβ were selected using a direct neutralization assay. The hybridomas were cloned by limiting dilution. One clone (2E8B3) was propagated in nude mice. The immunoglobulin fraction was isolated from sera of mice transplanted with this clone and immobilized by coupling to CNBr-activated Sepharose. Affinity chromatography analysis showed that a column prepared from this material was capable of complete binding of Mu IFNβ All Mu IFNa activity was present in the flow through fraction. Analysis of a [³⁵S]methionine-radiolabeled L cell interferon preparation over the monoclonal anti-Mu IFNβ agarose column resulted in a complete purification of Mu IFNβ SDS-polyacrylamide gel electrophoresis of the purified material showed that poly(I) · poly(C)-induced mouse L cell interferon contains one Mu IFNβ species with a molecular weight of 32,000 to 34,000.     

Properties of mouse leukemia viruses. XVII. Factors required for successful treatment of spontaneous AKR leukemia by antibodies against gp71

AKR mice were treated with heterologous anti-gp71 antibodies under various conditions in order to establish the optimal criteria for effective suppression of leukemia development. The strongest effect was observed when mice were treated at birth; and when this regimen was used, prior treatment of the mothers did not provide additional protection. If treatment was delayed until Day 3 (Schwarz et al., 1979), the beneficial effect of the serum diminished sharply, emphasizing the presence of a narrow window very early in the life of the AKR mouse when antibody must be present in order to have an effect on subsequent leukemia development. A number of parameters were examined in the experimental mice and as in our previous study (Schwarz et al., 1979), suppression of leukemia, which occurred in 68% of the animals, correlated with elimination of viremia and appearance of natural antiviral antibodies. Interestingly, our results suggest that antibody therapy is primarily effective against the thymic form of the disease. The availability of nonviremic, antibody-positive animals afforded us the opporounity to examine if these characteristics could be transmitted to the offspring. From selected mating crosses, we successfully derived both F1 and F2 generations of AKR mice which possessed high titers of antiviral antibodies and were nonviremic at 21–28 weeks of age. It appeared that a maternal effect may be responsible for this phenomenon. The implications of these findings are discussed in relation to the development of AKR leukemogenesis.     

Group-specific cytolytic antibody directed against the major glycoprotein (gp70) of murine leukemia viruses in serum of mice with dormant FLV infections.

Serum of mice with dormant Friend leukemia virus (FLV) infections contains cytolytic activity against FLC-745 cells, a Friend erythroleukemic cell line. FLC-745 cells express only one cell surface FLV-coded antigen, shown by competition radioimmunoassay experiments to be FLV virion gp70. AKR virus blocks completely the cytolytic activity of serum but cannot block the precipitation of gp70 from detergent-disrupted ¹²⁵I-labeled cells. These results indicate that the FLC-745 cytolytic antibody in serum from mice with dormant FLV infections is directed against virion gp70 and is group specific. Furthermore, this serum contains a type specific gp70 antibody which is not lytic for FLC-745 cells.     

Structural analysis of Rauscher virus Gp70 using monoclonal antibodies: sites of antigenicity and P15(E) linkage

A linear map of 19 monoclonal antibody-binding domains on Rauscher retroviral gp70 was generated using a technique we have designated PEC-MAP (partial enzymatic cleavage-monoclonal antibody precipitation). We used eight proteolytic enzyme preparations in limited digests to produce 39 gp70 fragments. Immune precipitation of these fragments by monoclonal antibodies from 51 cell lines allowed us to define 19 binding sites by virtue of overlapping fragments and differential precipitation patterns. The sites most accessible to proteolytic attack and antibody binding were then mapped by analyzing the apparent molecular weights of the various gp70 fragments. This analysis revealed three hyperreactive regions in gp70 located approximately within the first 2000 daltons of the amino end of gp70 as well as 18,000 and 38,000 daltons from the amino end of the M_r 47,000 deglycosylated gp70 molecule. In addition, we used monoclonal antibodies directed against the disulfide-linked p15(E) molecule to localize its linkage site to Domain XVII, estimated to be between 34,000 and 38,000 daltons from the amino end of deglycosylated gp70. These data place certain constraints on the tertiary structure of gp70 and suggest a mechanism for the generation of leukemogenic MCF recombinants.     

Genetic relationship of wild mouse amphotropic virus to murine ecotropic and xenotropic viruses.

The genomic relationship between amphotropic and ecotropic wild mouse (Mus musculus) leukemia viruses (MuLV) and between these viruses and the ecotropic and xenotropic MuLVs of inbred mice was determined by DNA-RNA hybridization using viral complementary DNA and polyA-containing 70S viral RNA. The results indicate that (a) the genomes of individual cloned amphotropic and ecotropic wild mouse virus isolates are closely related and (b) these nucleotide sequences are also related to, yet distinguishable from, the genomes of prototype ecotropic (Rauscher, Gross-AKR) and xenotropic (AT-124, NZB, AKR, Balb/c) MuLVs of inbred mice.     

Properties of mouse leukemia viruses XIII. Serum therapy of virus-induced murine leukemias

Treatment of STU mice with antiserum to the major glycoprotein (gp71) of Friend leukemia virus (FLV) was therapeutically active against massive infection with Friend or Rauscher viruses, whereas similar treatment with antisera to p12 and p15, two other proteins of the virion which are involved in surface reactions, were not effective. A more thorough study showed that the active principle is contained in the IgG fraction of gp71 antiserum and that treatment with this can lead to complete recovery of the mouse from FLV infection. As a consequence of the treatment, the host produces type-specific antibodies which are detectable by neutralization, radioimmunoassay with FLV gp71, and cytotoxic tests on FLV-infected cells. No significant change was observed in the pattern of autogenous antibodies directed against an AKR-type gp71 already present in normal mice. Treatment with immune serum instead of immune IgG or treatment with immune IgG at a later stage of infection with FLV (after 7 days p.i.) did not result in complete recovery of the mice. Paralysis of the host immune system by serum proteins and Friend virus, respectively, seems to be responsible for these phenomena. Some indication was obtained that the viral-induced paralysis can be overcome by combined inoculation of heterologous immune IgG and normal isogenic spleen and bone marrow cells.     

Characterization of ecotropic murine leukemia viruses in SJL/J mice

Backcross mice assorting for the two ecotropic murine leukemia virus loci of SJL/J mice (Emv-9 and Emv-10) were tested for production of infectious leukemia viruses. It was found that tail tissue from mice carrying Emv-9 could not be induced to produce ecotropic MuLV whereas tail tissue from mice carrying Emv-10 were inducible. No mice showed detectable levels of spontaneous virus production. Preliminary restriction enzyme analyses indicated Emv-9 contains a deletion within the polymerase region.     

Structural domains of endogenous murine leukemia virus gp70s containing specific antigenic determinants defined by monoclonal antibodies

Eight distinct antigenic determinants, or epitopes (labeled a-h) were identified on endogenous ecotropic murine leukemia virus (MuLV) gp70s using a series of murine and rat monoclonal antibodies. These epitopes were characterized by their distribution patterns in a panel of cloned MuLVs, and by their localization in fragments of gp70 generated either by spontaneous breakdown of purified gp70, or by controlled proteolysis of gp70 in solubilized virions. A major 32K carboxy terminal fragment was formed which contained epitopes b, c, and f. This fragment also possessed the p15(E) disulfide linkage site, and contained approximately four complex (type 1) carbohydrate chains. An amino terminal 35K fragment contained epitopes a, d, g, and h, and possessed two glycosylated sites, including a site which occasionally retained an endoglycosidase H-sensitive oligosaccharide chain. A related 49K fragment was also obtained which included the entire 35K region and contained an additional sequence bearing epitope e. In a series of dual-tropic MCF-type viruses studied, only those epitopes located in the 32K fragment were ever retained, indicating that for these recombinant viruses at least a portion of that domain was derived from the ecotropic parent. A model is presented indicating the likely orientation of these fragments and their structural characteristics.     

Isolation and mapping of cDNA hybridization probes specific for ecotropic and nonecotropic murine leukemia proviruses

To study the structure of murine leukemia proviruses in AKR mice by Southern hybridization, we have isolated and mapped ecotropic AKV and nonecotropic MCF-derived cDNA restriction fragments. The ecotropic-specific probes originate from four regions of the AKV genome which include the corresponding recombinant region of two MCF viruses (V1–36 and 247). We also isolated two nonecotropic probes from the recombinant region of MCP V1–36. The probes were characterized by (i) mapping of restriction fragments at the 3′ end of AKV and MCF V1–36 by a two-dimensional gel strategy, (ii) hybridization of restriction fragments to the related viral RNA genomes followed by electrophoresis, (iii) two-dimensional fingerprinting of single-stranded restriction fragments, and (iv) DNA sequence analysis of the ecotropic probes. The ecotropic AKV and nonecotropic MCF probes discriminate between two populations of endogenous murine leukemia viruses and show that the MCF viruses are not present in the germ line of AKR mice.     

Lysis of retroviruses with monoclonal antibodies against viral envelope proteins

Monoclonal antibodies identifying six independent antigenic determinants (epitopes) on the gp70 and p15(E) envelope proteins of murine leukemia virus were tested for their ability to lyse a panel of serologically different [³H]uridine-labeled retroviruses in the presence of complement. Antibodies against the gp70^a, gp70^b, and gp70^c epitopes were uniformly negative in virolysis assays, whereas antibodies against the p15(E)^a, p15(E)^b, and p15(E)^c epitopes lysed the viruses to high titer. The lytic activities of these p15(E)-specified antibodies paralleled their specific binding characteristics determined in previous assays with viral proteins. The p15(E) protein is known to be embedded directly into the viral membrane while the bulk of the glycoprotein (gp70) projects from the surface of the virion; thus our results indicate that the lytic activity of an antibody is related to the distance of the antibody:epitope complex from the membrane bilayer.     

Leukemogenic properties of AKR dualtropic (MCF) viruses: amplification of murine leukemia virus-related antigens on thymocytes and acceleration of leukemia development in AKR mice

The late preleukemic period in AKR mice (6–8 months of age) is characterized by amplified expression of murine leukemia virus (MuLV)-related cell surface antigens on thymocytes. Analysis of thymic biopsies of preleukemic AKR mice at 180 days of age showed that antigen amplification was prognostic of spontaneous leukemia development within approximately 100 days. In the present study we have investigated the viral basis of this preleukemic change in AKR thymus. Cloned isolates of ecotropic, xenotropic, and dualtropic MuLV that were derived from preleukemic or leukemic AKR thymus were tested by intrathymic injection of young AKR mice to determine which of the three classes of MuLV could induce antigen amplification, and, subsequently, accelerate leukemia development in the same animal. Only dualtropic MuLV exhibited these two activities in vivo. Considerable heterogeneity was observed among the 13 dualtropic MuLV isolates examined. In vitro three distinct serological phenotypes could be recognized on the basis of expression of MuLV gag gene-coded antigens GCSA and MuLV env gene-coded antigens. G_IX, G_(ERLD), G_(RADAI), G_(ARSL2) on the surface of infected cells. Virus isolates also differed in (i) relative ecotropic-xenotropic host range (dualtropism) as assayed by ratios of infectious titers on mouse and mink cells, (ii) induction of mink cell foci (MCF), and (iii) infectivity for NIH/3T3 cells. In vivo the two activities of antigen amplification and leukemia acceleration could be dissociated and thus represent distinct virus phenotypes. Seven isolates of dualtropic MuLV induced antigen amplification; these viruses encoded G_IX, G_(ERLD), and G_(AKSL2) antigens, were MCF inducing, had SC-1/mink titer ratios ≥ 0.4, and were infectious for NIH/3T3 cells. Only three of these virus isolates (MCF 247, MCF 69L1, and MCF 13) accelerated leukemia development. Analysis of dose-response relationships and kinetics of virus-induced antigen amplification by MCF 69L1 virus implicated virus infection of thymocytes in the preleukemic change. Moreover, the level of MuLV antigen expression on thymocytes at approximately 1 month postinjection of MCF 69L1 virus correlated directly with development of early leukemia. It is apparent that leukemia development in young AKR mice which can be induced experimentally by intrathymic injection of cloned isolates of dualtropic MuLV exhibits the hallmarks of spontaneous disease in this strain and thus can serve as a useful model for study of thymic leukemogenesis in mice.