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1.
Serological analysis of antigenic determinants on the env gene products of AKR dualtropic (MCF) murine leukemia viruses 总被引:6,自引:0,他引:6
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 gp70a, gp70d, and gp70e epitopes from all AKR dualtropic MuLV. In contrast to the common deletion of ecotropic-specific epitopes, the gp70b, gp70c, and p15(E)a epitopes were variably deleted in different recombinant viruses. 相似文献
2.
Vesicular stomatitis virus (murine leukemia virus) (VSV(MuLV)) pseudotypes containing a [3H]uridine-labeled VSV RNA genome and MuLV envelope glycoproteins (gp70) were produced by phenotypic mixing of the two viruses. In order to better detect such pseudotypes, an immunobiochemical (IB) technique was developed. [3H]Uridine-labeled virus progeny of the dual virus infection was immunoprecipitated by monospecific MuLV gp70 antibodies complexed with fixed Staphylococcus aureus. The immunoprecipitated 3H-labeled genomic RNA was identified as that of VSV by its sedimentation coefficient, by the lack of polyadenylate, and by molecular hybridization with complementary VSV RNA. By the IB technique, approximately 11% of the progeny of the dual virus infection were found to be VSV(MuLV). By neutralization and other biological assays, however, only 0.1% of the progeny were found to be VSV(MuLV) pseudotypes. Apparently, the IB technique is capable of detecting VSV pseudotypes encapsidated with only a few molecules of MuLV gp70. The IB technique, therefore, offers a quantitative and molecular technique for the detection of VSV(MuLV) pseudotypes and can be modified to detect other viral pseudotypes when other assays are lacking. In spite of its sensitivity however, the IB technique did not detect the formation of MuLV (VSV) pseudovirions among the virus progeny of the dual virus infection. These results confirmed a similar observation made previously using immunoelectron microscopy. 相似文献
3.
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 gp70a, gp70b, and gp70c 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. 相似文献
4.
Topological mapping of murine leukemia virus proteins by competition-binding assays with monoclonal antibodies 总被引:13,自引:0,他引:13
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. 相似文献
5.
Enzyme-linked immunosorbent assay for detection of retroviral gp70 and gp70-anti-gp70 immune complexes in sera from SLE mice. 总被引:1,自引:0,他引:1
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Since a retroviral envelope glycoprotein, gp70, present in sera is prominently involved in the pathogenesis of murine systemic lupus erythematosus (SLE), the detection of gp70-anti-gp70 immune complexes (gp70 IC) is particularly useful for the study of murine SLE. To facilitate the detection of gp70 and gp70 IC, we have developed a simple and sensitive enzyme-linked immunosorbent assay (ELISA). Using an affinity column coupled with whole mouse serum proteins containing serum gp70 or with Rauscher murine leukaemia virus (MuLV), antibodies specific to serum gp70 or to Rauscher MuLV gp70 were purified from hyperimmune goat anti-Rauscher MuLV gp70 antisera. Only affinity-purified anti-serum gp70 fraction, but not anti-Rauscher MuLV gp70 fraction, was able to detect serum gp70 efficiently in the ELISA, because only a minor fraction of goat anti-Rauscher MuLV gp70 antibodies is cross-reacting with serum gp70. This procedure could be applied to other antigen-antibody systems, in which only antibodies to heterologous cross-reacting antigens are available, to detect free and antibody-complexed antigens in pathological sera. 相似文献
6.
C3H mouse mammary tumor cells producing mouse mammary tumor virus (MMTV) were cocultivated with nonproducer mouse cells (KNIH) transformed by Kirsten sarcoma virus (KiSV). These cocultivated cells were then treated with mitomycin C and overlayed onto human embryonic skin and muscle cells. The virus resulting from this cocultivation could be titrated in a focus-forming assay on Fischer rat embryo (FRE) cells exhibiting one-hit kinetics. Furthermore, focus formation on FRE cells was neutralized specifically by antiserum directed against MMTV and the major MMTV external glycoprotein gp52, but not against a broadly reactive antiserum directed murine leukemia virus (MuLV) gp70 and MMTV gp36, p27, p14, and p10. These results demonstrate the generation of a KiSV(MMTV) pseudotype and further demonstrate that gp52 is a target antigen for neutralization of MMTV. This pseudotype possessed a wide host range, transforming cells of human, rat, mouse, mink, and rabbit origin. MMTV but not MuLV antigen expression was demonstrated in the KiSV(MMTV) pseudotype-infected cells. Analysis of intracellular MMTV protein synthesis in these in vitro-infected cells has indicated that the low yield of extracellular MMTV produced by the transformed cells may be the result of the poor expression of the MMTV gag precursor polyprotein relative to the expression of the env gene polyprotein. These studies thus provide the basis for an in vitro infectivity assay for neutralization and host range studies of MMTV. 相似文献
7.
C E Samuel 《Virology》1979,93(1):281-285
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. 相似文献
8.
Monoclonal antibodies against murine leukemia viruses: identification of six antigenic determinants on the p 15(E) and gp70 envelope proteins. 总被引:27,自引:0,他引:27
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. 相似文献
9.
Monoclonal antibodies to xenotropic and MCF murine leukemia viruses derived during the graft-versus-host reaction 总被引:12,自引:0,他引:12
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. 相似文献
10.
Martine Y.K. Armstrong Richard B. Weininger David Binder Carol A. Himsel Frank F. Richards 《Virology》1980,104(1):164-173
Previous work has shown that (BALB/c x A)F, (CAF1) mice with an immunological disorder, the graft-versus-host reaction (GVHR), express an oncogenic murine leukemia virus (MuLV) which appears to be responsible for the subsequent development of lymphoreticular tumors. A mink cell focus-inducing (MCF) virus has been isolated from a reticulum cell neoplasm induced in a BALB/c mouse by serially passaged B-tropic MuLV originating in a CAF, GVHR mouse. The cloned MCF virus has a dual host range. It grows both in mouse cells where it is XC negative, B tropic, and in mink lung cells where it induces characteristic foci. It is partially neutralized by xenotropic MuLV antiserum and it partially competes in a homologous ecotropic gp70 radioimmunocompetition assay. Both ecotropic and xenotropic MuLV interfere with its infectivity. In these biological properties, the GVHR MCF virus closely resembles recombinant AKR MCF virus. We discuss the possible origin and significance of a presumptive recombinant MuLV in a low virus model where lymphoreticular tumors are triggered by an immunological disturbance. 相似文献
11.
H-2 alloantisera have been previously reported to contain antibodies against murine leukemia viral antigens, but the nature of the viral antigens on mouse cells which interact with these antibodies has not been established. We have found that H-2 alloantisera recognize components of molecular weight 70 000-80 000 mouse lymphocytes and leukemia cells. These components were also detected by a goat antiserum against the murine leukemia virus (MuLV) glycoprotein (gp 70) and are therefore closely related to or identical with that viral protein. Although most H-2 alloantisera detected gp 70-like molecules on lymphocytes and leukemia cells from a great variety of mouse strains, only one H-2 alloantiserum was found to interact with a gp 70 component on cells from C57BL/10 and C57BL/6 mice. Animals such as C57BL/10 mice that lacked the component reacting with most H-2 alloantisera showed increased serum levels of anti-MuLV antibodies after injection of B10.A spleen cells having a gp 70 component detectable by other H-2 alloantisera. In contrast, strains with cells reactive to antiviral antibodies in the H-2 alloantisera had low responses to MuLV antigens after a similar immunization procedure. Serum levels of anti-MuLV antibodies in both groups of mice, however, were increased after injection of Freund's adjuvant. These observations suggest that anti-MuLV antibodies in mouse alloantisera may arise from a response to viral antigens on the immunizing cells and general stimulation of the immune system. 相似文献
12.
A quantitative estimation of retrovirus associated cell membrane antigens of murine and feline cells infected with their respective type C leukosis virus is presented. Using a radio-immune assay with three broadly reactive antisera, the minimum estimated number of retrovirus associated antigenic determinants on YAC [Moloney leukaemia virus (MuLV) infected murine] and FL-74 [feline leukaemia virus (FeLV) infected feline] cells was 1.3 x 10(6) and 1.6 x10(6) determinants per cell respectively. The virus structural proteins p27-30 and gp70 were detected by three component specific antisera on murine and feline cell surfaces in amounts which varied between cell isolates. MuLV infected cells produced as many as 1.9 x 10(5) p30 antigenic determinants and 7.5 x 10(5) gp70 determinants on infected cells. FeLV infected cells (FL-74) expressed 5.6 x 10(5) p27 and 7.5 x 10(5) gp70 antigenic determinants per single cell surface. The major core protein (p27-30) and the major envelope glycoprotein (gp70) antigens are sufficiently physically separated on cell surfaces so that binding of either of the membrane antigens with component specific antibodies does not interfere with binding of antibodies specific for the other. Despite the expression of interspecies determinants for p30, gp70, and other retrovirus associated antigens detected by antibody procedures, interspecies determinants of cell mediated immunity could not be demonstrated in immune mice bearing Moloney sarcoma virus (MSV) induced tumours. Furthermore, xenogeneic immunization of mice with FL-74 cells failed to protect mice against the growth of MSV induced lymphoma or sarcoma. 相似文献
13.
M N Robertson M Miyazawa S Mori B Caughey L H Evans S F Hayes B Chesebro 《Journal of virological methods》1991,34(3):255-271
Four monoclonal antibodies were selected for their ability to recognize the envelope protein of Friend murine leukemia virus (F-MuLV) in methanol-fixed tissue culture cells. Each of these monoclonal antibodies was found to react only with F-MuLV. By using recombinant retroviruses, it was determined that each of the monoclonal antibodies recognized the C-terminal one-third of the F-MuLV gp70 envelope protein. The monoclonal antibodies were effective in radioimmunoprecipitation of F-MuLV proteins, and one of the antibodies, 720, was also effective in Western blotting. The ability of antibody 720 to react with F-MuLV in methanol-fixed cells facilitated the use of a sensitive immunoperoxidase method with a focal virus infectivity assay. In immunohistochemical studies using light microscopy, antibody 720 could specifically label F-MuLV-infected cells in acetone-fixed tissue sections from F-MuLV-infected animals. Finally, in immuno-gold labelling studies using electron microscopy, antibody 720 could be used to distinguish F-MuLV from amphotropic MuLV. 相似文献
14.
15.
B. P. Croker M. Bourdon P. J. McConahey F. J. Dixon 《International journal of immunogenetics》1978,5(6):401-409
The levels of murine leukaemia virus (MuLV) proteins p30 and gp70, antinuclear antibody (ANA), anti-soluble nuclear protein, anti-single-stranded DNA, anti-double-stranded DNA and anti-histone antibodies were measured in B10.A and B10.A recombinant mice neonatally infected with MuLV-Scripps (Lerner et al., 1972). The incidence and latency of lymphoma and the incidence of glomerulonephritis were also determined. The mice studied could be divided into high-responder and low-responder groups. Most of the high ANA antibody could be attributed to anti-histone antibody. High response was associated with the H-2b haplotype and recombinant strains on the B10 background which were identical at the I-A subregion derived from the H-2b parental stock. In contrast, low ANA response was associated with the I-A subregion derived from the H-2k haplotype. In all groups of virus-inoculated animals, most animals developed serum elevations of p30 and gp70 and at least 72% of the inoculated animals developed lymphomas. High serum p30 levels correlated inversely with latency and directly with gp70 values. From 4 to 28% of the animals in each virus-inoculated group had histological evidence of glomerulonephritis, although no clear genetic basis could be ascribed to the incidence of glomerulonephritis, serum p30 or gp70 levels, or latency of lymphoma development. 相似文献
16.
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. 相似文献
17.
John Hilkens Alfonso Colombatti Mette Strand Elisabeth Nichols Frank H. Ruddle Jo Hilgers 《Somatic Cell and Molecular Genetics》1979,5(1):39-49
Mouse chromosome segregating somatic cell hybrids were established between a mouse thymic leukemia cell line (GRSL) and Chinese hamster E36 cells. The GRSL cells specifically bound purified Rauscher leukemia virus gp70 while the E36 cells exhibited no binding. The hybrids selectively bound Rauscher gp70 depending on the presence of a mouse cellular gene for the ecotropic murine leukemia gp70 receptor. A syntenic relationship was observed between the DIP-3 chromosome marker (on chromosome 5) and the gp70 receptor in primary clones and subclones of these hybrids; this was confirmed by chromosome analysis. The involvement of H-2 in the binding of Rauscher MuLV gp70 could be ruled out, because discordancies of the receptor presence and H-2 absence as well as of the receptor absence and H-2 presence type could be observed. Our results indicate that the Rec-1(replication ecotropic MuLV) gene of Gazdar et al. (4) may well be the receptor gene for the ecotropic murine leukemia virus. 相似文献
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20.
Neutralizing monoclonal antibodies directed to infectious bovine rhinotracheitis virus 总被引:1,自引:0,他引:1
Summary Infectious bovine rhinotracheitis virus (IBRV) has been shown in this report to have thirty-three polypeptides. Ten of the eleven polypeptides which can be labeled with (3H)-glucosamine are located on the surface of the virus since they can be surface labeled with sodium boro(3H)hydride. In order to define the immunologically important viral proteins, monoclonal antibodies were prepared against the virus and selected for their ability to neutralize infectivity. Four such hybridoma lines were obtained for characterization of the antigens that elicit neutralizing antibodies. The viral polypeptides were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and the specificity of each monoclonal antibody was determined by Western blot analysis and/or by immunoprecipitation of (35S)-methionine and (3H)-glucosamine labeled infected cell lysates by the monoclonal antibodies. One monoclonal antibody reacted with two glycoproteins, gp135 and gp78a, on the Western blot but immunoprecipitated three glycoproteins, gp135, gp78a, and gp54 from labeled infected cell lysates. The other three monoclonal antibodies immunoprecipitated a single glycoprotein, gp78b, from (3H)-glucosamine labeled infected cell lysates but not from (35S)-methionine labeled infected cell lysates.With 5 Figures 相似文献