Generation of a mouse mammary tumor virus (MMTV) pseudotype of Kirsten sarcoma virus and restriction of MMTV gag expression in heterologous infected cells.

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.     

Vicinal relationships between the major structural proteins of murine mammary tumor virus.

Spatial relationships between the major structural proteins of murine mammary tumor virus (MuMTV), a type-13 RNA tumor virus, were investigated. Untreated virions, when analyzed by diagonal gel electrophoresis, contained two major species of disulfide-linked protein oligomers, i.e., a high-molecular-weight aggregate of p18 and a dimer composed of gp34 and p28. These complexes are termed “native crosslinked oligomers.” In addition, induced crosslinking by dithiobis succinimidyl propionate (DTSP) of vicinal proteins of whole virions or rosettes (viral membranes with attached spikes or projections) was also investigated. Diagonal gel electrophoretic analyses of DTSP-crosslinked virions revealed dimers of gp55 · gp55 and gp55 · gp34 as well as the native crosslinked oligomers. No DTSP-induced crosslinking of internal viral proteins was observed. Similar studies with rosettes indicated the formation of DTSP-induced crosslinked dimers consisting of gp55·gp55 and gp34 · gp34 in addition to gp34 · p28. From these studies, we conclude that the MuMTV projection is a homooligomer of gp55 which is directly apposed to the transmembrane protein, gp34, on the external surface. In addition, a portion of the gp34 transmembrane protein interacts internally with the major core shell protein (p28) as a native crosslinked oligomer. The latter observation most probably accounts for the eccentric location of the MuMTV nucleoid. A model which derives from these studies is discussed vis-à-vis virus assembly and previous studies of MuMTV structure.     

Murine mammary tumor virus deficient in the major glycoprotein: biochemical and biological studies on virions produced by a lymphoma cell line

A cell line, designated MLA, was established from a spontaneous lymphoma of a DBA/2 mouse. These cells contained large amounts of intracytoplasmic type-A particles, the presumed precursors of mature MuMTV. Electron microscopy revealed that the MLA cells released MuMTV-like virions into the culture fluid, but these virions were devoid of the characteristic envelope “spikes” of MuMTV. Antigenic analysis of virions purified from the culture fluid of MLA cells showed that, by microimmunodiffusion, the glycoproteins of MuMTV, gp49 and gp36, were not detectable, nor was the glycoprotein gp70 of MuLV detectable. Internal structural proteins of MuMTV, p28 and p12, were however, readily detectable in these virions. By the more sensitive radioimmunoassay technique, it was found that the virions produced by MLA cells contained some gp49-related antigen, but the amount present was about 500-fold lower than in other tissue culture-derived MuMTV. MLA cells synthesized a 70,000-dalton precursor of MuMTV glycoproteins. Culture fluids from which the virions were removed by centrifugation did contain gp49, indicating processing of the precursor polypeptide, but this polypeptide was not incorporated into the virions. Molecular hybridization studies revealed that MLA cells contained a large amount of MuMTV-specific RNA; the amount of MuLV-related RNA was about 100-fold lower. Whereas 150–200 MuMTV particles were released into culture fluid per cell per day, few MuLV particles were released. Purified virions from the MLA cells were injected into BALB/c and C57BL weanling females. This resulted in mammary tumor development in BALB/c females, but not in C57BL. The tumors contained MuMTV with the normal complement of envelope spikes and gp49 was detectable in the virions.     

Expression and disposition of the murine mammary tumor virus (MuMTV) envelope gene products by murine mammary tumor cells

Three murine mammary tumor virus (MuMTV)-producing epithelial cell lines derived from murine mammary tumors were examined in order to identify the MuMTV-specific cell surface antigens and their distribution on the cell surface, to study the kinetics of the MuMTV envelope precursor processing, virus assembly, and release, and to characterize the soluble MuMTV antigens that are shed into culture medium. Cell surface labeling experiments showed that only the mature MuMTV envelope glycoproteins gp52 and gp36 were exposed on the cell surface, and that gp52 was more abundant than gp36. In cells producing large quantities of MuMTV, expression of gp52 on the cell surface was shown by immunoelectron microscopy to be localized predominantly on the surface of budding virions and not on smooth areas of the cell surface where virus was not budding. The cell surface associated gp36 was found not to be incorporated into budding virions. A few cells in all three cell lines were found to produce only a few or no MuMTV particles and in these cells, unlike in the high virus-producing cells, considerable quantities of gp52 were expressed on the surface membrane. All three cell lines were found to shed large amounts of the MuMTV env precursor polyprotein as well as the mature non-virion-associated glycoprotein, gp52, into the culture medium. The envelope precursor protein (P75^env) that was shed into the culture medium was found to differ from the predominant form of the cellular env precursor Pr70^env in that (1) P75^env migrated with an apparent higher molecular weight than Pr70^env in SDS gels; (2) Pr70^env contained only the core oligosaccharide, whereas P75^env contained fucose in addition to the core sugars; (3) two-dimensional gel electrophoretic analysis showed that Pr70^env could be resolved into three to four components migrating in the basic region of the isoelectric focussing gel (pH 7–8), whereas P75^env was resolved into 9–13 components migrating in a more acidic region of the gel (pH 5–7). The molecular structure of the exfoliated gp52 was found to be similar to that of the gp52 that was incorporated into the virions although the virion-associated gp52 was not the source of the gp52 in the medium. Our quantitative pulse-chase studies suggest that of the two populations of MuMTV env precursors that are present in MuMTV-producing cells, only Pr70^env is processed intracellularly to give rise to the mature MuMTV envelope proteins gp52 and gp36.     

Gene order of mouse mammary tumor virus precusor polyproteins and their interaction leading to the formation of a virus.

Mouse mammary tumor virus (MMTV) proteins are synthesized as two major precursor polyproteins; gPr75^env containing gp52 and gp36, and Pr75^gag containing p27, pp20, p14, and p10. The gene order for gPr75^env has been previously shown to be H₂N-gp52-gp36-COOH (Schochetman, et al., 1977). gag polyproteins undergo intracellular cleavage in cat cells infected with MMTV and GR mammary tumor cells. Based on immunoprecipitation studies with antisera against intermediate MMTV cleavage products we now report the gene order for Pr75^gag is H₂N-p10-pp20-p27-p14-COOH. These results were further substantiated by analyzing the binding to ssDNA of the intermediate cleavage products which contain p14. To analyze the interaction of MMTV proteins with the cell membrane leading to budding of a virus particle, we used (i) lactoperoxidase-catalyzed iodination of MMTV cell surface proteins, (ii) galactose oxidase-catalyzed radiolabeling of carbohydrates on cell surface MMTV glycoproteins, (iii) serum cytotoxicity based on [⁵¹Cr] release with monospecific MMTV antisera, and (iv) membrane immunofluorescence with monospecific MMTV antisera. Analysis of ¹²⁵I-labeled MMTV cell surface antigens by immune precipitation with MMTV anti-gp52, gp36, p27, p14, and p10 sera followed by SDS-PAGE revealed only ¹²⁵I-gp52. In contrast, cell surface glycoprotein labeling revealed [³H]gp52 and [³H]gp36, indicating that, although the protein portion of gp36 was buried, some carbohydrate regions were exposed. EDTA treatment of cells to alter cell membranes prior to iodination resulted in the labeling of both Pr75^gag and gp52 but not gPr75^env. Furthermore, anti-p10 but not anti-p27 serum was cytotoxic against EDTA-treated cells. Similar results were obtained when the same antisera were tested by membrane immunofluorescence, ruling out the possibility that anti-p27 serum was not cytotoxic because it was unable to fix complement. These results show that Pr75^gag molecules, presumably as MMTV cores, interact with cell membrane sites containing gp52 and gp36 via the hydrophobic p10 portion of the molecule.     

The structure of the mouse mammary tumor virus: isolation and characterization of the core.

A procedure was developed for preparing cell fractions rich in chloroplasts, nuclei, and pea enation mosaic virus (PEMV)-induced cytopathological structures (vesicles). Those fractions from infected pea plants which contained nuclei or vesicles also contained actinomycin D-insensitive RNA polymerase activity and PEMV-specific hybridizable RNA. The fraction from infected plants containing predominantly chloroplasts had little of this polymerase activity or RNA, as was the case with all fractions from healthy plants. The significance of the polymerase activity in the nuclei and vesicles is discussed, as well as the potential role of the vesicles in the virus infection cycle.     

Effect of dexamethasone on expression of endogenous mouse mammary tumor virus sequences in BALB/c tumor cell lines.

BALB/c mammary tumor cell lines which contain only endogenous murine mammary tumor virus (MMTV) sequences respond to dexamethasone (DXS) treatment with minimal (approximately 2-fold) increases in MMTV RNA. This is in marked contrast to the 10? to 20-fold increases observed with cell lines harboring exogenous MMTV variants. Comparison of hybridization results obtained with two complementary DNA probes representative of either the entire MMTV RNA genome or its poly(A)-adjacent sequences suggests that the DXS response of BALB/c lines is also qualitatively different from that of exogenous MMTV-producer cell lines. Thermal stability studies suggested a 2–3% divergence between the RNA sequences of endogenous BALB/c and exogenous C3H viruses, with the 3′-end of the viral RNA appearing to be conserved relative to the rest of the genome.     

Molecular basis of altered mouse mammary tumor virus expression in the D-2 hyperplastic alveolar nodule line of BALB/c mice

The preneoplastic D-2 hyperplastic outgrowth line, which was derived from a hormone-induced hyperplastic alveolar nodule (HAN) of a BALB/c mouse, was used for a detailed analysis of mouse mammary tumor virus (MMTV) expression. The D-2 HAN line has previously been shown to express viral RNA representative of the entire genome, although viral particles have been noted only rarely. The MMTV-specific mRNA, protein, and DNA content of the D-2 tissues was defined in an effort to better understand the molecular basis of the aberrant virus expression. Northern blotting techniques demonstrated the presence of properly processed 8.9 kb (genomic) and 3.6 kb (envelope) mRNA. Protein electroblotting procedures established the presence of properly processed viral core protein p28. In contrast, the envelope precursor polyprotein was not processed into detectable levels of gp52. Analysis of MMTV proviral content by Southern blot methodology revealed the presence of a newly acquired provirus which serves as a marker for the clonal nature of the D-2 line. The origin of the new provirus is unknown. Methylation studies established that the new proviral insert is hypomethylated and, therefore, is likely serving as the template for the MMTV expression observed in the D-2 HAN line. These characteristics of the D-2 line make it an excellent system in which to study the role, if any, of MMTV in the progression of D-2 preneoplastic tissues to the tumor phenotype.     

The interaction of antibody with the major surface glycoprotein of vesicular stomatitis virus. I. Analysis of neutralizing epitopes with monoclonal antibodies

Monoclonal antibodies reactive with the major surface glycoprotein (G-protein) of vesicular stomatitis virus serotypes Indiana and New Jersey (VSV-Ind, VSV-NJ) have been isolated and characterized. The reactivity of each monoclonal was determined by enzyme-linked immunosorbent assay (ELISA), competitive binding assay (CBA), and the ability to neutralize infectivity. It was found that the majority of the antibodies were of the IgG(2a) subclass. In the CBA, unlabeled monoclonal antibodies were used to compete for radiolabeled antibodies in binding to solid-phase immunoadsorbents. The VSV-NJ G-protein appears to contain four nonoverlapping epitopes by these analyses. However, the VSV-Ind G-protein is more complex since four epitopes were defined which exhibited varying degrees of overlap. In some cases, this overlap was defined by complete reciprocal competition between antibodies with different reactivity patterns. In other instances, partial or nonreciprocal competition between antibodies was observed. These results may indicate epitopes in close proximity or suggest allosteric modifications in the G-protein induced by antibody binding. A fifth epitope on the Ind G-protein was defined by a monoclonal antibody which could bind to the G-proteins of both VSV-Ind and VSV-NJ but could only neutralize infectivity of the VSV-Ind serotype.     

Organization and expression of endogenous murine mammary tumor virus genes in mice congenic at the H-2 complex

The organization and expression of endogenous murine mammary tumor virus (MuMTV) genetic information was examined in a number of mouse strains Congenic at the major histocompatibility complex (MHC). Analysis of restriction endonuclease-generated fragments of cellular DNA revealed no differences in the integration sites (EcoRI)or internal cleavage sites (PstI) of MuMTV proviruses in any of the (C7BL/10 (B10) Congenic strains tested. Similar studies indicated that the proviruses of BALB/c and BALB.B mice are indistinguishable, and that the restriction endonuclease-generated fragments of BALB/c mice are similar to those of the B10 strain. These proviruses behave as stable genetic elements which have been well conserved during the derivation of the Congenic strains. Also, since none of the characteristic MuMTV proviruses of the donor strains could be identified in the Congenic mice, they do not seem to be closely linked to the H-2 complex. When concentrations of viral RNA in lactating mammary glands (LMGs) were measured by molecular hybridization kinetics, comparable levels of MuMTV RNA were found in all the B10 Congenic mice; considerably less viral RNA was detected in mice with a BALB/c background. This viral RNA in mammary gland cells was not translated into detectable gp49, the major MuMTV glycoprotein. These results indicate that the differences observed in susceptibility to mammary tumorigenesis induced in H-2 Congenic mice by exogenous MuMTV are not influenced by the organization or expression of endogenous MuMTV proviral genes.     

In animals infected by bovine leukemia virus (BLV) antibodies to envelope glycoprotein gp51 are directed against the carbohydrate moiety

The immunological reactivity of the surface glycoprotein gp51 of bovine leukemia virus was examined by radioimmunoassay with sera from infected animals and sera obtained from rabbits after injection of the purified antigen. The respective influence of the protein and carbohydrate portions of the glycoprotein on the antigenic reactivity was investigated by digestions with glycosidases and proteases. Digestion of gp51 with a mixture of glycosidases abolished the reactivity of the antigen with sera of infected cattle or sheep. In contrast, the reactivity of gp51 with monospecific rabbit antisera was only slightly modified by the glycosidase treatment. Digestion of the same antigen with proteinase K and Pronase completely eliminated its reactivity with monospecific rabbit antiserum; sera from infected cattle or sheep still precipitated 10 to 15% of the same ¹²⁵I-antigen in direct radioimmunoassay. These results strongly indicate that natural immunity against BLV gp51 depends upon an intact carbohydrate side chain. Following purification of the antigen, protein antigenic sites are uncovered and reacted against by the injected rabbit. Solid phase radioimmunoassay furthermore showed that the carbohydrate antigenic site is probably unique as opposed to the probably multiple protein antigenic determinants. Antibody to gp51 prepared in the rabbit by injection of intact BLV-infected bovine lymphocytes reacted, in all tests performed, in much the same way as natural antibody found in BLV-infected cattle or sheep.     

The interaction of antibody with the major surface glycoprotein of vesicular stornatitis virus II. Monoclonal antibodies to nonneutralizing and cross-reactive epitopes of Indiana and New Jersey serotypes

Eleven monoclonal antibodies reactive with the major surface glycoprotein (G-protein) of vesicular stomatitis virus serotypes Indiana and New Jersey (VSV-Ind, VSV-NJ) were used to map antigenic sites found on one or both serotypes. The antibodies used were unable to neutralize infectivity of the virus in vitro although they were able to bind to the G-protein. Six of the antibodies bound to the G-proteins of both serotypes and delineated three nonoverlapping epitopes as determined by a competitive binding assay. In addition, one monoclonal antibody bound to both serotypes and could neutralize infectivity in vitro of only VSV-Ind. This antibody could compete with several cross-reactive nonneutralizing antibodies which could not neutralize either VSV-Ind or VSV-NJ. Three monoclonal antibodies were serotype specific for VSV-NJ and exhibited no overlap among themselves or with the cross-reactive antibodies. One VSV-Ind serotype-specific antibody was isolated which could compete with a cross-reactive antibody. Enhancement of antibody binding by the binding of a second antibody was observed in some cases. This phenomenon appeared to be due to an increase in availability of antigenic sites caused by allosteric modifications.     

Characterization of purified structural proteins of murine mammary tumor virus.

The major structural polypeptides of a type-B oncornavirus, murine mammary tumor virus (MuMTV), were isolated to homogeneity by ion-exchange and molecular sieving chromatography. Viral component proteins included the major envelope glycoproteins (gp68, gp55, and gp34) as well as the internal proteins (p28, p18, p14, and p12). Characterizations of these viral proteins included molecular weights, N-terminal amino acids, and quantitative amino acid analyses.     

Monoclonal antibodies define eight independent antigenic regions on the bovine leukemia virus (BLV) envelope glycoprotein gp51

Fifteen monoclonal anti-BLV gp51 antibodies are characterized. Competition antibody binding assays show that they are directed against eight independent antigenic regions on the BLV gp51 molecule. Conformation or accessibility of some of these gp51 epitopes change with the test system used, namely the liquid phase radioimmunoassay with radiolabeled antigen or the solid phase enzyme immunoassay with plastic bound gp51 or BLV particles. A two-site immunometric assay using monoclonal antibodies directed against two independent epitopes allows detection of isolated gp51 molecules at a minimal concentration of 0.4 ng/ml and is also suitable for the detection of BLV particles.     

Type-specific determinants on proteins of an endogenous C3H mouse mammary tumor virus (MMTV) distinguish this virus from highly oncogenic exogenous MMTVs

The genetically transmitted endogenous MMTV isolated from C3H mice after removal of the milk-transmitted virus by foster nursing is designated C3Hf MMTV to distinguish it from the highly oncogenic milk-transmitted exogenous virus designated C3H MMTV. We have isolated a MMTV-expressing C3Hf mammary tumor cell line which has no exogenous proviral sequences detectable by analysis of DNA fragments generated by Pst I restriction endonuclease. This cell line produced sufficient C3Hf MMTV to allow purification of the major proteins and an antigenic comparison of this virus with highly oncogenic exogenous MMTVs from C3H, GR, and RIII strains of mice. The envelope glycoproteins, gp52 and gp36, purified from the C3Hf MMTV, were found to have both group- and type-specific reactivities. Only C3H MMTV gave incomplete competition in the gp36 assay and, therefore, could be distinguished from C3Hf, RIII, and GR MMTVs which gave complete competition. Unique antigenic determinants exist on the gp52 of C3Hf MMTV since it is the only virus to give complete competition in the gp52 radioimmunoassay. GR MMTV competed only 60%, whereas C3H and RIII MMTVs gave 80% competition. This was the first demonstration that RIII and C3Hf MMTVs were immunologically distinct. Only group-specific reactivity was found with the gag-coded MMTV p27; however, group and class antigenic determinants were found on the gag-coded MMTV p10.     

Mammary tumors from GR mice contain more than one population of mouse mammary tumor virus-infected cells

Spontaneous mammary tumors in the GR mouse strain contain several acquired copies of mouse mammary tumor virus (MMTV) DNA that are not present in normal organ DNA and that are detectable by restriction endonuclease analysis and the Southern blotting procedure. Hormone-responsive and -independent cell populations were selected from spontaneous GR mammary tumors by grafting the tumors in castrated male GR mice in the presence and absence of female sex hormones. Analysis of the acquired MMTV DNA copies revealed differences between hormone-responsive and -independent cells derived from the same tumor; however, specific MMTV DNA fragments could not necessarily be correlated with the hormone responsiveness of the tumor. In some cases more than one proviral pattern could be detected for both hormone-responsive and hormone-independent cells. These results suggest that spontaneous GR mammary tumors are made up of more than one population of hormone-responsive and -independent cells which can be distinguished by their MMTV-specific proviral restriction fragments.     

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.     

Antibody-induced conformational changes result in enhanced avidity of antibodies to different antigenic sites on the tick-borne encephalitis virus glycoprotein

By the use of monoclonal antibodies we have recently defined eight distinct epitopes on the structural glycoprotein of tick-borne encephalitis (TBE) virus which differ with respect to location, function, or serological specificity (Heinz et al, Virology 126, 525-537, 1983). The present investigation reveals a complex network of interactions between antibodies directed against distinct nonoverlapping epitopes leading to enhanced binding of certain antibodies in the presence of bound second antibodies. The enhancement between antibody pairs can be either unidirectional or bidirectional. In addition, there are domains of predilection, which bind enhanceable antibodies (domain A) whereas others bind antibodies which preferentially induce enhancement (domain B). These domains represent structurally unrelated entities, domain A being sensitive to denaturation and fragmentation and domain B being resistant. Quantitative evaluation of binding data by Scatchard analysis revealed that the observed enhancement phenomenon is due to a two- to sixfold increase of antibody avidity. In the system described, enhancement of antibody binding is not dependent on antibody bivalency since it could also be demonstrated with purified Fab fragments acting either as enhanced or as enhancing antibody. It is therefore concluded that binding of antibodies to certain epitopes on the TBE virus glycoprotein induces conformational changes in distant parts of the molecule which can result in increased avidity of antibodies directed to conformationally changed epitopes. A possible explanation for the origin of this enhancement phenomenon is presented.     

Characterization of a terminal deoxynucleotidyl transferase activity in mouse mammary tumor virus.

Mouse mammary tumor virus (MMTV) contains an enzyme which catalyzes DNA synthesis in the presence of a preformed initiator without template direction. This type of enzyme activity is characteristic of the class of polymerases called terminal deoxynucleotidyl transferase. The MMTV enzyme is localized in the viral core and its activity depends upon the presence of detergent, reducing agent, preformed initiator, and divalent cation. Optimal conditions for activity include pH 6.85 and the use of a thymidine-containing substrate and initiator. The reaction product is predominantly single stranded. Concentration-dependent inhibition by several salts is observed. Manipulation of the reaction optima and salt concentration allows the functional separation of MMTV terminal transferase activity from RNA-dependent DNA polymerase activity.     

Gene order of the mouse mammary tumor virus glycoproteins

Immunochemical and tryptic peptide mapping techniques were used to show that the mouse mammary tumor virus (MMTV) envelope glycoproteins gp52 and gp36 are distinct components derived from a common glycosylated precursor polypeptide of 75,000 daltons (gPr75). Because both gp52 and gp36 are derived from a common precursor polypeptide and therefore have a common initiation site, we have been able to determine their gene order within the viral genome. The gene order was deduced from three different types of experiments. The first approach measured the differential inhibition by NaCl hypertonic shock on initiation of gp52 and gp36 synthesis. The second approach measured the kinetics of appearance of various MMTV proteins following the synchronized reinitiation of polypeptide synthesis resulting from NaCl hypertonic shock. The third approach analyzed polypeptides released from polyribosomes after a series of variable-length short pulses with [3H]amino acids. Our results indicate that the gene order for gPr75 is H2N-gp52-gp36-COOH and 5'-gp52-gp36-3' within the MMTV genome.