Murine Mammary Tumor Virus Research Articles

Unique association of three new type B retrovirus isolates with kinetochores and centrioles of the host cell

Three new virus isolates, M432, M832 and M960, obtained from the Southeast Asian mouse have been characterized morphologically in ultrathin sections and by negative staining with respect to their composition and intracellular assembly. The three isolates share certain morphological properties with the murine mammary tumor virus, a retrovirus of type B; yet previous investigations illustrated that they can readily be distinguished both from each other and from known retroviruses of the mouse, both of type B and C, by their RNA genome protein composition and reverse transcriptase activity.As illustrated in ultrathin sections (Fig. 1), the mature virions of about 100-120 nm in diameter contain a centrally located, electron dense nucleoid, a thin intermediate shell and an enveloping membrane derived from the plasma membrane. In negatively stained preparations (Fig. 1), the virions exhibit on their surface spikes which are best resolved on the isolate M432 and resemble the spikes present on the surface of retroviruses of type B.

Template-specific requirements for DNA synthesis by the Mason-Pfizer monkey virus DNA polymerase: Unique aspects

The biochemical properties of DNA polymerase purified from Mason-Pfizer monkey virus were studied, with respect to synthetic and natural template-primer utilization. These studies revealed the following new information about the Mason-Pfizer monkey virus enzyme: (a) Mason-Pfizer monkey virus polymerase was found to prefer template : primer molar nucleotide ratios of 2.5–5 : 1 for optimal rates of synthesis with poly(C) · (dG) 12–18 as template-primer. (b) Poly(A)-directed synthesis was stimulated by the addition of low concentrations of inorganic phosphate to the reaction mixture. (c) Poly(2′- O-methylcytidylate), poly(rCm), was the only template studied for which Mn 2+ proved the preferred divalent cation. Combinations of divalent cations stimulated rather than inhibited poly(rCm)-directed poly(dG) synthesis by the Mason-Pfizer monkey virus enzyme. (d) Heteropolymeric regions of rabbit globin mRNA and avian myeloblastosis virus 70 S RNA could be copied by the Mason-Pfizer monkey virus polymerase with oligo(dT), oligo(U) or in the case of avian myeloblastosis virus RNA, endogenous primers. In all such studies, Mg 2+ was the preferred divalent cation and a distinct preference for the DNA primer in the reverse transcription of natural RNAs was observed. These new findings necessitated comparative studies with the DNA polymerases from Rauscher murine leukemia virus and murine mammary tumor virus, as representative type C and type B retroviruses. Although the Mason-Pfizer monkey virus enzyme was found to share some properties in common with both type C and type B mammalian viral enzymes, certain of the above properties rendered it unique among the polymerases examined.

Type B and Type C RNA Tumor Virus Replication in Single Cells. Electron Microscopy With Tannic Acid2

Simultaneous replication of murine mammary tumor virus (type B) and murine leukemia virus (type C) was demonstrated electron microscopically along continuous stretches of the plasma membrane of single cells in cultures ofthe Mm5mt/c1 cell line. Types B and C virus buds were discriminated in thin sections with the aid of a tannic acid fixative that revealed the type B surface spikes as a homogeneous band of intermediate density and constant width on the surfaces of some buds (type B), whereas others (type C) remained with relatively smooth envelopes. Both types B and C buds may contain morphologically identical horseshoe-shaped nucleoids. Therefore, their identify (type B or C) could be ascertained in thin sections only on the basis of recognition of surface spikes.

Effect of a Mouse Mammary Tumor Virus-Derived Protein Vaccine on Primary Tumor Development in Mice 2

The vaccines used in this study were derived from purified murine mammary tumor virus (MuMTV) preparations. Approximately 60% of the protein fractions consisted of the major viral membrane glycoprotein gp52. Inoculation sc of 10 microgram MuMTV-S-derived vaccine significantly delayed the appearance of primary mammary tumors in GR and BALB/cfC3H mice (strains with high incidences of mammary cancer); in BALB/c and C3Hf mice, which have a moderate tumor incidence at an advanced age, this treatment resulted in a slight and substantial acceleration, respectively, of primary tumor development. The induced cellular immune reactivity for vaccination, as measured with the in vivo Winn test and the in vitro leukocyte adherence inhibition assay, was strongest in the GR strain as compared to the BALB/c strain. The titer of antibodies to tumor cells, as estimated by membrane immunofluorescence, was also higher in the GR strain. In BALB/cfC3H mice, the influence of different vaccination schemes with an MuMTV-O-derived protein vaccine on primary tumor development was studied. Before sc injection, the vaccine was precipitated on alum. A dose of 10 microgram vaccine resulted in a 61% decrease in tumor incidence. Two or five additional booster injections with 1 microgram protein vaccine had no beneficial effect, although the amount of antibody measured was increased after boosting.

The relative hydrophobicity of oncornaviral structural proteins

The interaction of the major structural polypeptides from the murine mammary tumor virus (MuMTV) and Rauscher murine leukemia virus (R-MuLV) with alkyl-agarose derivatives containing hydrocarbon arms of various lengths was examined. Both R-MuLV and MuMTV polypeptides were selectively adsorbed to octylimino (C 8)-agarose and decylimino (C 10)-agarose columns. Elution of proteins was accomplished using buffers containing 8.5 M ethylene glycol. In certain cases, polypeptides could only be removed from the columns by the addition of detergent to ethylene glycol-containing buffers. By examining the chromatographic behavior of polypeptides using (C 8)- and (C 10)-agarose columns and the conditions required for the elution of adsorbed proteins, we were able to determine the relative potential for hydrophobic interaction (degree of hydrophobicity) for each viral protein. The most hydrophobic polypeptide of MuMTV was found to be the glycoprotein gp34, while p15(E) and the gag precursor prp67 appeared to be the most hydrophobic of the R-MuLV proteins. The least hydrophobic polypeptides of MuMTV were p23 and p16, while those of R-MuLV were the two major internal proteins, p10 and p12. Four degrees of apparent hydrophobicity, from least hydrophobic to strongly hydrophobic, could be discerned by this procedure. The major core proteins (MuMTV p28, R-MuLV p30) of both viruses were slightly hydrophobic, while their major glycoproteins were moderately hydrophobic. The degree of hydrophobicity of oncornaviral polypeptides, as determined by hydrophobic chromatography, appeared to bear some relation to the different subviral components with which the proteins were associated. Our results indicate that hydrophobic chromatography may provide a new parameter for the characterization of oncornaviral polypeptides. This procedure should also prove applicable to the study of polypeptides from other viral systems.

Lysine tRNA is the predominant tRNA in murine mammary tumor virus

The method of aminoacylation and subsequent identification of the esterified amino acids was used to characterize the transfer RNAs in murine mammary tumor virus. Lysine tRNA was the major tRNA in both “free” 4S RNA and “7OS-associated” 4S RNA in virus derived from either tissue culture or mouse milk.

Oncogenicity of Murine Mammary Tumor Virus Produced in Tissue Culture: Brief Communication2

Murine mammary tumor virus (MuMTV), produced from a glucocorticoid-stimulated C3H mouse mammary adenocarcinoma cell line, was free of murine leukemia virus and oncogenic for weanling BALB/c mice. Adenocarcinomas were induced by MuMTV as early as 136 days post inoculation and with as low as 5 X 10(3) virus particles/mouse. Tumor incidence did not correlate directly with virus dose; rather, it was low at higher MuMTV concentrations (1.2 X 10(8) particles/mouse), reached and optimum at 1.3 X 10(5) particles/mouse, and decreased with virus dilution.

Retroviral “terminal deoxynucleotidyl transferase” activity is reverse transcription

Detergent-disrupted preparations of murine mammary tumor virus (MuMTV), Mason-Pfizer monkey virus (MP-MV), and Rauscher murine leukemia virus (RLV) were all found to be capable of catalyzing poly(dT) synthesis when only the exogenous primer oligo(dT) was supplied. Analysis of this apparent terminal deoxynucleotidyl transferase (TdT)-like DNA polymerase activity revealed that: (a) Optimal concentrations of divalent cations and the effect of KCl addition were similar or identical to those observed for reverse tranacriptase activity; (b) sensitivity to inhibition by inorganic phosphate for both TdT-like and reverse tranacriptase activity were identical and virion specific; (c) sedimentation coefficients obtained for TdT-like and reverse tranacriptase activity from each virion were identical, although almost all TdT-like activity was lost following velocity sedimentation of solubilized virions; the TdT-like activity could be restored through the addition of 70S RNA to reaction mixtures; (d) the products of TdT-like and reverse transcriptase reactions were found to be equally sensitive to S 1 nuclease treatment. Disruption of viral cores through the use of the ionic detergent sodium deoxycholate rendered TdT-like activity almost completely sensitive to RNase treatment. Additionally, antisera prepared against MP-MV DNA polymerase inhibited both TdT-like and reverse transcriptase activity to an identical degree. Our results therefore suggest that the “TdT” observed in MuMTV, MP-MV, and RLV is actually reverse tranacriptase activity directed by endogenous virion poly(A) annealed to the exogenously provided oligo(dT) primer.

Hydrophobic interaction of retroviral DNA polymerases with alkyl-agarose matrices

The chromatographic behavior of DNA polymerase activity in solubilized preparations of Rauscher murine leukemia virus (R-MuLV), murine mammary tumor virus (MuMTV), Mason-Pfizer monkey virus (MP-MV), and avian myeloblastosis virus (AMV) was examined on agarose derivatives containing hydrocarbon arms of various lengths. Ethyl-(C 2)- and hexylimino-(C 6)-agarose failed to bind significant quantitites of DNA polymerase activity. R-MuLV DNA polymerase bound to octylimino-(C 8)- and decylimino-(C 10)-agarose, and could be quantitatively recovered from these matrices by elution with buffers containing ethylene glycol alone and in the presence of detergents, respectively. The other viral polymerases demonstrated equal or greater affinities for these matrices. The potential for hydrophobic interaction by viral DNA polymerases revealed by our studies provides a mechanism whereby the specific complexing of reverse transcriptase with retroviral structural proteins in the absence of RNA may be explained.

Synthesis and processing of precursor polypeptides to murine mammary tumor virus structural proteins

Biosynthesis of murine mammary tumor virus (MuMTV) proteins was studied in the chronically MuMTV-infected epithelial cell line MuMT-73 by using monospecific antisera to the major MuMTV core protein p27 and the major envelope glycoprotein gp47. In pulse-labeling experiments using [35S]methionine, monospecific antisera to p27 precipitated a 75,000-molecular-weight protein as the major intracellular component. Analysis of the same cellular extracts with monospecific antisera to gp47 revealed that the gp47 precursor was a 70,000-dalton protein. After chase periods, there was a loss of label from the precursors and a concomitant increase of labeled extracellular mature viral proteins. The glycoprotein precursor incorporated labeled glucosamine and seemed to be processed more rapidly than the p27 precursor. Considerable amounts of apparently nonvirion-associated gp47 and glycoprotein precursor could be detected in the extracellular culture fluid.

Expression of murine mammary tumor virus-related antigens in human breast carcinoma (MCF-7) cells.

Tested by the indirect immunofluorescence technique, a human breast carcinoma cell in continuous in vitro cultivation (MCF-7) was found to express antigens cross-reactlve with the murine mammary tumor virus (MuMTV), but not with murine leukemia virus or Mason-Pfizer monkey virus. Antiserum dilution, antiserum absorption, and antiserum blocking experiments showed the cross-reactivity observed in MCF-7 cells to be MuMTV-specific. In long-term passage, only 4–6% of the total population of MCF-7 cells reacted specifically with anti-MuMTV sera. The number of specifically reactive cells could be increased up to 9–14% by treating MCF-7 cells with 10-7 or 10-am progesterone. Neither 5-iododeoxyuridine (IUdR), dexamethasone, nor IUdR plus dexamethasone, potent inducers of MuMTV expression in mouse cells, had any effect on MuMTV cross-reactive antligen expression in MCF-7 cells. Late-passage MCF-7 monolayers were phenotypic mixtures of cells. Small cells (S-cells) comprised 80–90% of the cells, and 10–20% were cells two to three times larger (L-cells). L-cells exclusively reacted with MuMTV antisera and responded to progesterone with augmented antigen levels. L-cells were separated from S-cells by cloning. Again, only L-cells In L-cell clones reacted with MuMTV antisera. The number of L-cell clones could be increased from 15% to about 35% by prolonged treatment of cells in the parent monolayer with IUdR; this resulted in a parallel increase in MuMTV-positive L-cells. However, the segregation of L-cells from S-cells and the increase in the number of L-cells by IUdR treatment did not permanently increase the number of MuMTV-positive cells. MuMTV-positive L-cells converted to MuMTV-negatlve L-cells rapidly during cell division such that the number of MuMTV-positive L-cells per clone was inversely related to clone size (I.e., number of cells per clone). To determine the sequence of events in dimunition of antigen synthesis in MCF-7 cell monolayers, earlier MCF-7 passages were examined. Passages 6–10 of one subllne were 50–60% positive in reactivity with anti-MuMTV sera. Approximately 80% of cells in these passages were L-cells. Within four subsequent passages, the percentage of MuMTV-positive cells diminished from 50–60% to 4–6%. During that time, the cultures remained at least 70% L-cells. Two models of growth dynamics were proposed to account for phenotypic Interconversions of MCF-7 cells during in vitro proliferation.

Is there a role for actin in virus budding?

Electrophoretic data from both sodium dodecyl sulfate-polyacrylamide gels (SDS-PAGE) and acid-urea gels reveal a protein in purified murine mammary tumor virus (MuMTV) which co-migrates with purified chick skeletal muscle actin. 125I-labeling of intact and disrupted virus preparations shows that the actin-like protein is not artifactually adsorbed to the outside of virions during isolation. Quantitative SDS-PAGE and examination of negatively stained preparations show that the actin cannot be accounted for by a contaminating population of virus-free vesicles. The ultrastructure of mammary epithelial cells and of Rous sarcoma virus-transformed chick embryo fibroblasts shows that virus extrusion is associated with filament-containing cellular processes. In particular, MuMTV is released from the ends of long microvilli which contain a bundle of 6-8-nm microfilaments and share other structural features with intestinal microvilli. We suggest that virus nucleoids require an interaction with host cell contractile proteins for their extrusion from the cell.

Transmission of Mammary Tumor Virus in Mouse Strain DD: Further Support for the Uniqueness of Strain GR

Outcrosses between high tumor mouse strain DD and low-incidence strains BALB/c and C57BL to produce reciprocal F1 susceptible offspring of both types resulted in high incidences of mammary tumors in (DD female X BALB/c male)F1 and (DD female X C57BL male)F1 females but not in the reciprocal hybrids with DD as the male parent. The finding that transmission of murine mammary tumor virus (MuMTV) in DD was through the milk, as most often observed in mouse strains, further supported the uniqueness of strain GR in which MuMTV is transmitted by either parent.

Mammary tumors and mammary tumor virus expression in hybrid mice of strains C57BL and GR.

Mammary tumorigenesis in genetic crosses between the high mammary tumor incidence GR and the low incidence C57BL mouse strains is highly correlated with murine mammary tumor virus expression in milk. Although the F1 and first backcross females had a mammary tumor incidence which was consistent with a single dominant gene segregation, the tumor incidence in the critical second backcross segregants disproved the single gene hypothesis. Genetic factors were clearly involved in regulation of virus expression which in turn correlated with both tumor incidence and tumor latency; these complex phenotypes are however best explained as threshold or quasicontinuous characters. As predicted from this model, the age specific incidence of mammary tumors showed a broad peak at 14-19 mo of age with no evidence of an early or late phase. Hematopoietic tumors showed no correlation with virus expression or mammary tumorigenesis suggesting different etiologies for these tumors.

Pseudotypes of vesicular stomatitis virus with envelope antigens provided by murine mammary tumor virus

Infection of two mouse mammary carcinoma cell lines with vesicular stomatitis virus (VSV) resulted in the formation of at least two types of particles containing the VSV genome but expressing different envelope characteristics (VSV pseudotypes). One of these VSV pseudotypes was infectious for a cell line derived from normal mouse mammary epithelial cells and mouse embryo cells but noninfectious for 3T3 cells, mink lung cells, and Vero cells. If mouse mammary tumor cells were treated with dexamethason some days prior to infection with VSV, the titer of this pseudotype was significantly increased. In contrast, the second pseudotype was infectious for mink cells, but not for the other cell lines tested, and the titer of this second pseudotype was unaffected by the presence of dexamethasone. The first pseudotype was found to be almost completely neutralized by anti-murine mammary tumor virus (MuMTV) serum whereas the second pseudotype was only partially neutralized at a higher antiserum concentration. Neither pseudotype showed the neutralization, host range, or interference properties of either ecotropic or xenotropic murine C-type viruses. These results suggest that the first pseudotype is VSV(MuMTV). The other pseudotype is less well defined but conceivably may represent a xenotropic MuMTV. In the course of these studies, a filterable agent was observed in GR mammary carcinoma cultures that reactivated the infectivity of VSV neutralized by antiserum. This agent was transmissible to mink cells.

Factors interfering with cellular immunological responses to the murine mammary tumor virus in tumor‐bearing mice

T-cell-enriched spleen cell fractions from BALB/c mice bearing virally induced mammary tumors become unresponsive with progressive tumor growth in the lymphocyte blastogenesis test, using purified mammary tumor virus (MTV) as an antigen. reactivity can be restored by mild trypsinization or extensive washing of the cells. When reactive cells from mice immunized with MTV were incubated with the wash fluid of the initially unresponsive cells. MTV-specific blastogenesis was inhibited. Washings of normal cells had no such effect. Leukocytes from tumor-bearing mice also become unresponsive to MTV in the leukocyte adherence inhibition (LAI) assay. Trypsinization or extensive washing does not restore reactivity. However, such treatment of reactive cells from immunized animals abolishes a positive reaction in the LAI assay, using MTV as an antigen. The washings of spleen cells from tumor-bearing mice inhibit the reactivity of leukocytes from immunized animals. Sera from these mice could block the reaction in both cellular immunological tests. The inhibiting factors appeared early after the onset of tumor growth. By means of the Sepharose bead immunofluorescence assay it was established that wash fluid and sera with blocking activity contain MTV antigens as well as anti-MTV antibodies.

Murine Mammary Tumor Virus Protein Vaccine: Induction of Antivirus Immunity and Inhibition or Acceleration of Growth of Transplanted Mammary Tumors2

Murine Mammary Tumor Virus Protein Vaccine: Induction of Antivirus Immunity and Inhibition or Acceleration of Growth of Transplanted Mammary Tumors2

The major structural proteins of murine mammary tumor virus: Techniques for isolation

We have utilized a variety of techniques of virus solubilization and protein separation for the isolation of the major structural polypeptides of murine mammary tumor virus, a B-type oncornavirus. Of these techniques, two isolation protocols proved to be effective and are reported here. In the first method, allowing the simultaneous purification of RNA-directed DNA polymerase and four major structural polypeptides (gp68, gp55, p28, and p12), the virions are disrupted with a nonionic detergent, and the solubilized proteins are separated by ion-exchange chromatography on DEAE- and phosphocellulose; final purification is achieved by molecular sieving chromatography. The second technique involves the solubilization of viral proteins with sodium dodecyl sulfate and chromatography on Sephadex G-200. The latter method is efficacious for the isolation of gp55, gp34, p28, and p12 after removal of the ionic detergent on Dowex columns, followed by final purification by molecular sieving chromatography.

Multiple antigenic determinants on the major surface glycoprotein of murine mammary tumor viruses.

The major external protein of murine mammary tumor viruses (MMTVs) is a glycoprotein of molecular weight 52,000 (gp52). We report here that the gp52s of MMTVs from different mouse strains contain both type-specific and group-specific antigenic determinants. Competitive radioimmunoassays with externally labeled virion preparations were used to demonstrate that MMTV virions from C3H, RIII, and GR mice competed identically in "group-specific" assays but either competed with altered slopes or competed incompletely in the appropriate "type-specific" assays. Similar results were obtained with purified gp52 preparations as competitors. MMTVs of RIII, C3H, and GR mice, grown in feline cells, competed similarly to the corresponding murine-derived MMTVs, thus demonstrating that the observed type-specific and group-specific antigenic reactivities are viral coded. In competitive radioimmunoassays using an antiserum against MMTV of C3H mice that was preabsorbed with MMTV of GR mice, type-specific reactivities were further magnified; MMTV of C3H mice competed completely whereas no competition was observed with MMTV of GR mice as a competitor. These studies are in agreement with previous biological and molecular hybridization studies on the diversity of MMTVs from different mouse strains.

Identification of the structural proteins of the murine mammary tumor virus that are serologically related to the antigens of intracytoplasmic type-A particles

The immunological relationship between intracytoplasmic A particles and B particles was investigated in order to determine whether A particles are the preformed cores of mature B particles. A serum prepared in rabbits against purified A particles (obtained from Leydig cell tumors) reacted, in immunodiffusion tests, to extracts from a variety of mouse tumors. It was found that tumors containing both A and B particles as well as those with only A particles contained A particle-associated antigens. Radioimmunoprecipitation and polyacrylamide gel electrophoretic (PAGE) techniques were used to identify those murine mammary tumor virus (MuMTV) proteins that were serologically related to the antigens of A particle. The MuMTV proteins were radiolabeled with either 14C-labeled amino acids in tissue culture or with 125I using the lactoperoxidase method. The solubilized viral proteins were tested with anti-A particle serum, or as controls, with antisera to MuMTV, MuMTV-gp55, and -p28, as well as normal rabbit and goat sera. Rabbit anti-MuMTV serum precipitated all the major polypeptides of MuMTV (gp55, gp36, p28, p18, and p12) when reacted with solubilized MuMTV proteins. However, rabbit anti-A particle serum precipitated only p28, p18, and p12. PAGE analysis of the MuMTV proteins precipitated by goat anti-MuMTV gp55 or by p28 sera showed only gp55 or p28; normal goat or rabbit serum did not precipitate detectable amounts of viral proteins indicating that the reactions were specific. These results suggest that A particles contain antigens which cross-react with the three major internal proteins of MuMTV and support the hypothesis that intracytoplasmic A particles are the preformed cores of B particles.