C3H Mouse Mammary Tumor Virus Research Articles

High prevalence of mouse mammary tumor virus-like gene sequences in breast cancer samples of Iranian women

ABSTRACTMouse mammary tumor virus (MMTV) is considered to be responsible for breast tumor development in mice. In several instances sequences homologous to the genome of this virus have been reported in human breast cancer samples.Here we aimed to evaluate MMTV involvement in human breast cancer development. DNA was extracted from 118 formalin fixed and paraffin embedded malignant (n = 59) and benign (n = 59) breast lesions. Using two sets of outer and inner MMTV-like envelope specific primers, in a nested PCR setup, MMTV genome was detected in 19 samples out of 59 (%32.2) cases of breast carcinomas, while in non-malignant breast tissue samples, only 3 samples out of 59 (%5) were positive. The difference was statistically highly significant (p < 0.001). Alignment of PCR amplified sequences with BR6, GR and C3H mouse mammary tumor virus strains emerged to be around 99% identical which is indicative of tumor tissue infection by an exogenous mouse MMTV genome.

Nuclear Matrix Binding Regulates SATB1-mediated Transcriptional Repression

Special AT-rich binding protein 1 (SATB1) originally was identified as a protein that bound to the nuclear matrix attachment regions (MARs) of the immunoglobulin heavy chain intronic enhancer. Subsequently, SATB1 was shown to repress many genes expressed in the thymus, including interleukin-2 receptor alpha, c-myc, and those encoded by mouse mammary tumor virus (MMTV), a glucocorticoid-responsive retrovirus. SATB1 binds to MARs within the MMTV provirus to repress transcription. To address the role of the nuclear matrix in SATB1-mediated repression, a series of SATB1 deletion constructs was used to determine protein localization. Wild-type SATB1 localized to the soluble nuclear, chromatin, and nuclear matrix fractions. Mutants lacking amino acids 224-278 had a greatly diminished localization to the nuclear matrix, suggesting the presence of a nuclear matrix targeting sequence (NMTS). Transient transfection experiments showed that NMTS fusions to green fluorescent protein or LexA relocalized these proteins to the nuclear matrix. Difficulties with previous assay systems prompted us to develop retroviral vectors to assess effects of different SATB1 domains on expression of MMTV proviruses or integrated reporter genes. SATB1 overexpression repressed MMTV transcription in the presence and absence of functional glucocorticoid receptor. Repression was alleviated by deletion of the NMTS, which did not affect DNA binding, or by deletion of the MAR-binding domain. Our studies indicate that both nuclear matrix association and DNA binding are required for optimal SATB1-mediated repression of the integrated MMTV promoter and may allow insulation from cellular regulatory elements.

The type B leukemogenic virus truncated superantigen is dispensable for T-cell lymphomagenesis.

Type B leukemogenic virus (TBLV) is a variant of mouse mammary tumor virus (MMTV) that causes T-cell lymphomas in mice. We have constructed a TBLV-MMTV hybrid, pHYB-TBLV, in which 756 bp of the C3H MMTV long terminal repeat (LTR) was replaced with 438 bp of the TBLV LTR. Intraperitoneal injection of pHYB-TBLV transfectants consistently resulted in T-cell lymphomas in 50% of injected weanling BALB/c mice with an average latency period of 5.7 (+/- 1.5) months. Transfectants of pHYB-TBLV containing a double-frameshift mutation in the truncated superantigen gene (sag) induced T-cell lymphomas with similar incidences, latency periods, and phenotypes, suggesting that cis-acting elements in the TBLV LTR determine disease specificity.

Type B leukemogenic virus has a T-cell-specific enhancer that binds AML-1.

Type B leukemogenic virus (TBLV) induces rapidly appearing T-cell tumors in mice. TBLV is highly related to mouse mammary tumor virus (MMTV) except that TBLV long terminal repeats (LTRs) have a deletion of negative regulatory elements and a triplication of sequences flanking the deletion. To determine if the LTR triplication represents a viral enhancer element, we inserted the triplication upstream and downstream in either orientation relative to the thymidine kinase promoter linked to the luciferase gene. These experiments showed that upregulation of reporter gene activity by the TBLV triplication was relatively orientation independent, consistent with the activity of eukaryotic enhancer elements. TBLV enhancer activity was observed in T-cell lines but not in fibroblasts, B cells, or mammary cells, suggesting that enhancer function is cell type dependent. To analyze the transcription factor binding sites that are important for TBLV enhancer function, we prepared substitution mutations in a reconstituted C3H MMTV LTR that recapitulates the deletion observed in the TBLV LTR. Transient transfections showed that a single mutation (556M) decreased TBLV enhancer activity at least 20-fold in two different T-cell lines. This mutation greatly diminished AML-1 (recently renamed RUNX1) binding in gel shift assays with a mutant oligonucleotide, whereas AML-1 binding to a wild-type TBLV oligomer was specific, as judged by competition and supershift experiments. The 556 mutation also reduced TBLV enhancer binding of two other protein complexes, called NF-A and NF-B, that did not appear to be related to c-Myb or Ets. AML-1 overexpression in a mammary cell line enhanced expression from the TBLV LTR approximately 30-fold. These data suggest that binding of AML-1 to the TBLV enhancer, likely in combination with other factors, is necessary for optimal enhancer function.

C3H mouse mammary tumor virus superantigen function requires a splice donor site in the envelope gene.

Mouse mammary tumor virus (MMTV) encodes a superantigen (Sag) that is required for efficient milk-borne transmission of virus from mothers to offspring. The mRNA used for Sag expression is controversial, and at least four different promoters (two in the long terminal repeat and two in the envelope gene) for sag mRNA have been reported. To determine which RNA is responsible for Sag function during milk-borne MMTV transmission, we mutated a splice donor site unique to a spliced sag RNA from the 5' envelope promoter. The splice donor mutation in an infectious provirus was transfected into XC cells and injected into BALB/c mice. Mice injected with wild-type provirus showed Sag activity by the deletion of Sag-specific T cells and induction of mammary tumors in 100% of injected animals. However, mice injected with the splice donor mutant gave sporadic and delayed T-cell deletion and a low percentage of mammary tumors with a long latency, suggesting that the resulting tumors were due to the generation of recombinants with endogenous MMTVs. Third-litter offspring of mice injected with wild-type provirus showed Sag-specific T-cell deletion and developed mammary tumors with kinetics similar to those for mice infected by nursing on MMTV-infected mothers, whereas the third-litter offspring of the splice donor mutant-injected mice did not. One of the fifth-litter progeny of splice donor mutant-injected mice showed C3H Sag activity and had recombinants that repaired the splice donor mutation, thus confirming the necessity for the splice donor site for Sag function. These experiments are the first to show that the spliced sag mRNA from the 5' envelope promoter is required for efficient milk-borne transmission of C3H MMTV.

Mutational and functional analysis of the C-terminal region of the C3H mouse mammary tumor virus superantigen.

The mouse mammary tumor virus (MMTV) encodes within the U3 region of the long terminal repeat (LTR) a protein known as the superantigen (Sag). Sag is needed for the efficient transmission of milk-borne virus from the gut to target tissue in the mammary gland. MMTV-infected B cells in the gut express Sag as a type II transmembrane protein that is recognized by the variable region of particular beta chains (Vbeta) of the T-cell receptor (TCR) on the surface of T cells. Recognition of Sag by particular TCRs results in T-cell stimulation, release of cytokines, and amplification of MMTV infection in lymphoid cells that are needed for infection of adolescent mammary tissue. Because the C-terminal 30 to 40 amino acids of Sag are variable and correlate with recognition of particular TCR Vbeta chains, we prepared a series of C-terminal Sag mutations that were introduced into a cloned infectious MMTV provirus. Virus-producing XC rat cells were used for injection of susceptible BALB/c mice, and these mice were monitored for functional Sag activity by the deletion of C3H MMTV Sag-reactive (CD4+ Vbeta14+) T cells. Injected mice also were analyzed for mutant infection and tumor formation in mammary glands as well as milk-borne transmission of MMTV to offspring. Most mutations abrogated Sag function, although one mutation (HPA242) that changed the negative charge of the extreme C terminus to a positive charge created a weaker Sag that slowed the kinetics of Sag-mediated T-cell deletion. Despite the lack of Sag activity, many of the sag mutant viruses were capable of sporadic infections of the mammary glands of injected mice but not of offspring mice, indicating that functional Sag increases the probability of milk-borne MMTV infection. Furthermore, although most viruses encoding nonfunctional Sags were unable to cause mammary tumors, tumors were induced by such viruses carrying mutations in a negative regulatory element that overlaps the sag gene within the LTR, suggesting that loss of Sag function may be compensated, at least partially, by loss of transcriptional suppression in certain tissues. Together these results confirm the importance of Sag for efficient milk-borne transmission and indicate that the entire C-terminal region is needed for complete Sag function.

Strain-Specific Expression of Spliced MMTV RNAs Containing thesuperantigenGene

The transmission of milk-borne or exogenous mouse mammary tumor virus (MMTV) requires infection of B cells in the gut-associated lymphoid tissue and expression of the superantigen (Sag) protein at the B-cell surface. Presentation of Sag at the B-cell surface is required for the transmission of MMTV to T cells and subsequent infection of the target mammary gland tissue. Because several different promoters have been reported for MMTVsagmRNA expression, we investigated whether the detection of splicedsagRNAs was dependent upon the cell type infected or the particular MMTV strain examined. In this study, we detected expression of splicedsagRNA from the standard promoter and from an internal U3 promoter in B-cell lines expressing endogenousMtv-6by RT-PCR, although expression from the standard promoter appeared to be at least 10-fold higher than that observed from the internal U3 promoter.SagRNA originating from exogenous C3H MMTV was not observed from either of the U3 promoters in any cell type examined. However, spliced mRNAs containing the exogenous C3H MMTV, endogenousMtv-8,or endogenousMtv-17 saggenes could be detected from a previously described promoter in theenvelopecoding region regardless of the cell type infected. Becausesag-specific RNAs can be initiated independently of the LTR promoters, there may be selection for independent control of MMTVsagand structural gene expression.

Major histocompatibility complex class II I-E-independent transmission of C3H mouse mammary tumor virus.

C57BL/6 mice are resistant to C3H mouse mammary tumor virus (MMTV)-induced mammary tumorigenesis and lack major histocompatibility complex class II I-E molecules that are essential for presentation of C3H superantigen to T cells. T cells are needed for transmission of milk-borne MMTV from the gut to the mammary gland. In this report, we show that infectious C3H MMTV is produced by C57BL/6 mice that nurse on C3H mothers but that virus production in the mammary gland is delayed compared with that in I-E+ mouse strains.

Exogenous Mouse Mammary Tumor Virus (MMTV) Infection Induces Endogenous MMTVsagExpression

The mouse mammary tumor virus (MMTV) superantigen (Sag) protein is involved in the transmission of milk-borne MMTV from virus-infected milk in the gut to the target mammary gland tissue. Using an RT–PCR assay forin vivoMMTV infection, BALB/c or C3H mice nursed on C3H MMTV-infected mothers showedsagmRNA expression in intestine, spleen, and thymus as early as 1 day after infection, whereas uninfected BALB/c control animals had approximately 10- to 30-fold lowersagexpression. Further fractionation experiments with small intestine indicated thatsagexpression occurred in gut-associated lymphoid cells. Restriction enzyme digestion of PCR products indicated that thesagmRNA detected was derived from the endogenous MMTVs, and sequencing analysis confirmed that the PCR products were derived from endogenousMtv-6.Expression of C3H-specific mRNA was detectable in BALB/cfC3H or C3H tissues by RNase protection or by RT–PCR. Endogenous MMTVsagexpression was low in spleen and undetectable in thymocytes of C3H MMTV-infected C57BL/6 mice, a strain resistant to C3H MMTV tumorigenesis and defective for MHC class II I-E molecules. The RT–PCR assay forsagmRNA appears to measure the Sag-induced stimulation previously predicted for milk-borne MMTV infection. Together these data suggest that exogenous MMTVsagexpression is minimal, but sufficient to rapidly stimulate transcription of endogenous MMTVsagmRNA in B- and T-cells in an MHC class II I-E-dependent manner. The endogenoussagexpression on maternal lymphocytes may increase the number of proliferating T-cells available for milk-borne MMTV infection.

Coexpression of exogenous and endogenous mouse mammary tumor virus RNA in vivo results in viral recombination and broadens the virus host range.

Mouse mammary tumor virus is a replication-competent B-type murine retrovirus responsible for mammary gland tumorigenesis in some strains of laboratory mice. Mouse mammary tumor virus is transmitted horizontally through the milk (exogenous or milk-borne virus) to susceptible offspring or vertically through the germ line (endogenous provirus). Exogenously acquired and some endogenous mouse mammary tumor viruses are expressed at high levels in lactating mammary glands. We show here that there is packaging of the endogenous Mtv-1 virus, which is expressed at high levels in the lactating mammary glands of C3H/HeN mice, by the virions of exogenous C3H mouse mammary tumor virus [MMTV(C3H)]. The mammary tumors induced in C3H/HeN mice infected with exogenous MMTV (C3H) virus contained integrated copies of recombinant virus containing a region of the env gene from an endogenous virus. This finding indicates that there was copackaging of the Mtv-1 and MMTV(C3H) RNAs in the same virions. Moreover, because Mtv-1 encodes a superantigen protein with a V beta specificity different from that encoded by the exogenous virus, the packaging of Mtv-1 results in an infectious virus with a broader host range than MMTV(C3H).

Expression of a MHC class II transgene determines both superantigenicity and susceptibility to mammary tumor virus infection.

Milk-borne mouse mammary tumor virus (MMTV) is a type B retrovirus that induces mammary carcinoma. Infectious MMTV, as well as genomically integrated mouse mammary proviruses, encode superantigens that are recognized by T cells that express appropriate T cell receptor V beta products. To determine the relationship between the superantigenic property of milk-borne MMTV and its in vivo infectivity, mice which were either positive or negative for expression of a transgene-encoded E alpha E beta class II major histocompatibility complex (MHC) product were exposed to milk borne C3H MMTV. Superantigen-mediated deletion of V beta 14-expressing T cells occurred only in E alpha transgene-positive mice, indicating that the deletion was E alpha E beta dependent. When mice were analyzed for viral infection by assaying viral p28 in the milk of recipient females, significant p28 levels were found only in E alpha E beta transgene-positive mice. Similarly, the presence of C3H MMTV LTR mRNA in mammary glands, as detected by PCR, paralleled p28 levels. These findings indicate that E alpha expression or the E alpha-dependent T cell response to viral superantigen is causally related to susceptibility to MMTV infection, and that lack of a permissive class II product can protect mice from virus infection.

Differential transport and processing of variant mouse mammary tumor virus glycoproteins.

The transport and proteolytic processing of two individual gene isolates of the mouse mammary tumor virus (MMTV) glycoprotein were compared in transfected rat HTC hepatoma cells. Plasmids were constructed such that the MMTV glycoprotein genes were constitutively expressed from the promoter within the Rous Sarcoma Virus 5' Long Terminal Repeat in the absence of other MMTV proteins. An isolate of the GR strain MMTV glycoprotein was efficiently transported and processed resulting in the localization of MMTV glycoproteins at the cell surface and in the extracellular environment. Moreover, the kinetics of acquisition of endoglycosidase H resistant oligosaccharide side chains and the rate of endoproteolytic cleavage of the glycosylated polyprotein expressed in transfected cells were virtually identical to that observed in viral-infected rat hepatoma cells. In contrast, a natural variant of the C3H strain MMTV glycoprotein expressed in transfected cells was retained in an intracellular compartment by a heavy chain binding protein (BiP)-independent pathway in an endoglycosidase H sensitive and uncleaved form. This MMTV glycoprotein isolate was retained early in the exocytic pathway and displayed a half-life of approximately 45 min in transfected cells. Only a minor fraction of the expressed C3H variant glycoprotein was detected at the cell surface but was not externalized. Our results suggest that the variant C3H MMTV glycoprotein contains one or more mutations that preclude its efficient transport through the exocytic pathway.

Mouse mammary tumor virus proviruses in T-cell lymphomas lack a negative regulatory element in the long terminal repeat.

The nucleotide sequences of long terminal repeats (LTRs) from several mouse mammary tumor virus (MMTV) proviruses acquired in mouse T-cell lymphomas were determined. All MMTV proviruses cloned from a C57BL/6 lymphoma contained an identical LTR deletion of 491 base pairs (approximately -655 to -165), whereas an MMTV provirus from a BALB/c T-cell lymphoma had a 430-base-pair deletion in the same U3 region. MMTV proviruses with LTR deletions were acquired in these tumors 10 times more frequently than proviruses with intact LTRs. Because the deletions removed a portion of the glucocorticoid response element or "regulated" enhancer, the transcriptional activity of the deleted MMTV LTRs was assessed in both transient expression and stable transfection experiments. Plasmids were constructed in which the deleted or full-length MMTV LTRs were placed upstream of the chloramphenicol acetyltransferase gene. Results from transfection experiments with these constructs showed that the basal expression of the deleted MMTV LTR in the absence of glucocorticoids was higher than that of the full-length Mtv-17 or C3H MMTV LTRs under the same conditions. Moreover, the C3H LTR with a similar deletion (-637 to -255) also promoted high basal levels of chloramphenicol acetyltransferase activity. These results, coupled with the observation in lymphomas of high basal levels of transcription from MMTV proviruses with deleted LTRs, suggested that these proviruses lack negative regulatory elements in their LTRs. Loss of the negative regulatory element may contribute to the selective propagation of proviruses with deleted LTRs.

Identification of a unique mouse mammary tumor virus in the BALB/cNIV mouse strain.

We examined the genetic structure, in terms of restriction endonuclease recognition sites, of the milk-transmitted, low-oncogenic mouse mammary tumor virus (MuMTV) of the BALB/cNIV mouse strain. An analysis with EcoRI documented the presence of acquired cNIV proviruses in the mammary tumor DNAs of BALB/cNIV animals. A comparison of tumor DNAs digested with PstI showed that both the cNIV MuMTV and C3Hf MuMTV proviruses lacked the 4.3- and 1.1-kilobase pair fragments characteristic of C3H MuMTV patterns. An examination of mammary tumor and normal, nonmammary tissue DNAs with BamHI supported the idea that the cNIV MuMTV is identical to the C3Hf MuMTV and demonstrated that these two low-oncogenic proviruses are identical to the high-oncogenic C3H MuMTV provirus with respect to a pair of BamHI sites which define a 1.3-kilobase pair fragment. For each of the three MuMTV strains, we also mapped DNAs generated in isolated virions by reverse transcription of their genomic RNAs. Our results showed that cNIV and C3Hf MuMTV are distinct entities by virtue of an additional PstI site within the cNIV long terminal repeat sequence. Another unique feature of cNIV MuMTV revealed by the analysis of virion-generated DNAs was the existence of a family of genomes within the cNIV population. We concluded that cNIV is distinct from its presumptive C3Hf MuMTV predecessor.

Processing and amino acid sequence analysis of the mouse mammary tumor virus env gene product

The envelope proteins of mouse mammary tumor virus (MMTV) are synthesized from a subgenomic 24S mRNA as a 75,000-dalton glycosylated precursor polyprotein which is eventually processed to the mature glycoproteins gp52 and gp36. In vivo synthesis of this env precursor in the presence of the core glycosylation inhibitor tunicamycin yielded a precursor of approximately 61,000 daltons (P61env). However, a 67,000-dalton protein (P67env) was obtained from cell-free translation with the MMTV 24S mRNA as the template. To determine whether the portion of the protein cleaved from P67env to give P61env was removed from the NH2-terminal end of P67env and as such would represent a leader sequence, the NH2-terminal amino acid sequence of the terminal peptide gp52 was determined. Glutamic acid, and not methionine, was found to be the amino-terminal residue of gp52, indicating that the cleaved portion was derived from the NH2-terminal end of P67env. The NH2-terminal amino acid sequences of gp52's from endogenous and exogenous C3H MMTVs were determined though 46 residues and found to be identical. However, amino acid composition and type-specific gp52 radioimmunoassays from MMTVs grown in heterologous cells indicated primary structure differences between gp52's of the two viruses. The nucleic acid sequence of cloned MMTV DNA fragments (J. Majors and H. E. Varmus, personal communication) in conjunction with the NH2-terminal sequence of gp52 allowed localization of the env gene in the MMTV genome. Nucleotides coding for the NH2 terminus of gp52 begin approximately 0.8 kilobase to the 3' side of the single EcoRI cleavage site. Localization of the env gene at that point agrees with the proposed gene order -gag-pol-env- and also allows sufficient coding potential for the glycoprotein precursor without extending into the long terminal repeat.

Topographical analysis of viral epitopes using monoclonal antibodies: Mechanism of virus neutralization

The mouse mammary tumor virus (MMTV) was used in neutralization studies with monoclonal antibodies (monoclones) to the major external glycoprotein gp52. Monoclones to MMTV gp52 showed that epitopes with different antigenic specificities were targets for virus neutralization. These epitopes were group specific (common to C3H, GR, RIII, and C3Hf MMTVs), class specific (common to C3H and GR MMTVs), and type specific (unique to C3H MMTV). Competition antibody binding assays using a radioiodinated C3H MMTV type-specific neutralizing monoclone revealed that all the monoclones that neutralized virus infectivity also blocked the binding of the type-specific monoclone to C3H MMTV. A topographically distinct site from this determinant was found using other monoclones which did not compete for the binding of the type-specific monoclone and their epitopes did not function as targets for neutralizing antibodies. However, these nonneutralizing monoclones could neutralize virus infectivity in the presence of antimouse IgG sera. Therefore, two topographically distinct sites could be identified on a single protein of the virus. One site functioned as a target for neutralization and the second site bound antibody but did not affect neutralization. The observation that virus bound to cells was no longer susceptible to neutralization by monoclones showed that neutralization affected virus prior to its binding to cell surface receptors. This result further indicated that antibody prevented virus adsorption to cells and that neutralization was mediated by steric hindrance from antibodies binding to epitopes adjacent to those determinants which functioned in virus binding to cell surface receptors.

Isolation and characterization of a new mouse mammary tumor virus from BALB/c mice

A novel mouse mammary tumor virus (MMTV) has been isolated from mice of a subline of the BALB/cCrl Med mouse strain designated BALB/cV. Whereas breeding females of the parent BALB/cCrl Med colony have a mammary tumor incidence of 1%, 47% of the breeding females of the BALB/cV subline develop mammary tumors before 10 months of age. Foster nursing experiments demonstrated this virus, termed MMTV (BALB/c), was transmitted only by milk. The novel MMTV isolate was shown to be immunologically related to, but distinct, from the MMTV variants of C3H, GR, and RIII mice by a series of competition radioimmunoassays for the MMTV 28,000-dalton major core protein (p28), and the 52,000 (gp52)- and 36,000-dalton (gp36) major envelope glycoproteins. Monoclonal antibodies directed against MMTV gp36 were also used to clearly distinguish MMTV(BALB/c) from MMTV(C3H), MMTV(RIII), MMTV(GR), MMTV(C3HfC57BL), and MMTV(A). MMTV-specific proviral DNA content of mammary adenocarcinomas arising in the BALB/cV subline was examined with restriction endonucleases and the Southern blot technique, and compared to the MMTV proviral DNA content of BALB/cAnDe mammary tumors. The virus arising from these latter tumors has been termed MMTV(O). Analysis of EcoRI digests of high-molecular-weight DNA from both types of mammary tumors demonstrated additional MMTV-related proviral sequences when compared to the DNA of normal BALB/c tissues. The patterns generated with the restriction endonuclease SacI distinguished the additional MMTV-specific proviral information in the mammary tumors of the BALB/cV mice from the proviral information in tissues containing the GR, C3H, or RIII MMTVs, as well as from the proviral information in the BALB/cAnDe mammary tumors. These immunological and molecular studies thus define MMTV(BALB/c) as a novel MMTV variant.

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.

Common surface receptors on both mouse and rat cells distinguish different classes of mouse mammary tumor viruses

Cell surface receptors which bind mouse mammary tumor virus (MMTV) were detected on mouse and rat cells. Virus binding was quantitated by measuring 125T-protein A binding to immune complexes composed of a C3H MMTV gp52 type-specific monoclonal antibody and receptor-bound MMTV. C3H MMTV binding to normal mouse mammary epithelial cells (NMuMG) was dose dependent and was ≥50% inhibited by GR MMTV, but not by endogenous C3Hf MMTV or Gross murine leukemia virus. These results were confirmed in [ 3H]leucine MMTV binding inhibition assays in which GR MMTV and C3H MMTV blocked C3H [ 3H]MMTV binding while C3Hf MMTV did not block. The affinities of C3H [ 3H]MMTV binding to receptors on NMuMG, NIH Swiss (SLP), and Fischer rat embryo (FRE) cells were identical by Scatchard analysis. C3Hf [ 3H]MMTV also bound these cells, but with an affinity approximately 10 times weaker than the C3H [ 3H]MMTV binding. Interference assays using a Kirsten sarcoma virus (C3H MMTV) pseudotype confirmed the importance of C3H MMTV specific binding to viral penetration and expression. Pretreatment of SLP or FRE cells with 100 μg of C3H MMTV or GR MMTV inhibited focus formation by ≥50% while C3Hf MMTV and RIII MMTV did not inhibit. Therefore, the functional C3H MMTV cell receptors on mouse and rat cells were related and were able to distinguish C3H MMTV and GR MMTV from C3Hf MMTV and RIII MMTV. These comparable receptors may represent evolutionarily conserved surface components of murine cells.

C3H/HeN mammary tumor-bearing mice develop type-specific neutralizing antibodies and group-specific precipitating antibodies for the mouse mammary tumor virus.

The development of mouse mammary tumor virus (MMTV)-neutralizing antibodies in various strains of mice was measured by their ability to neutralize the focus-forming capacity of a Kirsten sarcoma virus (C3H MMTV) pseudotype containing the MMTV envelope glycoprotein gp52. C3H/HeN, but not GR/N and RIII, mammary tumor-bearing mice were found to develop neutralizing antibodies to this pseudotype. In addition, non-tumor-bearing C3H/HeN, GR/N, RIII, NIH Swiss, C57BL/6, and BALB/c mice and 13 feral mice were also negative for neutralizing antibodies. The neutralization was immunoglobulin G mediated, and the antibodies of C3H/HeN mammary tumor-bearing mice were type specific and capable of distinguishing C3H and GR/N MMTVs from RIII and C3H/HeNf MMTVs. Precipitating antibodies were detected in sera from RIII and GR/N tumor-bearing mice, GR/N non-tumor-bearing mice, and six of the feral mice, although these same sera did not neutralize the Kirsten sarcoma virus (C3H MMTV) pseudotype. The results of this study and of a previous study demonstrate that C3H/HeN mammary tumor-bearing mice develop three functionally distinct antibody populations: (i) group-specific virus-precipitating antibodies; (ii) type-specific virus-neutralizing antibodies; and (iii) type-specific cytotoxic antibodies.