Mouse Mammary Tumor Virus Proteins Research Articles

Seroprevalence of Human Betaretrovirus Surface Protein Antibodies in Patients with Breast Cancer and Liver Disease.

Mouse mammary tumor virus (MMTV) is a betaretrovirus that plays a causal role in the development of breast cancer and lymphoma in mice. Closely related sequences that share 91–99% nucleotide identity with MMTV have been repeatedly found in humans with neoplastic and inflammatory diseases. Evidence for infection with a betaretrovirus has been found in patients with breast cancer and primary biliary cholangitis and referred to as the human mammary tumor virus and the human betaretrovirus (HBRV), respectively. Using the gold standard technique of demonstrating retroviral infection, HBRV proviral integrations have been detected in cholangiocytes, lymph nodes, and liver of patients with primary biliary cholangitis. However, the scientific biomedical community has not embraced the hypothesis that MMTV like betaretroviruses may infect humans because reports of viral detection have been inconsistent and robust diagnostic assays are lacking. Specifically, prior serological assays using MMTV proteins have produced divergent results in human disease. Accordingly, a partial HBRV surface (Su) construct was transfected into HEK293 to create an ELISA. The secreted HBRV gp52 Su protein was then used to screen for serological responses in patients with breast cancer and liver disease. A greater proportion of breast cancer patients (n = 98) were found to have serological reactivity to HBRV Su as compared to age- and sex-matched control subjects (10.2% versus 2.0%, P=0.017, OR = 5.6 [1.25–26.3]). Similarly, the frequency of HBRV Su reactivity was higher in patients with primary biliary cholangitis (n = 156) as compared to blood donors (11.5% vs. 3.1%, P=0.0024, OR = 4.09 [1.66–10.1]). While the sensitivity of the HBRV Su ELISA was limited, the assay was highly specific for serologic detection in patients with breast cancer or primary biliary cholangitis, respectively (98.0% [93.1%–99.7%] and 97.0% [93.4%–98.6%]). Additional assays will be required to link immune response to betaretrovirus infection and either breast cancer or primary biliary cholangitis.

Association of Mouse Mammary Tumor Virus With Human Breast Cancer: Histology, Immunohistochemistry and Polymerase Chain Reaction Analyses.

The purpose of this study is to determine whether mouse mammary tumor virus (MMTV)-associated human breast cancer has the same or similar histology to MMTV-associated mouse mammary tumors. Such associations may indicate a role for MMTV in human breast cancer. Immunohistochemical techniques (using antibodies directed against the signal peptide p14 of the envelope precursor protein of MMTV) and polymerase chain reaction (PCR) analyses were used to identify MMTV proteins and MMTV-like envelope gene sequences in a series of breast cancers from Australian women. The histological characteristics of these human breast cancer specimens were compared with MMTV positive mouse mammary tumors. The same methods were used to study benign breast tissues which 1-11 years later developed into breast cancer. MMTV p14 proteins were identified in 27 (54%) of 50 human breast cancers. MMTV env gene sequences were identified by PCR in 12 (27%) of 45 human breast cancers. There was a significant correlation between the presence of MMTV (identified by p14 immunohistochemistry) in human breast cancers and histological characteristics similar to MMTV positive mouse mammary tumors (p = 0.001). There was a non-significant correlation between the presence of MMTV env gene sequences (identified by PCR) in human breast cancers and histological characteristics similar to MMTV positive mouse mammary tumors (p = 0.290). MMTV p14 proteins were identified in 7 (54%) of 13 benign breast specimens that later developed into human breast cancers. MMTV by PCR was identified in two benign specimens one of whom later developed MMTV positive breast cancer. These observations offer evidence that MMTV may be associated with characteristic human breast cancer histology. p14-based immunohistochemistry appears to be a more reliable technique than PCR for the identification of MMTV in human breast cancer. Identification of MMTV-associated p14 proteins in benign breast tissues confirms prior PCR-based studies that MMTV infection occurs before the development of MMTV positive breast cancer. Many MMTV positive human breast cancers have similar histology to MMTV positive mouse mammary tumors. MMTV infection identified in benign breast tissues precedes development of MMTV positive human breast cancer. When considered in the context of prior studies, these observations indicate a likely role for MMTV in human breast cancer.

Mouse mammary tumor virus in anti-mitochondrial antibody producing mouse models

A human betaretrovirus resembling the mouse mammary tumor virus (MMTV) has been characterized in primary biliary cirrhosis (PBC) and associated with aberrant pyruvate dehydrogenase complex (PDC)-E2-like expression. As MMTV is prevalent in mice as either an exogenous or endogenous infection, we tested the hypothesis that MMTV is linked with anti-mitochondrial antibody (AMA) production in models with severe immune dysfunction. Evidence for MMTV was assessed in the liver and spleen of mice by PCR and immunochemistry and PDC-E2-like protein by immunochemistry. ELISA and Western blot were used to investigate AMA and anti-MMTV antibody production. Increased MMTV gag or env expression was detected in the livers of AMA producing mice including NOD.c3c4, CD4 directed dominant negative TGF-β receptor II and IL-2 receptor α knockout mice as well as the NOD parental strain when compared to healthy strains and biliary disease control mice. The NOD.c3c4 mice expressed MMTV surface and capsid proteins and aberrant PDC-E2-like protein in the bile ducts, whereas IL-2 receptor α knockout mice, NOD.c3c4 and the NOD mice expressed MMTV proteins and aberrant PDC-E2-like protein in the spleen. A significant correlation between anti-MMTV antibody production and AMA production was observed in the sera of NOD and NOD.c3c4 mice (p<0.0001). The association of betaretroviral protein production and aberrant PDC-E2-like protein expression in the NOD.c3c4, NOD, and the IL-2 receptor α knockout mice is comparable to observations in patients with PBC. The correlation of AMA and anti-MMTV suggests the hypothesis that MMTV infection may trigger the production of AMA.

Detection of human mammary tumor virus proteins in human breast cancer cells

Mouse mammary tumor virus (MMTV) has been proven to induce mammary cancer in mice. MMTV-like env gene sequences have been detected in one-third of the human breast tumors studied. The whole proviral structure with 95% homology to MMTV was found in two human breast tumors and was designated as human mammary tumor virus (HMTV). HMTV viral particles with betaretroviral features have been isolated. In addition, a retrovirus called human betaretrovirus (HBRV), homologous to the mentioned retroviruses, has been isolated from tissues of patients with primary biliary cirrhosis. In this report, the expression of HMTV envelope (Env) and capsid (Ca) was detected in 10 primary cultures of human breast cancer containing HMTV sequences (MSSM) by Western blot and fluorescence activated cell sorting (FACS), using a panel of antibodies against HMTV Env, HBRV Env and Ca and the MMTV Env Gp36 and Ca P27 proteins. By contrast, HMTV proteins did not react with antibody against the MMTV Env Gp52 protein. All the antibodies detected MMTV proteins with exception of two out of four monoclonal antibodies against HMTV Env. Approximately 13% of the MSSM cells showed HMTV protein expression by FACS analysis. This report shows the expression of HMTV proteins for the first time in human breast cancer cells using a panel of antibodies against HMTV, HBRV and MMTV proteins. This should be taken into consideration when MMTV antibodies are used to detect HMTV proteins in human tissues.

Stimulation of basal transcription from the mouse mammary tumor virus promoter by Oct proteins.

The steroid hormone-inducible promoter of mouse mammary tumor virus (MMTV) contains three overlapping sequences related to the consensus octamer motif ATGCAAAT. Basal promoter activity in the absence of hormone induction from a template in which all three octamer elements were mutated was decreased by two-to threefold in in vitro transcription assays. Oct-1 protein purified from HeLa cell nuclear extracts, as well as recombinant Oct-1 expressed in bacteria, recognized MMTV octamer-related sequences, as shown by DNase I footprinting. Furthermore, rabbit polyclonal antiserum directed against recombinant Oct-1 completely inhibited the formation of specific complexes between MMTV octamer-related sequences and proteins present in nuclear extracts of HeLa cells, indicating that Oct-1 is the major protein in HeLa nuclear extracts that recognizes octamer-related sequences in the MMTV promoter. In addition, depletion of Oct-1 from the nuclear extract by using Oct-1-specific antiserum or a sequence-specific DNA affinity resin decreased in vitro transcription from the wild-type MMTV promoter to a level identical to that obtained from a promoter in which all three octamer-related sequences were mutated. Addition of purified HeLa Oct-1 or recombinant Oct-1 to the depleted extract selectively increased transcription from the wild-type relative to the mutated promoter, demonstrating that Oct-1 transcription factor stimulates basal transcription from the MMTV promoter. A similar effect was observed when purified recombinant Oct-2 was added to the Oct-1-depleted extract, suggesting that Oct-2 may play an important role in MMTV transcription in B cells.

Glucocorticoid-regulated trafficking of mouse mammary tumor virus proteins in permeabilized hepatoma cells. Requirements of intracellular membrane transport for maturation of the cytoplasmic phosphorylated polyprotein.

Glucocorticoids coincidentally regulate the localization of mouse mammary tumor virus (MMTV) glycoproteins and maturation of viral phosphoproteins in viral infected rat hepatoma cells. To test for a functional interaction between MMTV transmembrane glycoproteins and cytoplasmic phosphoproteins, the bacterial cytolysin streptolysin-O was utilized to selectively permeabilize the plasma membrane and reconstitute exocytic trafficking. Streptolysin-O-permeabilized M1.54 cells pretreated with glucocorticoids retained the capability for proteolytic processing, cell surface delivery, and externalization of MMTV glycoproteins as determined by immunoprecipitation and immunofluorescence microscopy. The efficient maturation of MMTV phosphoproteins indicated that these viral proteins are properly transported near or to the plasma membrane in permeabilized cells. These maturation events in semi-intact cells were dependent on the addition of cell cytosol and were specifically inhibited by the membrane impermeant GTP analog guanosine 5'3-O-(thio)triphosphate, an agent known to impede vesicular transport of membrane proteins, but which has not previously been shown to alter cytoplasmic protein maturation or transport. The addition of anti-MMTV antibodies directed against the cytoplasmic domain of the glycoprotein precursor to transport competent semi-intact M1.54 cells resulted in the dramatic inhibition of both MMTV glycoprotein and phosphoprotein maturation. These results were not obtained using either preimmune sera or antiserum specific for the luminal portion of the glycoprotein precursor. Our findings suggest that the functional interaction of cytosolic MMTV phosphoproteins with the cytoplasmic domain of the viral membrane glycoprotein is required for the efficient transport and processing of each class of proteins in glucocorticoid-treated cells and provides the first evidence for the involvement of vesicular transport in the delivery and maturation of cytoplasmic viral proteins at the plasma membrane or the pericellular region.

Purification of immature cores of mouse mammary tumor virus and immunolocalization of protein domains.

The immature capsids of the mouse mammary tumor virus (MMTV), known as intracytoplasmic A particles, have been isolated from murine L1210 leukemia cells. The diameter of the isolated particles was 80 nm as determined by negative staining. Two polypeptides of 77 and 110 kDa were found to be their major polypeptide components, in agreement with the expected sizes of the Gag and Gag-Pro precursor polypeptides of the mature MMTV proteins. Both polypeptides were recognized by antibodies directed toward the matrix (p10) and capsid (p27) proteins of MMTV. Immunogold labeling of p10 on isolated A particles, visualized by negative staining, showed that this protein is located at the surface of the immature capsids, whereas p27 can be detected only in broken or disrupted particles, suggesting that it has an internal location. These observations were confirmed by immunolabeling of both proteins on thin sections of A particle-producing cells. In addition, the viral protease had a more internal position than p27. Since the sequential order of the viral proteins in the Gag precursor is p10-pp21-p27-p14 and that in Gag-Pro is p10-pp21-p27-p30-protease, our results demonstrate the radial organization of the polypeptide precursors forming the intracytoplasmic A particles.

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.

Disruptions in intracellular membrane trafficking and structure preclude the glucocorticoid-dependent maturation of mouse mammary tumor virus proteins in rat hepatoma cells.

We have previously shown that glucocorticoids regulate the trafficking and processing of mouse mammary tumor virus (MMTV) proteins in viral-infected M1.54 rat hepatoma cells. To examine the role of intracellular membrane integrity on MMTV protein maturation, brefeldin A (BFA) was utilized to disrupt membrane flow between the endoplasmic reticulum and Golgi. Immunoprecipitation and immunofluorescence microscopy revealed that in the presence of dexamethasone, BFA inhibited the proteolytic processing, cell surface delivery, and externalization of MMTV glycoproteins. Glycosidase digestion and inhibitors of protein glycosylation confirmed that the observed differences in apparent sizes of MMTV glycoprotein products are due to BFA-induced changes in oligosaccharide processing. BFA treatment inhibited the proteolytic processing of the MMTV phosphoprotein precursor, which normally associates with the cytoplasmic face of intracellular membranes. Similarities in salt extraction efficiency revealed that BFA did not affect the membrane affinity of the uncleaved phosphorylated precursor. In a complementary approach, proteolytic processing of the phosphorylated polyprotein did not occur in glucocorticoid-treated HTC cells transfected with a mutant MMTV provirus encoding a normal phosphorylated precursor, but which express a truncated MMTV glycoprotein missing its transmembrane domain and cytoplasmic tail. These results suggest that the MMTV glycoproteins and phosphoproteins may interact at a late step in the transport pathway in a manner required for their mutual processing in response to glucocorticoids and establishes the importance of functional interactions with intracellular membranes for maturation of the cytoplasmic MMTV phosphoproteins.

Two different genes coding for processable and nonprocessable forms of a viral envelope protein can account for the apparent hormonal stimulation of protein processing in W7MG1 lymphoma cells.

In the mouse mammary tumor virus (MMTV)-infected mouse T-lymphoma cell line W7MG1, glucocorticoid hormone regulates two aspects of MMTV gene expression: hormone stimulates MMTV gene transcription and increases the ratio of mature envelope proteins to envelope precursor protein produced. To separate these two effects and determine the mechanism by which hormone regulates the conversion of the envelope precursor Pr74 to the mature cleaved products gp52 and gp33, we constructed expression vectors in which the envelope gene is constitutively transcribed. Surprisingly, the envelope precursor protein Pr74 encoded by two independently isolated, allelic envelope genes behaved differently. Pr74-P (encoded by the ENV/P gene) was processed efficiently to the mature products gp52 and gp33, independently of the level of expression, hormonal induction of cellular genes, or the presence of other MMTV proteins. In contrast, under the same conditions, Pr74-N (encoded by the ENV/N gene) was not processed further despite being relatively stable. In sucrose gradient analyses, Pr74-P sedimented as monomers, whereas Pr74-N was found in high mol wt aggregates of heterogeneous size. Coimmunoprecipitation analysis determined that Pr74-N associated with BiP, whereas Pr74-P did not. This is indicative of improper folding of Pr74-N in the endoplasmic reticulum.(ABSTRACT TRUNCATED AT 250 WORDS)

Influence of mammary cell differentiation on the expression of proteins encoded by endogenous BALB/c mouse mammary tumor virus genes

The interactions between differentiation-associated cellular events in the intact mammary gland or in cultured mammary cells and the post-transcriptional activity of the endogenous mouse mammary tumor virus (MMTV) loci were investigated. The transcriptional activities of the endogenous MMTV proviruses of the BALB/c mouse strain ( Mtv-6, Mtv-8 and Mtv-9) appear to be regulated differentially during pregnancy-induced mammary gland development (J.E. Knepper D. Medina and J.S. Butel J. Virol. 59, 518–521, 1986). Analysis of MMTV-specific proteins at various stages of mammary gland development (virgin, midpregnant, lactating, regressing) established the presence of steady-state levels of a 67,000- M r env precursor-type polypeptide at all physiological stages. However, processing to lower-molecular-weight env-specific proteins, including a predominant 50,000- M r species, was detected only with the transition to the functional mammary gland phenotype. The contributions of cell proliferation, cell-matrix interactions, and modulation of functional activity to the pattern of endogenous MMTV protein expression were investigated using a 3-dimensional collagen type I culture system. Growth and cell-matrix interactions (cell polarization, lumen formation) leading to formation of 3-dimensional duct-like structures were permissive for the synthesis and processing of MMTV-specific proteins; accumulation of high levels of the 50,000- M r env-specific polypeptide was associated with the onset of the fully functional mammary cell phenotype. Expression of MMTV-specific proteins was not due to amplification of a specific cell subpopulation. The potential of the full-length Mtv-8 and Mtv-9 proviruses to be transcribed, as indicated by their methylation status, was not dramatically different between differentiated and undifferentiated mammary cells in culture. This study indicates that MMTV transcriptional activity is reflected at the protein level in mammary tissue of BALB/c mice and that viral protein synthesis and processing may serve as important markers of different physiological stages of mammary epithelial cells. These observations also suggest a general approach to the examination of potential modulatory effects of cellular interactions (cell-cell, cell-matrix or both) known to be important in various differentiated epithelial cell systems for the expression of viral genes.

Glucocorticoid-resistant lymphoma cell variants that contain functional glucocorticoid receptors.

A mouse T-lymphosarcoma cell line stably infected with mouse mammary tumor virus (MMTV) was used as the parent line for a genetic analysis of two glucocorticoid hormone responses, hormone-induced cytolysis and stimulation of viral gene expression. Variants were selected for survival and elevated expression of MMTV proteins in the presence of the steroid. The MMTV marker provided a sensitive test for glucocorticoid receptor (GR) function in the hormone-resistant variants. This strategy resulted in the isolation of two novel types of hormone-resistant variants. One type of variant with only about 25% of the level of GR found in the parent line was resistant to the cytolytic effects of glucocorticoid but produced increased levels of MMTV gene products in response to the hormone. This variant phenotype demonstrated that the MMTV response requires fewer GR than the cytolytic response. Another variant, which required approximately 100-fold higher concentrations of hormone than the wild-type cells for both responses, apparently contained GR with altered hormone-binding properties.

Glucocorticoid-resistant lymphoma cell variants that contain functional glucocorticoid receptors.

A mouse T-lymphosarcoma cell line stably infected with mouse mammary tumor virus (MMTV) was used as the parent line for a genetic analysis of two glucocorticoid hormone responses, hormone-induced cytolysis and stimulation of viral gene expression. Variants were selected for survival and elevated expression of MMTV proteins in the presence of the steroid. The MMTV marker provided a sensitive test for glucocorticoid receptor (GR) function in the hormone-resistant variants. This strategy resulted in the isolation of two novel types of hormone-resistant variants. One type of variant with only about 25% of the level of GR found in the parent line was resistant to the cytolytic effects of glucocorticoid but produced increased levels of MMTV gene products in response to the hormone. This variant phenotype demonstrated that the MMTV response requires fewer GR than the cytolytic response. Another variant, which required approximately 100-fold higher concentrations of hormone than the wild-type cells for both responses, apparently contained GR with altered hormone-binding properties.

Characterization of mouse mammary tumor virus gag-pro gene products and the ribosomal frameshift site by protein sequencing.

The synthesis of retroviral polyproteins that are the translational products of the genome-size mRNA is initiated in the upstream gag gene. The synthesis of the products of the protease gene (pro) and polymerase gene (pol) is regulated by translational suppression (in-frame read-through or frameshift) of termination codons as a strategy developed for controlling the level of replicative enzymes required only in catalytic amounts. In mouse mammary tumor virus (MMTV), three overlapping reading frames are utilized for the synthesis of gag-encoded Pr77, gag-pro-encoded Pr110, and gag-pro-pol-encoded Pr160 polyproteins. To characterize pro gene products and to determine the site of frameshift required for the synthesis of Pr110, we purified and sequenced three MMTV proteins: p14, p30, and p13. Sequence analysis showed that p14 is the basic nucleic acid-binding protein derived entirely from gag, and p13 is a product of the pro gene and has characteristic sequences of protease. A comparison of the amino acid sequences of p30 with the corresponding nucleotide sequence of proviral DNA allowed the delineation of the frameshift site utilized in vivo for the synthesis of the gag-pro-encoded fusion polyprotein Pr110. The results showed that: (i) the N-terminal 94 residues of p30 are translated from the gag frame; (ii) residue leucine-95 is specified by either the last UUG codon of gag or the overlapping CUU codon in the pro frame; and (iii) the elongation of the peptide chain from residue 96 continued to be encoded in the pro frame to the pro terminator. The possible mechanisms of frameshift and of the tRNAs involved are discussed.

Chromatographic purification and immunological analysis of viral polypeptides of mouse mammary tumor virus

The seven mouse mammary tumor virus (MMTV) structural polypeptides (GP52, GP36, P28, P23, P14, P12, and P10) were purified to apparent homogeneity. An improved and reproducible Chromatographic method was used to obtain high yields of viral proteins from the same batch of isopycnically purified MMTV. The viral structural proteins were first extracted repeatedly with a high salt and high pH buffer in the presence of 1% Triton X-100. Loss of the minor viral proteins during column purification was minimized by first purifying the smaller molecular weight viral proteins (P14, P12 and P10) by oligo-d(T) column chromatography from the dissociated virions. The other four major viral polypeptides (GP52, GP36, P28 and P23) were then fractionated by affinity and ion-exchange columns. High titer polyclonal antibodies were produced against all of the seven structural proteins except P12 and P10. These antisera were monospecific and showed no cross-reactivity in radioimmunoassay towards other MMTV proteins. With minor modifications, this method has also been applied to purify other structural proteins from several different C-type retroviruses.

Mammary Cancer Stages in BALB/cV Mice: Mouse Mammary Tumor Virus Expression and Virus-Host Interactions&lt;xref ref-type="fn" rid="FN2"&gt;2&lt;/xref&gt;

A unique subline of BALB/c mice, designated "BALB/cV," exhibits an intermediate mammary tumor incidence (47%) and harbors a distinct milk-transmitted mouse mammary tumor virus (MMTV). Virus expression and virus-host interactions were examined during the different stages of mammary tumorigenesis (normal, preneoplastic, and neoplastic) in the BALB/cV system. Protein immunoblot analyses established the presence of correctly processed (BALB/cV)MMTV structural proteins in all types of BALB/cV mammary tissues. Competition enzyme-linked immunosorbent assays demonstrated that cells from each biologic phenotype were capable of supporting high levels of (BALB/cV)MMTV protein expression. However, mammary epithelial cells that spontaneously underwent the inappropriate pathway of squamous metaplasia did not contain detectable levels of (BALB/cV)MMTV structural proteins. Iodination experiments revealed the presence of a 68K env-related protein on the surface of BALB/cV mammary cells. Nevertheless, sera from 40 mice bearing BALB/cV-positive mammary tissues did not contain detectable levels of anti-env antibodies. Metabolic labeling experiments showed that the half-life of transformation-related, host cell protein p53 (approximately equal to 60 min) in the distinct BALB/cV mammary cell populations was similar to that reported for normal mouse 3T3 cells. It appears that p53 is not stabilized by protein interactions involving any MMTV-encoded or MMTV-induced protein in mammary tumor cells. These characteristics of the BALB/cV system are compatible with the hypothesis that MMTV is only one of two or more cooperating factors required to mediate complete mammary transformation.

Glucocorticoid-dependent maturation of viral proteins in mouse lymphoma cells: isolation of defective and hormone-independent cell variants.

Maturation of mouse mammary tumor virus proteins is dependent on glucocorticoid hormones in W7MG1, a stably infected mouse T-lymphosarcoma cell line derived from WEH17. We used an immunological procedure to select variant cell lines with altered levels of viral glycoproteins on the cell surface. One variant, W7M329a, expressed lower-than-normal levels of the major viral glycoprotein, gp52env, on the cell surface before and after exposure to hormone. Two other variants, W7M302b and W7M326.4, expressed elevated levels of gp52env on the cell surface even in the absence of hormone. Analysis of the levels and/or rates of synthesis of viral RNA and glycoproteins before and after hormone treatment indicated that the variant phenotypes resulted from changes in posttranslational steps of viral gene expression. The hormone-independent maturation of MMTV proteins is a novel variant phenotype that has not previously been reported.

Antibodies interacting with mouse mammary tumor virus proteins in sera of healthy women

Antibodies interacting with mouse mammary tumor virus proteins in sera of healthy women

Highly sensitive immunoadsorption procedure for detection of low-abundance proteins

A procedure that virtually eliminates nonspecific adsorption of radiolabeled proteins during immunoprecipitation was devised utilizing staphylococcal cells containing protein A (Staph A). Immunoprecipitates (antigen-antibody complexes) were solubilized from Staph A pellets into detergent micelles by incubation in a small volume of 1% sodium dodecyl sulfate (SDS) at 23°C for 10 min. To allow re-formation of immunocomplexes and rebinding to new Staph A, the SDS-solubilized material was diluted 20-fold in buffer containing 1% Triton X-100 and 0.5% sodium deoxycholate. Specific conductance measurements revealed that this solubilization and subsequent reimmunoadsorption of antibody-antigen complexes occur at SDS concentrations that are first above and then below its critical micelle concentration. This procedure lowered the nonspecific background from approximately 2250 parts per million (ppm) to less than 25 ppm with a final recovery of 30–50% depending on the antigen and antibody. Chaotropic agents such as 2 m urea, 0.2 m KOH, and 3.5 m MgCl 2 (as well as combinations of urea and SDS) can substitute for 1% SDS, although the final recovery is somewhat lower. Fluorography of radiolabeled proteins obtained in this manner displays virtually undetectable background even for exposures as long as 2 months. These methods allowed the unambiguous detection of low-abundance antigens at a high level of sensitivity, for example, mouse mammary tumor virus protein products and epidermal growth factor receptor.

Identification and characterization of a mouse mammary tumor virus protein uniquely expressed on the surface of BALB/cV mammary tumor cells.

A unique subline of BALB/c mice, designated BALB/cV, exhibits an intermediate mammary tumor incidence (47%) and harbors a distinct milk-transmitted mouse mammary tumor virus (MMTV). The BALB/cV subline was used to study the molecular basis of potential virus-host interactions involving cell surface-expressed MMTV proteins. Cell surface iodination identified virus-specific proteins expressed on BALB/cv primary mammary tumor cells grown in culture. In contrast to (C3H)MMTV-producing cell lines which expressed MMTV gp52, BALB/cV tumor cells lacked gp52 and expressed instead a 68K, env-related protein. The 68Kenv protein was also detected on the surface of metabolically labeled BALB/cV tumor cells by an external immunoprecipitation technique. The expression of 68Kenv was restricted to mammary tissues of BALB/cV mice that also expressed other MMTV proteins. Biochemical analysis established that 68Kenv was not modified by N-linked glycosylation. 125I-labeled 68Kenv was rapidly released into the media of tumor cell cultures and was recovered both in the form of a soluble protein and in a 100,000 g pellet. The biologic function of this cell surface-expressed viral protein remains unknown.