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.     

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.     

Purification and properties of murine mammary tumor virus DNA polymerase.

The surface tubule protein p58 was found to be a useful marker to follow vaccinia membrane biogenesis as it is related to virus assembly. Synthesis of p58 does not occur in the absence of DNA replication and is maximal between 8–12 hr postinfection. During normal vaccinia development, p58 as well as other proteins located at or near the surface is among the last components to be integrated into developing particles. Although envelopes can be assembled around immature particles in the absence of DNA and p58 synthesis, combined biochemical and electron microscopic autoradiography studies reveal that such envelopes are either not utilized at all or only inefficiently in the formation of mature virions, once DNA replication has been restored. These observations imply that vaccinia assembly is a tightly coupled process in which internal components are integrated before the completion of the envelope.     

Mammary tumors from BALB/c mice with a reported high mammary tumor incidence have acquired new mammary tumor virus DNA sequences

Various laboratories have reported differences in the mammary tumor incidence caused by the endogenous mouse mammary tumor virus (MMTV) in BALB/c mice. In order to resolve these differences, we have compared the MMTV specific nucleic acids extracted from BALB/c normal organs and tumor tissue obtained from laboratories reporting either a high or low BALB/c mammary tumor incidence. Hybridization kinetics and restriction endonuclease analysis indicate that mammary tumor tissue from laboratories reporting a high mammary tumor incidence contains integrated MMTV-specific DNA that is not found in normal organs from these mice and is therefore not in the germ line. Furthermore we cannot detect these acquired MMTV DNA sequences in a BALB/c mammary tumor from a laboratory reporting a low mammary tumor incidence.     

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.     

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.     

Production of mouse mammary tumor virus upon transfection of a recombinant proviral DNA into cultured cells

We have investigated the intracellular proteins synthesized in rat XC and feline kidney cells transfected with endogenous mouse mammary tumor virus (MMTV) proviral DNA. The endogenous provirus GR40, associated with the Mtv-8 locus, directs the synthesis of gag proteins indistinguishable from those found in MMTV-infected cells. The env precursor Pr73env and the mature gp52 proteins could not be detected in these cells. Instead an env-related protein of 68K is synthesized. In contrast to this endogenous provirus, a cloned exogenous proviral variant directs the synthesis of apparently normal env proteins upon transfection into the same cell lines. These results suggest that the env gene of the endogenous MMTV provirus GR40 is defective. The exogenous proviral variant is not expected to synthesize virus particles since it carries a rearrangement in the gag gene. In order to obtain an MMTV provirus capable of correctly expressing both gag and env functions, we have constructed a hybrid endogenous-exogenous provirus containing the 5' long terminal repeat (LTR)-gag of GR40 and the pol-env-3' LTR of the exogenous provirus. Upon transfection into feline kidney cells, this hybrid provirus directed the synthesis of apparently authentic gag and env proteins. Further, virus particles can be detected in the culture medium of the transfected cells by electron microscopy. Viral proteins obtained from viral particles banded in a sucrose gradient were detected by immunoprecipitation.     

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.     

In vitro transformation with avian myelocytomatosis virus strain CMII: characterization of the virus and its target cells

Avian myelocytomatosis virus strain CMII induced an in vitro transformation in cells from various hematopoietic tissues and could be quantitated by focus and soft agar colony assay techniques. The CMII-transformed bone marrow cells had a high proliferative capacity in comparison to uninfected controls. The cells closely resembled hematopoietic cells transformed by strain MC29 myelocytomatosis virus, but differed from avian myeloblastosis virus (AMV)-transformed cells. They were phagocytic, became adherent under certain conditions of culturing, and required colony-stimulating factor for colony formation in semisolid medium. These properties are characteristic for cells of the granulocyte/macrophage lineage of differentiation. In contrast to avian erythroblastosis virus, CMII effectively transformed macrophage cultures suggesting that the target cell belongs to the corresponding differentiation lineage. That it is not identical to normal granulocyte/macrophage colony-forming cells was demonstrated by cell separation experiments. In addition to hematopoietic cells, CMII induced a morphological transformation in chicken fibroblasts. CMII was found to consist of a mixture of a transforming component and an associated nontransforming virus of subgroup B or D. The transforming component is defective for replication and could be complemented by standard helper viruses of subgroups B, C, and D.     

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.     

Endogenous mouse type-C RNA virus of SWR cells: Inducibility locus containing structural information for a new endogenous virus class

A type-C RNA virus with a host range preference for BALB/c mouse embryo cells is chemically inducible from embryo cells of the inbred SWR strain. The SWR virus is shown to be readily distinguishable from previously identified endogenous viruses of mouse cells by nucleotide sequence homology analysis. A single dominant gene for SWR virus inducibility is shown to segregate in crosses involving the inducible SWR and non-virus-inducible NIH Swiss strains. Moreover, nucleotide sequences specific for the SWR virus are demonstrated to be present only in cellular DNAs of SWR virus-inducible embryo lines. These findings establish that the SWR inducibility locus contains structural information of this virus.     

Endogenous mouse mammary tumor virus DNA is distributed among multiple mouse chromosomes.

We have examined the distribution of endogenous mouse mammary tumor virus-specific DNA in the genome of A/HeJ mice by using molecular hybridization and restriction endonucleases to analyze DNA from mouse-hamster hybrid clones that segregate mouse chromosomes. We have found that MMTV sequences are located on at least three separate chromosomal pairs, including chromosome number four.     

Expression of type C virus p30 in mouse cells infected with herpes simplex virus.

Expression of type C virus p30 in BALB/c and NIH Swiss mouse cells infected with live (productive infection) or uv-irradiated herpes simplex virus (uv-HSV) types 1 and 2 was studied by use of either immunofluorescent (FA) or radioimmunoassay (RIA) procedures. No evidence for enhanced expression of p30 was obtained with either of these procedures although activation of type C virus in uv-HSV-infected BALB/c cells was readily demonstrated at low frequencies (～10^?4) by infectious center assay. While FA staining with rabbit anti-murine leukemia virus (MuLV) p30 serum was observed in mouse cells productively infected with HSV or infected with uv-HSV, this was shown to be nonspecific. It was concluded that activation of type C virus in uv-HSV-infected BALB/c cells does not occur in significantly more cells than detected by infectious center assay.     

Identification of the target cells in human B lymphocytes for transformation by Epstein-Barr virus.

Lymphocytes from human umbilical cord blood were purified and then separated into rosette-forming cells (T cells) and non-rosette-forming cells (non-T cells). Non-rosette-forming cells were further divided by the technique of rosette formation, after neuraminidase treatment, into surface immunoglobulin (SIg)-carrying cells (B cells) and cells lacking SIg but carrying Fc receptors (null cells). The three cell subsets, T, B, and null cells, were examined for susceptibility to transformation by the B95-8 strain of Epstein-Barr virus (EBV) using the criteria of colony formation by transformed cells and/or transformation efficiency as judged by the days required for the first appearance of transformation. Not only the T cells but also the null cells were unsusceptible to transformation by EBV. In contrast, B cells were highly susceptible. In a study of the quantitative relationship between the target cells for viral transformation and those B cells which possessed SIg after an acid pH treatment, 10 lymphocyte preparations from three cord blood samples and seven adult peripheral blood samples were tested individually. The fraction size of transformable cells was determined by the growth-curve procedure for transformed cells while the procedure of direct membrane immunofluorescence with anti-IgM (μ-specific) serum and polyvalent anti-Ig serum was used to determine the fractions of SIg and SIg(M) cells. The actual fraction of EBV target cells was nearly equal to that of SIg(M) B cells but not to that of the total SIg B cells. Thirty-four lymphocyte preparations from eight cord and 26 adult peripheral blood samples were examined for the percentage of SIg B cells and for susceptibility to EBV transformation as assayed by the colony-formation procedure. EBV susceptibility and the fraction of SIg(M) B cells, but not of total SIg B cells, correlated nicely, suggesting that SIg(M)-bearing cells were probably the major target among the B lymphocytes for transformation by EBV.     

Studies on the role of M protein in virus assembly using a ts mutant of HVJ (Sendai virus).

A temperature-sensitive mutant of HVJ, HVJ cl.151, was isolated from BHK cells persistently infected with HVJ and characterized. HVJ c1.151 virion had an M polypeptide different in apparent molecular weight from that of HVJ wild-type, that is 36,000 and 34,000 daltons, respectively. HVJ c1.151 was blocked in a late function required for virus maturation. M protein antigen of HVJ c1.151 was detected in infected cells by immunofluorescent microscopy only at permissive temperature but not at nonpermissive temperature, although GP and NP antigens were detected at both temperatures. Further, analysis of the infected cells by SDS-polyacrylamide gel electrophoresis showed that viral structural polypeptides P, F₀, NP, and F and nonstructural polypeptide C were synthesized in infected cells at nonpermissive temperature and these structural polypeptides were incorporated into virions upon temperature shiftdown, whereas polypeptides HN and M, which may be synthesized at nonpermissive temperature, were not able to be incorporated into virions upon temperature shift down. Thus, the temperature-sensitive lesion of HVJ c1.151 is considered to be in HN and M proteins. Membrane fluorescense, immunoferritin electron microscopy, and SDS-polyacrylamide gel electrophoresis of plasma membranes showed that migration of F₀, and F to the cell surface occurred normally even at nonpermissive temperature. Immunoferritin electron microscopy also demonstrated that the viral glycoproteins which arrived at the plasma membrane were dispersed on the entire surface of the membrane at the nonpermissive temperature and that viral components synthesized at this temperature could not assemble at the plasma membrane. In addition, it was found that antibody-induced redistribution of viral glycoproteins on the surface of cells infected with HVJ c1.151 and incubated at nonpermissive temperature occurred very rapidly; in contrast, such redistribution of viral glycoproteins occurred more slowly and less completely in cells incubated at permissive temperature, suggesting that viral glycoproteins on the plasma membrane of cells at nonpermissive temperature have a high degree of mobility in the plane of the membrane as compared with those on the plasma membrane of cells at permissive temperature. These results suggest strongly that a function which fixes the viral glycoproteins at restricted areas of plasma membrane to form a viral envelope is blocked in HVJ c1.151-infected cells at nonpermissive temperature. Analysis of plasma membrane by SDS-polyacrylamide gel electrophoresis showed that viral glycopolypeptides but no NP were present on membranes isolated from HVJ cl.151-infected cells at nonpermissive temperature in spite of the presence of a large amount of NP in the whole cells, whereas plasma membranes isolated from cells at permissive temperature contained all viral structural polypeptides. The possible roles of M protein of HVJ in formation of the viral envelope and association of nucleocapsid with the envelope during assembly are discussed based on these results.     

Domains of simian virus 40 large T-antigen exposed on the cell surface

The orientation of SV40 large T on the surface of SV40-transformed mouse cells and of human cells infected with nondefective adenovirus 2 SV40 hybrid viruses has been studied. Using antibodies against a synthetic peptide corresponding to a region of 11 amino acids at the carboxyterminus of large T, the surface of formaldehyde-fixed SV40-transformed cells could be specifically stained by indirect immune fluorescence. Staining was inhibited by an excess of the peptide. These data suggest that the carboxyterminus of large T is exposed on the surface of formaldehyde-fixed cells. Antibodies against the carboxyterminus of large T also stained the surface of cells infected with the hybrid viruses Ad2⁺ND1, Ad2⁺ND2, and Ad2⁺ND4. Thus, the carboxytermini of the SV40-specific proteins synthesized in hybrid virus-infected cells are also exposed on the cell surface. When analyzed with an antiserum against purified denatured large T, which among many other determinants also recognizes the large T carboxyterminus, surface fluorescence was observed in cells infected by all three hybridviruses. The surface fluorescence of Ad2⁺ND1-infected cells, expressing an SV40-specific protein of 28 K, and Ad2⁺ND2-infected cells expressing SV40-specific proteins of 42 K and 56 K molecular weight, was completely inhibited by carboxyterminal peptide. However, the surface fluorescence of Ad2⁺ND4-infected cells, expressing SV40-specific proteins up to nearly full size large T, was unaffected by carboxyterminal peptide. Our data suggest that a major portion of large T, located between a region near the carboxyterminus and a region corresponding to the aminoterminus of the 56 K protein, is not exposed on the surface of hybridvirus-infected cells. However, some parts of the aminoterminal one-third of large T appear to be exposed again. We conclude that SV40 large T on the surface of SV40-transformed cells is oriented in a specific manner, suggesting that it is specifically associated with the plasma membrane.     

Structural components of mouse mammary tumor virus. I. Polypeptides of the virion.

Mouse mammary tumor virus (mMTV) obtained from MJY-alpha cell cultures and analyzed by polyacrylamide gel electrophoresis possesses four major polypeptides and six to eight minor components. Three of the major proteins, with estimated molecular weights of 60,000, 52,000 and 37,000, are glycoproteins, as demonstrated by labeling with [³H]glucosamine. Labeling with [³⁵S]sulfate revealed significant amounts of sulfate associated with the 60,000 (gp60) and 52,000 (gp52) glycoproteins, whereas sulfate was minimally incorporated into the 37,000 (gp37) dalton glycoprotein. At least three glycopeptide species containing both [³H]glucosamine and [³⁵S]sulfate labels were obtained after extensive Pronase digestion of mMTV, indicating that [³⁵S]sulfate is covalently linked to the carbohydrate component of mMTV glycoproteins. Variations in the polypeptide pattern of mMTV were observed when virions were grown and labeled under different culture conditions. The amount of gp60 decreased substantially, with an apparent increase in a minor polypeptide at 33,000 daltons, if virions were harvested from stationary cultures or if culture medium was not changed daily during viral harvests. When virions containing gp60 were incubated with medium from stationary cultures or with stationary cell layers, the level of gp60 decreased with a corresponding increase in the amount of radioactivity at 33,000 daltons. Cleavage of gp60 and gp52 was demonstrated by treatment of purified mMTV virions with trypsin or alpha-chymotrypsin (1–150 μg/ml) for 1 min to 3 hr. Virions were morphologically unaltered after incubation with either protease; however, both gp60 and gp52 were absent from the polypeptide patterns. The data suggest that gp52 is cleaved to form 33,000, 22,000, and possibly 37,000 dalton glycoproteins which remain associated with virions. Other mMTV virion polypeptides were resistant to treatment with protease.     

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.     

Self-assembly of eggplant mosaic virus protein.

Purified bottom component of eggplant mosaic virus (EMV) was dissociated in cold 66% acetic acid in the presence of 0.01 M dithiothreitol (DTT). EMV coat protein free from residual RNA could be obtained by dialysis against 0.02 M sodium acetate buffer, 0.001 M DTT, pH 4.0. The aggregation states of this protein were investigated after dialysis against 0.02 M sodium acetate buffers, 0.001 M DTT, pH 4.0 to 5.5. At pH 4.0, the protein reassembled essentially into threads and small aggregates sedimenting at about 8–9 S. Increasing numbers of isometric shells formed with increasing pH. At pH 5.5, they represented the totality of soluble material and sedimented at about 50 S. Once formed, these shells were stable at pH 7.0, unlike the aggregates formed at pH 4.0, which precipitated if dialyzed directly to pH 7. Conditions required for assembly and some possible implications for the assembly mechanism are discussed.