Alterations of a mouse mammary tumor virus glycoprotein with interferon treatment.

The effects of exogenous mouse interferon on the MJY-alpha mammary tumor cell line chronically infected with mouse mammary tumor virus (MMTV) were examined. Interferon at concentrations of 25 to 2,000 IU/ml in culture medium did not alter the growth rate or morphology of the cell layers. Electron microscopic examination of interferon-treated cells indicated a decrease in the numbers of A-type and budding B-type particles of MMTV. However, the levels of extracellular MMTV virions in the culture supernatants were not significantly reduced. Profiles of MMTV glycoproteins and nonglycosylated polypeptides obtained by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of virions purified from interferon-treated cultures revealed increases in the relative levels of the 60,000-dalton glycoprotein, gp60.     

Genetic marker patterns and endogenous mammary tumor virus genes in inbred mouse strains of Japan

In order to establish the genetic relatedness of the inbred mouse strains kept in Nara, genetic marker patterns were determined in conjunction with a study on endogenous mammary tumor viral genes in these strains. Isoenzyme patterns combined with patterns of other genetic markers, show that the unrelatedness between various inbred strains of the dd stock is as high or even higher as between strains of known different origin and geneology. Based on endogenous viral gene patterns the dd stock derived mice can be subdivided into three group, DDD, DDN, DDO, KF and DD/Tbr. The DD/Tbr and its foster-nursed substrain (DD/Tbrf) have the lowest number of endogenous viral genes, i.e. two, while the other strains carry 4-6 such genes. The SLN and SHN strains, derived from a Swiss stock, have a similar pattern of viral genes different that of all other strains studied, also strains of Swiss origin from other sources, such as the NFS and the GR.     

Glucocorticoid regulation of mouse mammary tumor virus gene expression.

Glucocorticoid hormones act rapidly and specifically to stimulate the synthesis of mouse mammary tumor virus RNA in a variety of mouse mammary tumor cells and infected heterologous cells. The increase in viral RNA production appears to be mediated by receptor proteins and requires the presence of basal levels of viral RNA. Infection of heterologous cells with MMTV may alter host cell responses to glucocorticoids; in addition, production of unintegrated viral DNA in these cells has provided reagents required for studying the structure and function of the viral DNA itself. The advent of new techniques for genetic manipulation of eukaryotic cells and for isolation of large amounts of specific DNA sequences should now permit detailed analyses of steroid hormone action in this system.     

Amplification and novel locations of endogenous mouse mammary tumor virus genomes in mouse T-cell lymphomas.

Endogenous mouse mammary tumor virus genomes are amplified and located in novel cell DNA sequences in many mouse T-cell lymphomas. Transplanted tumors recovered from the same mouse strain and shown to be of independent origin by chromosomal analysis, by the presence of JH immunoglobulin gene rearrangements, or by the integration patterns of exogenous Moloney MuLV genomes frequently showed similar patterns of novel mouse mammary tumor virus-containing cell DNA fragments. This process of amplification and relocation can occur within a limited number of cell generations and in C57BL/6 mice does not lead to the synthesis of mature virus-encoded proteins. In some instances, amplified mouse mammary tumor virus genomes contained novel restriction cleavage sites in the gag-pol region. The restricted time course of occurrence, lack of synthesis of mature virion proteins, and apparent site specificity indicate that this process of retrovirus amplification differs significantly from virus replication after exogenous infection.     

The endogenous proviral mouse mammary tumor virus genes of the GR mouse are not identical and only one corresponds to the exogenous virus.

The endogenous proviral copies of mouse mammary tumor virus (MMTV) were selected from a gene library of GR mouse DNA. We obtained five different lambda. MMTV recombinant clones. Four of them correspond to the 3' Eco RI fragments of the endogenous proviruses an one comprises an intact MMTV provirus with 2 to 3 kb of flanking mouse genomic DNA. Heteroduplex formation followed by S1 digestion under stringent conditions shows that there is nucleotide sequence heterology among the cloned endogenous proviral copies. Only one endogenous proviral copy, associated with the mtv-2 locus, was found to be totally homologous to the exogenous proviral DNA.     

Characterization of endogenous and exogenous mouse mammary tumor virus proviral DNA with site-specific molecular clones.

Restriction fragments of the mouse mammary tumor virus (MMTV) proviral DNA were obtained by molecular cloning procedures. A 4-kilobase fragment delimited by two PstI sites was isolated from unintegrated, linear MMTV DNA and amplified in the pBr322 plasmid vector. EcoRI fragments of proviral DNA, integrated into the genome of a GR mammary tumor cell line, were isolated as lambda recombinant molecules. Five different recombinant phages which contained the 3' region of the MMTV proviral DNA and adjacent host DNA sequences were isolated. Heteroduplex analysis and S1 nuclease digestion suggested that there is no extensive sequence homology in the host DNA flanking the different proviral genes. The cloned DNA was fractionated into site-specific restriction fragments which served as molecular probes in the analysis of the endogenous MMTV proviral copies of C3H, GR, BALB/c, and feral mice. This allowed the correlation of MMTV-specific EcoRI fragments obtained from genomic DNA of these strains with the 5' and 3' ends of the proviral gene. Restriction fragments of two clones which contained the proviral sequences adjacent to the flanking host DNA as well as 1 to 2 kilobases of host DNA were used as hybridization probes, and the results allow the following conclusions: the proviral DNA of both clones contains nucleotide sequences complementary to the 5' and 3' ends of proviral DNA; and the host DNA flanking one clone belongs to the unique class of genomic DNA, whereas the DNA flanking the second clone is reiterated at least 15 times within the mouse genome.     

Transgenic mouse mammary tumor virus superantigen expression prevents viral infection.

Endogenous mouse mammary tumor virus (MMTV) proviruses have recently been shown to cosegregate genetically with the minor lymphocyte-stimulating loci, also termed self-superantigens. The antigenic activity has been localized to the open reading frame (ORF) protein encoded in the long terminal repeat of MMTV. We show here that unlike their nontransgenic littermates, transgenic mice expressing high levels of an ORF protein derived from the C3H exogenous MMTV specifically delete their V beta 14+ T cells and do not become infected with this virus when it is present in their mother's milk. Thus, it appears that MMTV utilizes cells of the immune system in its infection pathway, and mice that retain endogenous MMTVs should be immune to infection by exogenous virus. These results offer possible new approaches to anti-viral therapy or immunization.     

Cell culture factors influencing in vitro expression of mouse mammary tumor virus.

Several cell culture factors were found to influence in vitro expression of mouse mammary tumor virus (MMTV) in the mouse adenocarcinoma cell line Mm5mt/c1. Cells were propagated in a variety of commercially available cell culture media to which dexamethasone (DXM) was added as a stimulator of MMTV production. Culture seeding density, culture medium type, and glucose concentration each influenced MMTV production when expressed on a per cell basis. Maximum cell growth occurred in cultures grown in RPMI-1640 medium containing insulin. Those media which provided good cell growth were not necessarily optimal for virus expression. Addition of insulin did not stimulate MMTV synthesis although dexamethasone alone was stimulatory in all media used; however, maximum MMTV expression was obtained when dexamethasone and insulin were used in concert. Equivalent levels of MMTV-specific cell membrane antigen, MMTV-specific protein, and virus particles were produced at incubation temperatures of 32 degrees, 34 degrees or 37 degrees C; however, higher levels of virus-related RNA-dependent DNA polymerase (RDDP) activity were recovered from cultures incubated at 32 degrees and 34 degrees C than at 37 degrees C. Decreased levels of RDDP were attributed to enzyme thermolability at 37 degrees C incubation.     

ML antigen of DBA/2 mouse leukemias: expression of an endogenous murine mammary tumor virus

Spontaneous, transplantable leukemias of DBA/2 mice express an antigen (ML) which cross-reacts with antigens of murine mammary tumor virus (MuMTV). The MuMTV cross-reactive antigen of the DBA/2 leukemias (ML cells) was found to be a glycoprotein of 78,000 molecular weight containing antigenic determinants of the major MuMTV glycoprotein gp52. No MuMTV particles were produced by the ML cells, although they did contain type A particles--the pronucleocapsids of MuMTV. The ML antigen appeared to be an aberrant form of the intracellular MuMTV env precursor molecular prgp70, which was not processed properly but instead acquired extra carbohydrate groups and was expressed in uncleaved form on the cell surface. Isolation of MuMTV core protein p28 from the leukemic cells and subsequent tryptic peptide mapping analysis showed that the p28 from leukemia cells differed from the p28 of MuMTV isolated from DBA/2 mouse milk. These observations indicate that the MuMTV expressed in DBA/2 leukemic spleen cells is of a different strain than the virus secreted in lactating mammary glands of DBA/2 mice and probably represents the expression of an endogenous DBA/2 provirus.     

An endogenous mouse mammary tumor virus genome common in inbred mouse strains is located on chromosome 6.

We have examined EcoRI-restricted cellular DNA from mouse-hamster somatic cell hybrids. Results of this analysis show that the unit II mouse mammary tumor virus proviral genome is located on mouse chromosome 6. Restriction analysis of cellular DNA from (C3H/OuJ X Czech II) X Czech II backcross mice showed a strong linkage between unit II and Igk. The gene order of these markers on chromosome 6 relative to the Raf and Kirsten murine sarcoma virus ras-2 proto-oncogenes was established.     

A standardized nomenclature for endogenous mouse mammary tumor viruses.

We propose a revised standardized nomenclature for endogenous mouse mammary tumor viruses based on characterization by molecular cloning techniques and genetic segregation data.     

Nucleotide sequence of human endogenous retrovirus genome related to the mouse mammary tumor virus genome.

We determined the complete nucleotide sequence of the human endogenous retrovirus genome HERV-K10 isolated as the sequence homologous to the Syrian hamster intracisternal A-particle (type A retrovirus) genome. HERV-K10 is 9,179 base pairs long with long terminal repeats of 968 base pairs at both ends; a sequence 290 base pairs long, however, was found to be deleted. It was concluded that a composite genome having the 290-base-pair fragment is the prototype HERV-K provirus gag (666 codons), protease (334 codons), pol (937 codons), and env (618 codons) genes. The size of the protease gene product of HERV-K is essentially the same as that of A- and D-type oncoviruses but nearly twice that of other retroviruses. A comparison of the deduced amino acid sequences encoded by the pol region showed HERV-K to be closely related to types A and D retroviruses and even more so to type B retrovirus. It was noted that the env gene product of HERV-K structurally resembles the mouse mammary tumor virus (type B retrovirus) env protein, and the possible expression of the HERV-K env gene in human breast cancer cells is discussed.     

Asymmetric transcription of mouse mammary tumor virus genes in vivo and in vitro

Mouse mammary tumor virus (MMTV) DNA fragments were cloned into M13 bacteriophage, and the single-stranded recombinant phage DNAs were used as strand-specific nucleic acid hybridization probes to measure synthesis of plus (genomic) and minus strands of MMTV RNA in cultured cell lines and in cell-free preparations of nuclei. Pulse-labeling studies showed that synthesis of MMTV RNA in three different cell lines was highly asymmetric. In nuclear preparations from a cloned line of MMTV-infected rat hepatoma cells, elongation of nascent MMTV RNA chains and initiation of new MMTV RNA chains with nucleoside (beta-S)triphosphates were also highly asymmetric.