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Mouse mammary tumor virus (MMTV)-induced mammary adenocarcinomas can develop from several different premalignant precursors common in GR mice. Insertion mutagenesis of the mammary protooncogenes int-1 and int-2 was studied in this multistep system by analyzing samples from various stages of neoplastic development for novel int-1 and int-2 restriction fragments generated by MMTV provirus integration. int-1 and int-2 insertion mutations were observed in both premalignant lesions and malignant tumors. Some of the tumors with insertion mutations were experimentally derived from insertion mutation-free premalignant precursors. Each class of neoplasm examined had a characteristic frequency of int-1 and int-2 insertion mutations; however, no correspondence was observed between neoplasm morphology and mutation of either gene. These results indicate that insertion mutation of the int-1 and int-2 loci by MMTV provirus can be involved in the earliest identifiable stages of neoplastic development as well as during progression of premalignant lesions to tumors. Insertion mutation of int-1 and int-2 is therefore not stage specific in this system.

Antigenic determinants of mouse mammary tumor virus (MuMTV) from the low-mammary-tumor-incidence strain BALB/cNIV were compared by competition radioimmunoassay with those of MuMTV's isolated from several high- and low-mammary-tumor-incidence mouse strains, using rabbit hyperimmune sera against BALB/cNIV MuMTV and against MuMTV from the high-mammary-tumor-incidence strain BALB/cfC3H. Using anti-BALB/cfC3H serum in competition radioimmunoassay, BALB/cNIV MuMTV lacked antigenic determinants present on MuMTV's from the BALB/cfC3H, C3H, and GR strains. With anti-BALB/cNIV serum in competition radioimmunoassay, type-specific antigenicity was detected with BALB/cNIV MuMTV. We found class-specific antigenicities on BALB/cNIV MuMTV that were shared with RIII and C3Hf MuMTV's BALB/cNIV MuMTV was reportedly derived from the C3Hf strain by infection of BALB/c mouse mammary tissue after transplantation into a (BALB/c X C3Hf)F1 hybrid followed by retransplantation into the BALB/c strain (D. R. Pitelka, K. B. DeOme, and H. A. Bern. Proc. Am Assoc. Cancer Res. 6:51, 1965). However, BALB/cNIV MuMTV contained type-specific antigens not present on C3Hf MuMTV. The possible origin of these determinants is discussed.

The polypeptide, antigenic, and morphological structure of the mouse mammary tumor virus was studied following protease digestion of intact virions. Intact, untreated virions (ρ = 1.17 g/ml) had characteristic envelope spikes, five major polypeptides, and were precipitated by antisera against gp52. Two of the major polypeptides, with molecular weights of 52,000 (gp52) and 36,000 (gp36), had carbohydrate moieties. Protease treatment resulted in spikeless, “bald” particles (ρ = 1.14 g/ml), which had altered surface antigenicity and which contained neither gp52 nor gp36. These data indicated that gp52 and gp36 were on the viral envelope. Bald particles retained a 28,000 dalton polypeptide (p28) which was proposed as the major internal polypeptide.

We have examined an assortment of preneoplastic and neoplastic mouse mammary tissues for the presence of an mRNA which could encode the putative long terminal repeat gene product of mouse mammary tumor virus. We report here the detection of a novel mouse mammary tumor virus-specific, polyadenylic acid-containing transcript in certain preneoplastic and neoplastic mammary tissue of BALB/c mice. The molecule is 1.6 kilobases in length and contains sequences from the transcriptional leader and the U3 region of the proviral DNA. The upstream terminus of the 3' information lies 75 to 80 nucleotides from the beginning of the long terminal repeat open reading frame, in close proximity to a consensus splice acceptor in the DNA. The transcript was detected in hormonally or chemically induced neoplastic, preneoplastic, and lactating mammary tissue of BALB/c mice, but not in preneoplastic or tumor tissue induced by exogenous viruses in any strain of mice examined. This implies that the RNA we observed is transcribed from an endogenous provirus template.

DNAs extracted from the mammary tumors of GR mice were analyzed for mouse mammary tumor virus proviral sequences by the restriction enzyme-Southern blot procedure. The tumor DNAs contain more proviral copies of mouse mammary tumor virus than DNA from a nonmalignant tissue. The degree of proviral amplification is small (ca. one to five additional copies) and appears to be variable from tumor to tumor. The restriction patterns of the amplified proviral sequences suggest a clonal origin for the tumor mass. In addition, the restriction patterns observed after digestion with the enzymes BglII and SacI indicate that only one of the proviruses endogenous to GR mice is amplified. The amplified provirus found in GR mammary tumors is identical to the provirus that is missing in GR-Mtv-2- mice, a congenic line exhibiting a low mammary tumor incidence.

A dominant restriction allele, Akvr-1r, from California wild mice (Mus musculus domesticus) confers resistance to exogenous ecotropic murine leukemia virus (MuLV) infection. The presence of an ecotropic MuLV envelope-related glycoprotein in uninfected virus-resistant cells suggests that viral interference is a possible mechanism for this resistance. We molecularly cloned the ecotropic MuLV envelope-related sequence from the genomic DNA of a wild mouse homozygous for the Akvr-1r locus. The cloned provirus was defective and contained a C-terminal end of the pol gene, a complete envelope gene, and a 3' long terminal repeat. The presence of this provirus was directly correlated with Akvr-1r-mediated virus resistance in cell cultures and hybrid mice. The Akvr-1r provirus restriction map and partial DNA sequence were identical to those of the Fv-4r allele, an ecotropic MuLV resistance locus from Japanese feral mice (M. musculus molossinus), which was previously shown to be allelic with the Akvr-1r gene. The 3' host flanking sequences of Fv-4r and Akvr-1r also had identical restriction maps. These findings indicate that Akvr-1r and Fv-4r are the same gene. It was probably acquired by interbreeding of these feral species in recent times. Conservation of this locus might be favored by the useful function that it performs in protection against ecotropic MuLV infection endemic in both populations of wild mice.

We used a two-dimensional Southern blotting procedure to analyze the tissue-specific methylation patterns of the five endogenous mouse mammary tumor viruses in the GR/A mouse strain. Our findings suggest that in certain tissues (brain, kidney, and liver) all proviruses are extensively methylated. In other tissues (spleen, placenta, and testes) all proviruses are hypomethylated to some degree. In these tissues individual proviruses display both quantitative and qualitative differences in methylation. We interpret the general patterns of tissue-specific hypomethylation in terms of a "hitch-hiker" model: mouse mammary tumor virus proviral methylation patterns reflect the tissue-specific activity of neighboring sequences. The observation that certain sites on particular proviruses are differentially methylated in a tissue-specific fashion may reflect tissue-specific differences in the makeup or conformation, or both, of proviral-containing chromatin.

The polypeptide and glycoprotein compositions of the mouse mammary tumor virus virion from primary monolayer cultures of BALB/cfC3H mouse mammary tumor cells were studied by polyacrylamide gel electrophoresis by using internal and external labeling and Coomassie blue and periodic acid Schiff (PAS) staining. Twelve polypeptides were reproducibly resolved by the combined methods. Five major polypeptides were demonstrable with estimated molecular weights of 52,000, 36,000, 28,000, 14,000, and 10,000. Seven minor polypeptides were also consistently detected and had estimated molecular weights of 70,000, 60,000, 46,000, 38,000, 30,000, 22,000, and 17,000. Carbohydrate was associated with five of these polypeptides as measured by PAS stain or [ 3 H] glucosamine labeling, or both. These glycoproteins had estimated molecular weights of 70,000, 60,000, 52,000, 36,000 and 10,000. The majority of the PAS stain and glucosamine was found in the 52,000 and 36,000 dalton peaks.

The temporal relationships of the production of infectious dengue-2 virus and its antigens were investigated in intracerebrally infected suckling mice. Infectious virus, a slowly sedimenting noninfectious hemagglutinin (SHA), and a noninfectious soluble complement-fixing antigen (SCF) were found in the brain. Serum contained high concentrations of SCF antigen relative to infectivity when compared to SCF to infectivity ratios in the brain. Degradation of virions by Tween-80 and ether produced two antigens with sedimentation characteristics similar to the noninfectious antigens occurring naturally in infected tissues. However, the virionderived SHA differed from native SHA when examined by electron microscopy and by equilibrium centrifugation in cesium chloride. Virion-derived SCF (as well as the virions and both SHA antigens) was denatured by sodium lauryl sulfate (SLS) and 2-mercaptoethanol (2-ME), whereas native SCF retained its complement-fixing activity. SLS and 2-ME treatment of dengue-1 and dengue-2 sucrose-acetone antigens increased their serotypic specificity. The hemagglutinin present in sucrose-acetone antigens was predominantly native SHA.

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.

Exogenous mouse mammary tumor virus (MMTV) was cloned from a GR mammary tumor. Clone lambda GRT39 contained a full-length integrated MMTV(GR) provirus and both 5' and 3' host flanking DNA. The lambda GRT39 provirus had no apparent structural changes associated with cloning and retained the exogenous MMTV gag gene poison sequence. When introduced into rat mammary adenocarcinoma LA7 cells, the lambda GRT39 provirus was fully expressed. lambda GRT39-transfected LA7 cells made MMTV RNA, had gp52 SU protein on the cell surface, and produced B-type retrovirus particles characteristic of MMTV. Mammary tumors developed in hormone-stimulated BALB/c females injected with MMTV from lambda GRT39-transfected LA7 cells [MMTV (lambda GRT39)]. The tumors had new, clonally integrated copies of the MMTV(lambda GRT39) provirus and were expressing MMTV antigen. These data indicate that the lambda GRT39 provirus is biologically active and pathogenic.

Antigenic compositions of slowly sedimenting dengue-2 hemagglutinin (SHA) and soluble complement-fixing antigen (SCF) were compared with the virion (rapidly sedimenting hemagglutinin, RHA) by radioimmune precipitation (RIP), RIP inhibition, kinetic neutralization, and neutralization blocking tests with the use of hyperimmune mouse ascitic fluids. RHA and SHA were unable to inhibit completely the RIP of each other by anti-RHA, and neutralization by anti-RHA was not blocked by SHA. This indicated that SHA is serologically related, but not identical, to RHA. SHA differed from RHA in that SHA lacked the “core” polypeptide but contained the two envelope polypeptides. In addition, SHA contained a polypeptide with a molecular weight of 16,500 daltons and a suggestion of several other proteins. These data, when considered with other evidence, suggest that SHA is a special form of “incomplete virus.” SCF was unable to inhibit the RIP of SHA or RHA or to block neutralizing antibodies. Further, anti-SCF did not neutralize RHA or precipitate significant levels of SHA or RHA. Polyacrylamide gel electrophoresis separated SCF from structural polypeptides by molecular size. This evidence suggests that SCF is a nonstructural antigen.