Donald L. Fine

Propagation of mouse mammary tumor cell lines and production of mouse mammary tumor virus in a serum-free medium.

SummaryFive different mouse mammary tumor cell lines were propagated in a serum free medium. Evaluation of growth characteristics, including logarithmic growth, cell population increase, protein production and days to confluency, showed serum-free medium comparable to serum-containing medium. Mouse mammary tumor virus expression and production, in C3H and GR tumor cell lines, as determined by virus particle counting and RNA dependent DNA polymerase assays, subsequent to dexamethasone stimulation revealed equivalent to higher levels of virus in serum-free medium as compared to serum-containing medium.

Isolation of separate precursor polypeptides for the mouse mammary tumor virus glycoproteins and nonglycoproteins.

Abstract We have employed monospecific antisera to the major glycoproteins (gp52 and gp36) and the major nonglycoprotein (p27) of the mouse mammary tumor virus (MMTV), and we now report the first isolation of an intracellular MMTV precursor polypeptide to p27. The precursor polypeptide to p27 (Pr75) binds to single-stranded DNA (ssDNA) and can be easily separated from the precursor to gp52 and gp36 (gPr75) by ssDNA-Sepharose column chromatography. [35S]Methionine-labeled Pr75 contained tryptic peptides of p27 and p14 of MMTV. Protein p14 has previously been shown to be capable of binding to ssDNA. In contrast, [35S]methionine-labeled gPr75 contained tryptic peptides of only gp52 and gp36, neither of which binds to ssDNA.

Coexistence of the mouse mammary tumor virus (MMTV) major glycoprotein and natural antibodies to MMTV in sera of mammary tumor-bearing mice.

Abstract Sera of mammary tumor-bearing mice contain the major envelope glycoprotein (gp52) of mouse mammary tumor virus (MMTV) and autogenous antibodies to MMTV. Although antibodies to MMTV are readily demonstrable in sera of both tumor-free and tumorbearing animals, detection of the viral glycoprotein appears to be dependent on the presence of a palpable mammary tumor. MMTV gp52 was detected both as the free protein and in a high molecular weight complex as demonstrated by velocity sedimentation centrifugation. MMTV gp52 was also detected in both mammary tissue and lymph nodes of female C3H/HeN mice. The reproductive organs of the male C3H/HeN mice, such as the vas deferens and vesicular, coagulating, and prostate glands, contained gp52, while no MMTV gp52 could be demonstrated in the testes, epididymis, or preputial glands. This antigen was not found in any tissues of male or female BALB/c mice.

Expression of natural antibodies against endogenous and horizontally transmitted macaque retroviruses in captive primates

To determine the prevalence of retrovirus antibodies in captive primate populations and to identify factors that influence antibody responses, we analyzed sera from seven geographically separated primate colonies for antibodies to the genetically [baboon endogenous virus (BaEV), squirrel monkey retrovirus (SMRV), and macaque endogenous viruses (MMC-1 and MAC-1)] and horizontally [Mason Pfizer monkey virus (MPMV) and gibbon ape lymphoma virus (GaLV)] transmitted primate retroviruses. Antibodies were detected with a solid-phase radioimmunoassay in which iodinated staphylococal protein A is used for precipitation of immunoglobulins. Naturally occurring antibodies to MPMV were found in sera of Macaca mulatta from seven geographically separated primate colonies and in the sera of three different macaque species. The predominant reactivity of these natural sera was directed against antigens shared by MPMV, BaEV, and SMRV. Several of these sera were found to have MPMV-neutralizing activity. Antibodies to the endogenous Macaca mulatta virus, MMC-1, were found in three species of macaques and in Macaca mulatta sera from five of the seven colonies tested. These positive sera were directed predominantly against a 69,000 molecular weight protein and against determinants shared by both MMC-1 and MAC-1, an endogenous virus of Macaca arctoides. Antibody expression to MMC-1 appears to be both sex and age related, whereas antibody expression to MPMV in the same monkey population was not.

Mouse mammary tumor virus and murine leukemia virus cell surface antigens on virus producer and nonproducer mammary epithelial tumor cells

Abstract Mouse mammary tumor virus (MMTV)- and murine leukemia virus (MuLV)- specific cell surface antigens (CSA) on virus producer and nonproducer mammary epithelial tumor cells were studied using the techniques of lactoperoxidase catalyzed iodination of cell surface proteins followed by radioimmune precipitation with monospecific antisera to the major MMTV proteins gp52, gp36, p27, and p10 and to the major MuLV proteins gp70 and p30. The incorporation of iodinated CSA into extracellular virus was determined by analyzing labeled proteins in purified virus. On cells producing only MMTV both gp52 and gp70 were present on the cell surface. Furthermore, gp52 was the only labeled protein in extracellular MMTV produced by these cells. On cells producing both MMTV and MuLV, both gp52 and gp70 were present on the cell surface, and were the only labeled proteins present in their respective extracellular viruses indicating that gp70 and gp52 are present on mutually exclusive cellular viral budding sites. In addition, MuLV anti-p30 serum precipitated two iodinated proteins with molecular weights of 85,000 and 95,000 daltons, analogous to the Gross cell surface antigen (GCSA). Labeled gp52 and gp70 represent true CSA as demonstrated by the fact that they were also present on the surface of cells producing no virus, but producing large amounts of MMTV glycoproteins and nonglyco-proteins. These results further demonstrate that the precursor to the MMTV glycoproteins (gPr75-MMTV env) is cleaved prior to the appearance of gp52 on the cell surface.

Simian Tumor Virus Isolate: Demonstration of Cytopathic Effects in vitro

Several cell lines that were derived from primates and inoculated with virus originally obtained from a spontaneous mammary carcinoma showed cytopathic effects characterized by multinucleation. These cytopathic effects appeared as early as 24 holurs after inoculation. Multinucleated cells contained virus particles characteristic of the original virus isolate.

Type D retroviruses: Occurrence of natural antibodies to Mason-Pfizer monkey virus in rhesus monkeys

Abstract Sera from rhesus monkeys maintained at three separate geographic regions were found to possess naturally occurring antibody to the Mason-Pfizer monkey virus (MPMV) major structural protein. The specificity of this reactivity was established by absorption with purified viral protein. While animals from two of these colonies were housed in association with MPMV-infected monkeys, no such prior exposure to virus-infected animals could be traced in the case of the third colony. These results in combination with the previous inability to detect full complements of MPMV genetic sequences in the cellular DNA of the normal rhesus monkeys provide evidence for horizontal transmission of MPMV. Alternatively, the present findings could reflect humoral immune response to expression of an endogenous type D retrovirus highly related to MPMV.

Demonstration of components of serum-free culture medium effecting maximum in vitro expression of mouse mammary tumor virus

SummaryBy using a chemically defined serum-free (SF) medium for propagation of Mm5mtc1 mouse adenocarcinoma cell cultures and clonal derivatives, medium components including hormones, glucose and individual amino acids were evaluated as to modulation of mouse mammary tumor virus (MMTV) production. Insulin, hydrocortisone and dexamethasen each increased MMTV production on a per cell basis over constitutive expression that ocures in SF medium devoid of hormones. Maximum production occurred when all three hormones were present. Hormone-stimulated virus expression also was influenced by glucose concentration. Cell growth and maximum MMTV expression increased when thyroxine, asparagine, proline and serine were omitted from the medium formulation. The resulting modified SF medium provides and ideal system for the propagation of high MMTV-producer clones and for the study of the biochemical regulation of MMTV expression.

Functionally conserved determinants on gp70s of endogenous primate retroviruses.

Abstract Endogenous viruses of Old and New World monkeys are divided morphologically into type C isolates of baboons, macaques, and owl monkeys and type D isolates of squirrel monkeys and langurs. Mason-Pfizer monkey virus (MPMV), a type D virus, is horizontally transmitted in rhesus monkeys and is highly related to the langur isolate. Although these endogenous type C and D viruses possess little nucleic acid sequence homology and are antigenically distinct, they do possess cross-reactive determinants in their envelope gp70 antigen, as shown by sensitive interspecies radioimmunoassays. The ability of MPMV to induce syncytia in KC (Rous sarcoma virus-transformed human glioma) cells allowed for investigation of the role that these conserved determinants play in recognition of cellular receptors. The capacity of other viruses to bind to common cellular receptors and to inhibit MPMV-induced syncytia was measured in a quantitative assay using a high syncytia-inducing variant of MPMV. Pretreatment of KC cells with disrupted MPMV, baboon endogenous virus (BaEV), squirrel monkey retrovirus, and the BaEV-related endogenous cat virus RD114 blocked syncytia formation by MPMV. Partial competition was observed with endogenous macaque isolates MAC-1 and MMC-1. However, no inhibition was observed using feline leukemia, murine leukemia, equine infectious anemia, or murine mammary tumor viruses, none of which possess antigenic determinants which are cross-reactive with the endogenous primate retroviruses. Syncytia formation was also blocked by purified gp70s of MPMV and BaEV, demonstrating that inhibition was due to competition for viral gp70 cellular receptors.

Comparative large-scale propagation of retroviruses from old world (mason-pfizer monkey virus) and new world (squirrel monkey virus) primates

SummaryA cell culture method is described for the large-scale (50 to 150 1) production of Mason-Pfizer monkey virus and squirrel monkey virus, two primate retroviruses. Virus production was achieved with suspension cultures of chronically infected A204 human rhabdomyosarcoma cells harvested and clarified in the logarithmic stages of cell culture growth. Methods for the subsequent purification and concentration of virus material utilizing zonal centrifugation also are described. Applications of these methodologies resulted in products that afforded biochemical comparisons of these agents in a manner such that host cell-derived variations were minimized. These data indicated that high levels of production and efficient recovery and purification of virus material were achieved.