Surface localization of virus production on a glucocorticoid-stimulated oncornavirus-producing mouse mammary tumor cell line by scanning electron microscopy.

A chronically infected continuous mouse mammary tumor cell line containing virus particles of type B morphology, free of contaminating type C virions, has been grown in tissue culture. These cells were treated with dexamethasone, a synthetic glucocorticoid, a potent stimulator of mouse mammary tumor virus expression. Surfaces of untreated and dexamethasone-treated cells were investigated by scanning electron microscopy. Untreated cells demonstrated a moderate expression of mouse mammary tumor virus (80 particles/cell) distributed diffusely over the cell surface. However, virions on dexamethasone-treated cells were localized in clusters of 100 to greater than 2000 virus particles, often with more than one cluster per cell. Dexamethasone-treated cells typically showed a 10-fold increase in cell-associated virus over untreated cells. Concentrated extracellular fluids from untreated and dexamethasone-treated cultures were quantitated for free virus. Dexamethasone-treated culture fluids demonstrated a similar 10-fold increase of extracellular particles, in contrast to untreated cultures. This increase in virus particles on the cell surfaces as well as in the extracellular fluids supports the theory that dexamethasone has a stimulatory effect on viral replication, not just on the release of budding particles. The ultrastructure of budding mouse mammary tumor virus during dexamethasone stimulation, determined by scanning and transmission electron microscopy, and the significance of such an in vitro system for viral immunodiagnosis are discussed.     

Immunocolloidal gold electron microscopy of viral antigens and cellular actin in C3H/He mouse mammary tumors

Mouse mammary tumor virus (MMTV) antigens and cellular actin were visualized with Protein A-coated colloidal gold at the ultrastructural level in C3H/He mouse mammary tumors using various antisera. Observations with the postembedding method and statistical analyses of these data showed: (a) a glycoprotein with a molecular weight of 52,000 resided on the envelope of mature virions (B-particles), on the surface of budding particles, and on the vacuolar membrane, but not on intracytoplasmic A-particles (A-particles); (b) B-particle antigens were shared by A-, B-, and budding particles with more reacting gold particles on B-particles; and (c) a protein with a molecular weight of 27,000, p27 and A-particle antigens were also shared equally by all MMTV-related particles with preferential localization on the nucleoid of B-particles and the double ring of A-particles. All of these observations are consistent with and further confirm the proposal that A-particles are pronucleocapsids of MMTV. Cellular actin was concentrated in the outermost thin cytoplasmic layer and in microvilli. Single gold particles were also found on budding and B-particles implying that actin plays a role in the MMTV budding process.     

亲代L7811及体外培养小鼠白血病细胞系L7811—85...

L7811 was an ascitic form of lymphocytic leukemia induced by myleran in 615 mice. Under electron microscope, cytoplasmic and intracisternal type A virus-like particles were observed. L7811-85 was a cell line established in vitro from the parental in vivo L7811 line. Instead of type A particles, mature and immature type C murine virus-like particles could be observed budding from the plasma membrane to extracellular space. With the in vitro subpassage of L7811-85 cells, more intracellular and extracellular type C particles appeared with an increase in its tumorigenicity. When L7811-85 cells, were inoculated IP to normal 615 mice, ascites tumor developed. The type A virus particles appeared again budding into cisterna of endoplasmic reticulum but not to extracellular space. The mechanism of virus particle type transformation remains to be studied.     

A new virus in a spontaneous mammary tumor of a rhesus monkey

This study investigated the ultrastructure and development of virus particles detected in a spontaneous mammary tumor of an 8 year old rhesus monkey. During a 3-month period of tumor growth, several biopsies were performed. Thin section electron microscopy of the tumor tissue revealed 2 types of particles: 1) an intracytoplasmic, electron-dense, ring-shaped particle, and 2) an extracellular particle with an outer unit membrane and a central dense nucleoid. The intracytoplasmic development and virus maturation by a process of budding at the level of the cell membrane was reconstructed.     

Conventional and immunocolloidal gold electron microscopy of eight simian retroviruses closely related to human T-cell leukemia virus type I

Simian retroviruses closely related to human T-cell leukemia virus type I (HTLV-I) were isolated from 8 species, examined by both conventional and thin section immunocolloidal gold electron microscopy, and compared with HTLV-I. Mature forms of simian viruses were found in extracellular aggregates and within cytoplasmic vacuoles. They were morphologically similar to each other and to HTLV-I. They consisted of a seemingly smooth envelope and a centrally located nucleoid. Their size varied considerably among species and also within the same species; this is characteristic of this group of retroviruses. No budding particles of simian viruses were observed. Thin section immunocolloidal gold electron microscopy using various human and simian sera showed that simian viruses were antigenically related to each other and to HTLV-I. One drawback of this otherwise very useful technique was the difficulty in identifying virions because of the poor preservation of their fine structure by fixation with glutaraldehyde alone. This was overcome by using materials prepared for conventional electron microscopy, in which virions showed weak but specific reactions with gold particles after deosmification and antigen restoration with sodium metaperiodate.     

In vitro susceptibility of mink lung cells to the mouse mammary tumor virus.

Lung cells from mink embryos were infected in vitro with a purified mammary tumor virus isolated from RIII mouse milk. Specific virus antigen at the cell surface was detected by membrane immunofluorescence; B-type virions budding from the cell membrane were seen by electron microscopy. Nucleic acid hybridization confirmed replication and specificity of the virus produced.     

Oncornavirus-like particles in baboon type C virus-infected chimpanzee lung cells (SFRE:CL-1).

Intracytoplasmic type A particles were observed in a fetal chimpanzee lung culture (SFRE:CL-1) inoculated with type C virus-containing supernatants from a coculture of baboon placenta and SFRE:CL-1 cells. Budding, immature, and mature type C particles were also noted. In thin section, spike-like structures were rarely detected on budding intracytoplasmic type A particles but were occasionally observed on some immature and mature virus particles. Unlike mouse mammary tumor virus or Mason-Pfizer monkey virus, infected SFRE:CL-1 cells contained no eccentric or rodshaped nucleoids.     

Morphologic and antigenic properties of mouse mammary tumor virus produced in a hormone-responsive fashion by C57Bl/10 mammary tumors of non-viral origin

Bl-MaTU/A1 mouse mammary tumor cells, derived from a C57B1/10 mammary adenocarcinoma induced by dimethylbenzanthracene and mammotropic hormones, express virus particles and proteins related to mouse mammary tumor virus (MMTV). Immunocytochemical analysis by means of monospecific and monoclonal anti-gp52 sera revealed a different localization of the main structural proteins of MMTV in Bl-MaTU/A1 and GR cells (the latter used as a positive virus-producing control). Immunoelectron microscopy of B-type particles budding from the microvilli of dexamethasone-stimulated Bl-MaTU/A1 cells showed remarkably weak reactivity of the viral envelope with anti-gp52-protein A-gold complexes as compared with that of dexamethasone-stimulated GR cells. Since Bl-MaTU/A1-associated MMTV originates from the amplified unit II of endogenous MMTV, which is altered probably within the env gene, the observed antigenic difference in the Bl-MaTU/A1-associated MMTV may be due to altered synthesis of gp52 glycoprotein in these cells.     

Identification of the virions in the in vitro L1210(V) leukemia cell lines by morphological, virological, and immunological techniques.

In vitro L1210 (V) cell lines contained abundant intracytoplasmic A-particles, numerous C-type particles, a small number of B-type particles, and occasional intracisternal A-particles. The intracytoplasmic A-particles were incorporated into both spiked (B-type) and smooth-surfaced (C-type) particles formed at the budding site.Both B-and C-type particles also developed by gradual accululation of neucleooid material. The particles, particularly the C-type, exhibited a wide range of densities. The cells showed strong surface immunofluoresence for both murine mammary tumor virus and Gross murine leukemia virus antigens and variable degrees of cytoplasmic immunoflurescence for the protein antigens (p1 to p6) of Rauscher leukemia virus. The cells, the culture supernatant, and the purified virus each gave positive reactions with murine mammary tumor virus and murine leukemia virus antisera by immunodiffusion. The viral particles failed to infect C57BL, C57BL/6 X DBA/2F1 (hereafter called BD2F1), BALB/c, Af,and RIIIf mice. Howver, the cells were highly tumorigenic in BD1F-1 mice, moderately tumorigenic in BALB/c mice, but not tumorigenic in C57BL, Af, and RIIIf mice.     

Ultrastructural cytochemistry of enzymes associated with murine leukemia viruses. I. Adenosine triphosphatase

Ultrastructural cytochemistry of plasma-virus pellets from mice injected with Rauscher or Moloney virus showed a phosphatase activity specific for adenosine triphosphate and diphosphate, and for inosine triphosphate and diphosphate. This enzymatic activity was localized at the viral envelope and was associated with only part of the virus population. Moloney virus showed a smaller proportion of active virions than Rauscher virus. The adenosine triphosphatase activity associated with the murine leukemia viruses was far less pronounced than that associated with avian myeloblastosis virus. The origin of this enzymatic activity has not been clarified. Since the envelope of the budding viruses reflects some of the properties of the budding cell membrane, the adenosine triphosphatase of the blood-forming organs was extensively investigated. Most of the virus-replicating cells were adenosine triphosphatase-negative in these organs. The viral enzyme might be adsorbed at the surface of the virus by electrostatic forces.     

Demonstration of type-R and type-C virus particles in hamster pancreatic adenocarcinomas

Two transplanaable solid tumors (CBP and PDP) and two continuous cultured cell lines (CBP-TC and PDP-TC) were established from nitrosamine-induced pancreatic ductal adenocarcinomas in inbred Syrian hamsters. Electron microscopic examination of both CBP and PDP solid tumors as well as CBP-TC cultured cells revealed the presence of type-R virus particles, which consisted of a 100 nm envelope containing a central 50 nm nucleoid with characteristic radiating ‘spokes’. Type-R particles were abundant in cultured CBP-TC cells, moderately frequent in CBP solid tumors, and rare in PDP solid tumors; they were not present in PDP-TC cultured cells. The CBP and PDP solid tumors and cultured PDP-TC cells exhibited type-C viral particles, having a 150 nm envelope containing a 75 nm nucleoid. Mature, immature and budding forms of Type-C viruses were present and quite prominent in PDP tumors. Type-C particles were present but rare in CBP solid tumors and in PDP-TC cells. C-particles were not observed in cultured CBP-TC cells. Examination of normal hamster pancreas, liver, duodenum, muscle and connective tissue failed to reveal evidence of type-R or type-C virus particles. It is important to recognize the presence of virus particles in hamster pancreatic carcinoma lines, since viruses could potentially affect biochemical or immunological studies on the carcinomas through the production of viral proteins that could be mistaken for tumor-associated antigens.     

An experimental animal model for the study of human scirrhous carcinoma.

Scirrhous-like carcinomas were induced by the inoculation of cloned mouse mammary carcinoma cells into the cleared fat pads of BALB/c mice. The inoculated cells induced palpable tumors in 100% of the animals within 60 days. The epithelial cells in vivo grew in rows and clusters and were attached by short developing desmosomes. Microvilli and organelles were scarce; intracisternal type A particles were observed in all the epithelial cells. Carcinoma cells invaded the dermis and muscle. The stroma was formed by morphologically normal fibroblasts and large masses of collagen. No virus particles were observed budding from the epithelial cell surface or in the stromal fibroblasts. The similarities between the scirrhous-like carcinoma in BLAB/c mice and the juman scirrhous carcinoma suggest this animal system as a model for the study of the human disease.     

Pathogenesis of oncogenic simian adenoviruses. IV. The histopathology and ultrastructure of intraperitoneal neoplasms induced by SV20

The histopathology and ultrastructure of SV20-induced intraperitoneal (IP) neoplasms is described and compared to neoplasms induced by this virus administered by the intracranial and subcutaneous routes. SV20-induced IP neoplasms are relatively undifferentiated and contain two types of tumor giant cells. Dilated rough endoplasmic reticulum was increased, but annulate lamellae were rarely observed in IP neoplasms. Virus-like particles within the cytoplasm and nuclei of neoplastic cells resembled the original inoculated SV20 virus. Virus particles associated with cisternae of endoplasmic reticulum, nuclear projections, and unusual cytoplasmic bodies with many smaller dense structures were observed. These latter dense structures were attached to the inner and outer surface of cytoplasmic bodies and resembled the nucleoid of virus particles.     

Immunologic, virologic, and genetic aspects of mammary tumor virus-induced cell-surface antigens: presence of these antigens and the Thy 1.2 antigen on murine mammary gland and tumor cells.

The distribution of the normal differentiation antigen Thy 1 and the mammary tumor virus (MTV)-induced antigens or antigen complexes MLm and MLr were studied in mouse mammary gland cells, mammary tumor cells, and other cell types, by use of ascites leukemia cells of the GR mouse strain as target cells in the cytotoxicity test. The Thy 1.2 antigen was detected by an AKR antiserum to C3Hf thymocytes. MLm was shown by a homologous C57BL antiserum to GRSL2 leukemia (absorbed in vivo in GR mice); MLr was detected by a rabbit heterologous antiserum (absorbed in vivo in C57BL or GR mice and in vitro with BALB/c milk) prepared against Tween 80- and ether-treated purified B particles. Sera from Sprague-Dawley rats bearing murine leukemia virus (MuLV)-producing syngeneic tumors were not cytotoxic or only slightly cytotoxic for GR leukemias transplanted in vivo, which indicated that MuLV-induced antigens were absent or present in very low quantity in such leukemias. The MLr and MLn antigens or antigen complexes were possibly identical to the mammary leukemia (ML) antigen, since they could be detected not only on GR but also on DBA/2 leukemia cells and since their distribution was exactly the same as that of MTV. Both the MLr and MLm antigens were present in purified B particles, and antigenic activity were present in purified B particles, and antigenic activity was enhanced by destruction of the purified virus particles. The antigens were about eightfold enriched in a preparation of B-particle envelopes, as shown by quantitative cytotoxicity absorption (CYTA) tests. Purified nucleoid fractions of B particles were only lightly positive for the antigen, probably due to envelope contamination. One dominant gene was responsible for the expression of MLr, as shown by CYTA tests with mammary glands of individual animals of segregating crosses between the GR strain with high mammary cancer incidence and strains with low incidence. This gene was closely linked with or was possibly identical to 1) the gene for cytoplasmic MTV gs antigen expression as seen by fixed cell immunofluorescence, and 2) the gene causing mammary tumors in the GR mouse strain.     

Effects of ribonuclease and deoxyribonuclease on a murine leukemia virus (Rauscher)

Plasma pellets and femoral bone marrow from BALB/cJ mice infected with the Rauscher leukemia virus were fixed, embedded, and sectioned. The thin sections were incubated in ribonuclease and deoxyribonuclease solutions, stained, and examined in the electron microscope. Specific attention was paid to the action of the nucleases on characteristic virus particles in the plasma preparations and on viruses being produced by the "budding" phenomenon in the femoral bone marrow. Ribonuclease solutions digested the nucleoids of the virus particles in the plasma preparations from mice infected with Rauscher leukemia virus. The nucleoid portion of the "budding" virus particles in bone marrow, and the connecting cytoplasm of the stalks were also digested by ribonuclease solutions. In addition, the outer coat of the "budding" particles was affected in a nonspecific manner. The centers of the "budding" particles in the bone marrow and the nucleoids of viruses in plasma preparations were not digested by deoxyribonuclease solutions. Influenza virus, a known ribonucleic acid (RNA) virus, was used as a control for nuclease activity. The nucleoids of influenza virus particles were digested by ribonuclease but not by deoxyribonuclease solutions. After coriphosphine staining of the plasma virus preparations, the fluorescence was quenched in preparations treated with ribonuclease, but did not appear to be diminished in those treated with deoxyribonuclease. This study suggests that infection of mice with Rauscher leukemia virus produces virus particles in plasma and "budding" particles in bone marrow, both of which contain RNA.     

Search for retrovirus-like particles in human breast cancer cells in culture

We have recently established four new human breast cancer cell lines that were characterized as being of human mammary origin. We examined these cell lines for particles morphologically resembling retroviruses by electron microscopy, for extracellular and intracellular particles containing high-molecular-weight RNA and RNA-directed DNA polymerase by biochemical assays, and for mouse mammary tumor virus (MMTV)-related sequences in the cell genomes by molecular hybridization. An extensive search for budding particles by thin-section electron microscopy of cells did not provide evidence for retrovirus-like particles. Similarly, 1000- to 2000-fold concentrated samples of medium harvested from 10(8) cells did not contain particles of a density of 1.14 to 1.16 g/ml containing RNA-directed DNA polymerase. Compared with DNA polymerase activity of MMTV, and taking into account the particle weight and protein content of retroviruses, we estimate that, if these cells produce retrovirus-like particles, this production would be less than 1.6 particles/cell every 24 to 72 hr. The hybridization of cell DNA with MMTV complementary DNA also did not show detectable amounts of virus-related sequences in the cell genome. Analysis of the hybridization results suggested that, if the human breast cells contained MMTV-related sequences, they must be present in less than one copy per 100 cells. Thus, we have obtained no convincing evidence for the presence of retrovirus-like particles or subviral components in these cells. It is of course possible that these cells contain virus information but at levels below the sensitivity of our assay procedures.     

Ultrastructural comparison of Oncovirinae (type C), Spumavirinae, and Lentivirinae: three subfamilies of Retroviridae found in farm animals

The successive steps of maturation of seven retroviruses from five species of farm animals and one retrovirus from a mouse were compared in cell cultures. The viruses included three type C oncoviruses, one spumavirus, and three lentiviruses. Although members of the 3 subfamilies shared some gross morphologic features such as budding on plasma membranes, core, and surface projections, differences were noted in the ultrastructural detail of these features. Type C oncoviruses did not show any structural differentiation in identifiable form in the cytoplasm as opposed to characteristic features observed in the spumavirus and lentivirus subfamilies, respectively. Budding viruses were distinct among the 3 subfamilies. The type C bovine leukemia virus budding on vacuole membranes differed from the two other type C viruses by lacking an electron-lucent intermediate layer as did the lentiviruses. Differentiation between type C oncoviruses and lentiviruses could be confusing because of the similarity of the fully mature virions appearing in the intercellular space. However, each subfamily of retroviruses can be readily differentiated from one another when each morphologic stage of virus replication is examined by electron microscopy.     

Spontaneous appearance of cytopathology and rat C-type virus (WF-1) in a rat embryo cell line

An established line (WF-1) of Wistar/Furth rat embryo cells spontaneously developed cytopathology after about 2 years in vitro. The cellular changes appeared within 5–7 days after subculturing of the cultures and consisted of altered rounded and fusiform cells with dark, prominent nuclei. These changes increased after removal of bovine amniotic fluid from the culture nutrient but were not apparent if the cultures were subcultured at 3–4 day intervals. Most of the altered cells were viable, but they failed to multioly under the conditions employed. Electron microscopical examination of the WF-1 cells revealed numerous extracellular and budding C-type virus particles. The cells, when inoculated into rats, produced progressively growing fibrosarcomas which also contained C-type particles. Initially the WF-1 virus failed to produce visible effects in the REL line of Sprague-Dawley rat embryo cells. Several months later, upon concentration and banding at 1.16 g/cm³ in a sucrose gradient, low-titered infectious virus of about I TCID₅₀ per 10,000 virus particles was recovered. This produced foci of predominantly viable rounded cells in REL cultures similar to those induced by the C-type virus (RMTDV or BV-1) previously isolated from chemically induced rat mammary tumors. Immune BV-1 serum prevented the WF-1 virus from producing these cellular effects. Tests for properties characteristic of murine leukemia viruses (gs-1 antigen, gs-3 interspecies antigen, and the XC reaction) were negative indicating that the WF-1 virus was not a mouse virus. These observations suggested that upon prolonged cultivation in vitro the WF-1 rat embryo cells spontaneously liberated a rat C-type virus related to the BV-1. Whether the cell-altering and partially lethal effects produced by the WF-1 virus were due to some cytopathic C-type particles in a generally non-cytopathic population or reflected the concentration of only one biological type of particles needs further study.     

Viral inclusions and other cytoplasmic components in a Leydig cell murine tumor: an electron microscopic study

Cytoplasmic inclusion bodies which were clearly visible under light microscopy have recently been described in estrogen-induced testicular interstitial cell tumors of mice. An ultrastructural study has revealed that the inclusions were formed of considerable numbers of type A virus particles which measured approximately 76 mμ in external diameter. Despite the abundance of intracytoplasmic viral particles, there was no sign of extracellular release or budding of mature particles from cellular membranes. Among the unusual cytoplasmic components of the tumor cells were bundles of filaments with a wavy appearance and a distinctive cross-sectional pattern. These fibrillar structures may be a cellular manifestation of the presence of virus. In discussion, the inclusions of the testicular intestitial cell tumors are compared to inclusions found in other murine tumors.