The co-carcinogenic activity of 4-nitropyridine-1-oxide (4-npo) and prevention of transformation by type-specific anti-viral antibodies

Summary Fischer rat embryo cells chronically infected with Rauscher murine leukemia virus, and known to be sensitive to transformation by potent chemical carcinogens, were transformed by the weak carcinogen 4-nitropyridine-1-oxide. Transformed cells grew in semi-solid agar and produced tumors in newborn Fischer rats. Transformation was inhibited by antisera specific for the ecotropic Rauscher murine leukemia virus, but not by antisera of equal toxicity specific for xenotropic Swiss mouse AT-124 virus. This work was supported by contract NO1-CP-43240 within the Virus Cancer Program of the National Cancer Institute.     

Structural Proteins of Mammalian Oncogenic RNA Viruses: Murine Leukemia Virus Neutralization by Antisera Prepared Against Purified Envelope Glycoprotein

Goat and rabbit antisera prepared against a purified Rauscher murine leukemia virus glycoprotein (gp69/71) rapidly neutralized spleen focus-forming virus in Rauscher and Friend virus preparations. Absorption studies revealed that most of the neutralizing activity of goat anti-Rauscher virus gp69/71 serum was directed against type- and group-specific determinants.     

Structural studies on Rauscher murine leukemia virus: isolation and characterization of viral envelopes.

A preparative method for isolating pure viral envelopes from a type-C RNA tumor virus, Rauscher murine leukemia virus, is described. Fractionation of virions of Rauscher murine leukemia virus was studied after disruption of the virions with the detergents sodium dodecyl sulfate of Nonidet P-40 in combination with ether. Fractionation was performed through flotation in a discontinuous sucrose gradient and, as appeared from electron microscopic examination, a pure viral envelope fraction was obtained in this way. By use of sensitive competition radioimmunoassays or sodium dodecyl sulfate-polyacrylamide gel electrophoresis after immunoprecipitation with polyvalent and monospecific antisera directed against Rauscher murine leukemia virus proteins, the amount of the gag and env gene-encoded structural polypeptides in the virions and the isolated envelope fraction was compared. The predominant viral structural polypeptides in the purified envelope fraction were the env gene-encoded polypeptides gp70, p15(E), and p12(E), whereas, except for p15, there was only a relatively small amount of the gag gene-encoded structural polypeptides in this fraction.     

Genes controlling receptors for ecotropic and xenotropic type C virus in Mus cervicolor and Mus musculus.

Gene loci controlling cell surface receptors for murine leukemia virus were studied by using murine X Chinese hamster hybrid cells. Hybrids which exclusively segregate murine chromosomes were made by fusing Mus cervicolor and Mus musculus lymphocytes to hamster fibroblasts. Sensitivity to Moloney murine leukemia virus infecotion and specific binding of the envelope glycoprotein of Rauscher murine leukemia virus (gp70) cosegregate and isozyme analysis show an association with chromosome 5 in both species. With the possible exception of one clone, no evidence was found for a proviral integration site independent of chromosome 5. Evidence is presented for additional unlinked ectropic and xenotropic receptors independent of chromosome 5.     

Identification of an FMR cell surface antigen associated with murine leukemia virus-infected cells.

FMR antigens are found on the surface of cells infected with Friend, Moloney, and Rauscher murine leukemia viruses (MuLV). These antigens are serologically distinct from the G cell surface antigens that are found on cells infected with endogenous MuLV (AKR and Gross virus). Cell surface antigens of both virus groups are immunogenic in mice, and immunization with appropriate virus-infected cells leads to the production of cytotoxic antisera. The cytotoxic activity of FMR antisera can be absorbed by disrupted preparations of Rauscher MuLV, but not by AKR MuLV. FMR antisera precipitate the viral envelope proteins gp70, pl5(E), and p12(E) from detergent-disrupted preparations of [3H]leucine-labeled MuLV. The reaction of these antisera with p15(E) and p12(E) proteins is directed against group-specific antigens and can be absorbed with AKR MuLV; in contrast, the reaction of these antisera with gp70 is directed against type-specific antigens and is absorbed only by viruses of the FMR group. In immune precipitation assays with detergent-disrupted 125I surface-labeled cells, FMR antisera react only with type-specific antigens of the viral envelpe protein. On the basis of these findings we conclude that the FMR cell surface antigen is a determinant on the MuLV env gene product.     

Plasma membrane glycoproteins encoded by cloned Rauscher and Friend spleen focus-forming viruses.

Rauscher spleen focus-forming virus (SFFV) was cloned free of its helper virus into normal rat kidney and mouse fibroblasts, and the resulting nonproducer fibroblast clones were analyzed. Our results suggested that Rauscher SFFV encodes a glycoprotein with an apparent Mr of 54,000 (gp54) that reacts with antisera made to the envelope glycoprotein (gp70) of ecotropic murine leukemia viruses, as well as with a rat antiserum that reacts with the gp70's of dual-tropic mink cell focus-inducing and HIX viruses but not with the gp70's of ecotropic viruses. In these respects and in its tryptic peptide map, Rauscher SFFV-encoded gp54 is nearly identical to the gp55 glycoprotein which we previously reported to be encoded by Friend SFFV (Dresler et al., J. Virol. 30:564--575, 1979). However, gp54 is slightly smaller, and it lacks one methionine-containing tryptic peptide that occurs in gp55. Studies with cytotoxic antiserum in the presence of complement and with a rosetting technique which employed sheep erythrocytes coupled to protein A suggested that the gp54 and gp55 glycoproteins are weakly expressed on the surface membranes of SFFV-infected cells. In addition, the Rauscher SFFV genome also encodes gag polyproteins which appear to be identical to the gag polyproteins encoded by helper Rauscher murine leukemia virus, but differ from the antigenically related polyproteins encoded by some but not all clones of Friend SFFV. Furthermore, the glycosylated gag polyproteins encoded by Rauscher SFFV and by some Friend SFFVs also appear to be expressed on the surface membranes of infected cells. These results suggest that similar env gene recombination and partial deletion events were involved in the independent origins of two different strains of acute erythroleukemia virus.     

Glycoproteins of friend murine leukemia virus: separation and NH2-terminal amino acid sequences of gp69 and gp71.

The NH2-terminal amino acid sequences (initial 23 residues) of Friend murine leukemia virus gp71 and gp69 were determined and found to be different but highly related. Friend murine leukemia virus gp71 differed from Rauscher murine leukemia virus gp70 in only one position. Friend murine leukemia virus gp69 showed approximately 41% homology to these glycoproteins but lacked the glycosylation site (sequon) occurring at position 12 in Rauscher murine leukemia virus gp70.     

Persistent infection of a rat kidney cell line with Rauscher murine leukemia virus

Duc-Nguyen, Huu (National Cancer Institute, Bethesda, Md.), Edith N. Rosenblum, and Robert F. Zeigel. Persistent infection of a rat kidney cell line with Rauscher murine leukemia virus. J. Bacteriol. 92:1133-1140. 1966.-The propagation of a murine leukemia virus (Rauscher) in a kidney cell line, derived from a rat with lymphoid leukemia, was studied. A complement-fixing (CF) antigen reacting with Rauscher immune sera was detected at various passage levels, which correlated with the visualization by use of electron microscopy of viral buds and viral particles in different stages of maturation in all passages. Five-month-old monolayers continued to shed virus and to yield high CF antigen titers. The cell-free supernatant fluid from cultures of the 14th passage was shown to be infectious for a normal rat kidney cell line, as evidenced by the appearance of the CF antigen in this line. Interferon production was not demonstrated in infected cultures. The overall data indicated that rat kidney cells could be used to propagate Rauscher virus in a carrier state.     

Spontaneous Release of Endogenous Ecotropic Type C Virus from Rat Embryo Cultures

Type C viruses were isolated from embryo cultures of two different rat strains, Sprague-Dawley and Fischer. Both viruses (termed rat leukemia virus, RaLV) were released spontaneously from rat embryo cells, have a density of 1.14 to 1.15 g/cm(3) based on equilibrium sedimentation in sucrose gradients, contain 60-70S RNA, RNA-directed DNA polymerase, and rat type C virus-specific 30,000 molecular-weight-protein determinants. Molecular hybridization studies using the Sprague-Dawley RaLV 60-70S RNA show that the virus-specific nucleotide sequences are present in the DNA of rat embryos. Both Sprague-Dawley and Fischer RaLV can rescue the murine sarcoma virus genome from Kirsten murine sarcoma virus-transformed nonproducer cells and are neutralized by antisera to the RPL strain of RaLV. In contrast to previous RaLV's, these viruses propagate in their own cells of origin as well as in cells of heterologous rat strains.     

Impaired processing of precursor polypeptides of temperature-sensitive mutants of Rauscher murine leukemia virus.

The synthesis and processing of virus-specific precursor polypeptides in NIH/3T3 cells infected at the permissive temperature (31 degrees C) with temperature-sensitive (ts) mutants of Rauscher murine leukemia virus was studied in pulse-chase experiments at the permissive and nonpermissive (39 degrees C) temperatures. The newly synthesized virus-specific polypeptides were analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis after immunoprecipitation with polyvalent and monospecific antisera against Rauscher murine leukemia virus proteins. In cells infected with ts mutants defective in early replication steps (the early mutants ts17 and ts29), and ts mutants defective in postintegration steps (the late mutants ts25 and ts26), the processing of the primary gag gene product was impaired at the nonpermissive temperature. gag-pr75 of all four mutants was converted into gag-pr65; however, gag-pr65 accumulated at the nonpermissive temperature, and the main internal virion polypeptide p30 was not formed. Therefore, the proteolytic cleavage is blocked beyond gag-pr65. Concomitantly, the formation of the env gene-related polypeptide p12(E) of all four mutants was blocked at the restrictive temperature. In contrast, cells infected with the late mutant ts28, which produced noninfectious virions at 39 degrees C, showed a normal turnover of the gag and env precursor polypeptides.     

Sialylatin of glycoproteins of murine mammary tumor virus, murine leukemia virus, and Mason-Pfizer monkey virus.

Neuraminidase treatment of mouse mammary tumor virus, Rauscher murine leukemia virus, and Mason-Pfizer monkey virus resulted in loss of their capacity to inhibit hemagglutination of influenza virus. Hemagglutination-inhibition activity of these RNA tumor viruses could be restored by in vitro resialylation catalyzed by sialyl transferase. The major glycoprotein in the intact envelope of desialylated and, to some extent, native virions could be specificallly labeled in vitro with CMP-(14C) sialic acid. These studies further characterize the individual glycoproteins of mouse mammary tumor virus, Rauscher murine leukemia virus, and Mason-Pfizer monkey virus.     

5''-terminal nucleotide sequences of the Rauscher leukemia virus and gibbon ape leukemia virus genomes exhibit a high degree of correspondence.

The 5'-terminal regions of gibbon ape leukemia virus-Hall's Island and Rauscher murine leukemia virus have been completely sequenced. The chain length for the 5'-terminal region of Rauscher murine leukemia virus is 140 nucleotides, and that for gibbon ape leukemia virus-Hall's Island is 144 nucleotides. An alignment of the sequences maximizing the number of ocrrespondences with the minimum introduction of gaps shows 81% nucleotide matches. From the complementary RNA, secondary structures of this region have been proposed. These data demonstrate the conservation of the 5'-terminal genetic sequences of these viruses and strongly reinforce the concept that viruses of murine origin and viruses of the gibbon ape leukemia virus-Simian sarcoma-associated virus group are closely related.     

Recovery of Rauscher Leukemia Virus from Large Volumes of Seeded Cow's Milk and from Infected Murine Spleens

Rauscher murine leukemia virus was used as an indicator agent to develop a methodology for the extraction and concentration of a theoretical leukemia virus from bovine milk and tissues. The indicator virus was seeded into cow''s milk or was recovered from infected murine spleens. The tissue homogenates and the defatted milk were processed in a B-XVI rotor of a Spinco L-4 ultracentrifuge at a flow rate of 3 liters/hr. The efficiency of Rauscher virus recovery was greatest when the rotor was used without a gradient. A loss of between 0.6 and 0.7 log of total infectious virus, as determined by the spleen assay method, resulted when the seeded milk and murine spleens were processed. The procedures developed are presently being used in transmission experiments in an attempt to induce leukemia in the bovine.     

Recovery of Rauscher Leukemia Virus from Large Volumes of Seeded Cow''s Milk and from Infected Murine Spleens

Rauscher murine leukemia virus was used as an indicator agent to develop a methodology for the extraction and concentration of a theoretical leukemia virus from bovine milk and tissues. The indicator virus was seeded into cow's milk or was recovered from infected murine spleens. The tissue homogenates and the defatted milk were processed in a B-XVI rotor of a Spinco L-4 ultracentrifuge at a flow rate of 3 liters/hr. The efficiency of Rauscher virus recovery was greatest when the rotor was used without a gradient. A loss of between 0.6 and 0.7 log of total infectious virus, as determined by the spleen assay method, resulted when the seeded milk and murine spleens were processed. The procedures developed are presently being used in transmission experiments in an attempt to induce leukemia in the bovine.     

Cell-Cycle Dependent Appearance of Murine Leukemia-Sarcoma Virus Antigens

Normal rat kidney (NRK) cells, NRK cells infected with Rauscher murine leukemia virus, and NRK cells infected with Kirsten murine sarcoma-leukemia virus (NRK-K) were synchronized by a double thymidine block. At intervals after release from thymidine blockage, the cells were examined for the presence of viral antigens in the cytoplasm and on the cell surface by immunofluorescent microscopy by using goat anti-Rauscher murine leukemia virus and goat anti-Moloney leukemia virus (Tween-ether disrupted) sera. Detection of viral antigens in the cytoplasm was periodic during the cell cycle. Antigens were detected first during the S phase, increased during the G2 phase, and disappeared during the M and G1 phases. A similar pattern of surface immunofluorescence was observed. Infectious virus was detected in culture fluids from synchronized cells during the M phase. Surface immunofluorescence was detected in NRK-K cells with anti-Rauscher murine leukemia virus and may represent the presence of group-specific antigens on the cell surface. Control, uninfected NRK cells, which did not normally fluoresce, showed weak immunofluorescence during the S and G2 phases after synchronization. Synchronization can be used to amplify latent oncornavirus expression.     

Isolation of Naturally Occurring Viruses of the Murine Leukemia Virus Group in Tissue Culture

A tissue culture cell system for isolation and identification of members of the murine leukemia virus group (the complement fixation for murine leukemia test) was modified to permit the isolation of naturally occurring virus from leukemic and normal mice. The important factors for increasing the sensitivity of the test were the use of National Institutes of Health (NIH) strain Webster Swiss embryo cell cultures and the selection of rat-immune sera having complement-fixing antibodies to tissue culture antigens of both the Gross and FMR subgroups. In all, 163 strains of mouse leukemia virus, from 11 inbred mouse strains, have been isolated. Representative virus isolates were shown to possess the properties of the murine leukemia virus group; i.e., they were chloroform-sensitive, noncytopathic agents which replicated in mouse embryo tissue culture and produced group-reactive, complement-fixing antigen and budding C-type particles visible by electron microscopy. These viruses could serve as helpers in the rescue of Moloney sarcoma virus genome from non-producer hamster sarcoma cells, yielding pseudotypes. All of the 19 field isolates tested were neutralized by Gross passage A antiserum but not by potent antisera to the Moloney, Rauscher, and Friend strains. Virus was recovered regularly from embryos and from the plasma and spleen of adult mice of high leukemic strains. In low leukemic mouse strains, different patterns of virus detection were observed. In C3H/He mice, virus was occasionally present in embryos and was found in 40% of adult spleens. BALB/c mice were virus-negative as fetuses or weanlings, but spleens of more than half of the mice over 6 months of age yielded virus. NIH mice have never yielded virus. In reciprocal matings between AKR and BALB/c mice, virus recovery from embryos was maternally determined. The development of tissue culture isolation procedures made possible for the first time the application of classical infectious disease methods to the study of the natural history of murine leukemia virus infection.     

Host Effect on Arbovirus Replication: Appearance of Defective Interfering Particles in Murine Cells

Serial passage of Semliki Forest virus (SFV) in chicken embryo cells had little effect on SFV yield; however, high multiplicity infection of murine cells with one of the late passage pools (passage 9 SFV) resulted in a virus yield 10- to 20-fold lower than that obtained with earlier passage virus and 80-fold lower than the corresponding yield in chicken cells. This effect was accompanied by a striking decrease in the levels of 42S and 26S RNA and by increased proportions of a small single-stranded viral RNA (molecular weight, 9 x 10(5)) and of a low-molecular-weight replicative form. There was also a reduction in the number of specific membranous structures previously associated with the group A arbovirus replication complex. These results suggested that passage 9 SFV contained defective interfering particles which were detected more readily after one passage in a murine indicator host cell. Identical results were obtained with two different murine cell lines: one a leukemia virus-free clone of AKR cells and the other JLS-V9 cells chronically infected with Rauscher leukemia virus. Host production of RNA tumor virus particles apparently did not affect arbovirus replication.     

Membrane receptors for murine leukemia viruses: characterization using the purified viral envelope glycoprotein, gp71.

The 71,000 dalton glycoprotein (gp71) purified from Rauscher murine leukemia virus (R-MuLV) by affinity chromatography specifically binds to murine but not other mammalian cells in culture. Binding is prevented by specific antiserum raides to gp71 (anti-gp71). The binding assay as described in this report can detect receptors on as few as 300 murine cells, and with 1 X 10(5) cells gives significant binding with 30 sec. The results show that the purified glycoprotein retians biologic activity and can form a stable complex with specific receptors on mouse cell membranes. The assay can therefore be used to characterize the nature of the cellular receptors that are essential for leukemia virus infection. Purified gp71 binding to mouse cells is prevented if the cells are actively producing related ecotropic type C viruses, presumably because the receptors are occupied and are not available to bind exogenously applied gp71. The binding of gp71 to murine cells is enhanced by the presence of calcium ions and low pH. Binding studies performed using an excess of 125I-gp71 indicate the NIH/3T3 cells bind approximately 5.3 X 10(5) molecules of 125I-gp71 per cell.