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.     

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.     

Type C RNA virus proteins: lack of binding specificity to host cell chromosomal DNA in vitro.

Through the use of two different DNA-protein reconstitution methods, we examined the potential role of type C RNA tumor virus proteins as putative regulatory agents in the control of virus expression. Rauscher murine leukemia virus, purified from chronically infected rat cell cultures, was iodinated in vitro with [125I], and dissociated in a nondetergent high-ionic-strength urea-containing buffer. The chemically separated [125I]-labeled viral polypetides were reconstituted with purified DNA by affinity column chromatography and by gradient dialysis renaturation. In both instances, no detectable amount of radioactivity specifically bound to double-stranded DNA of any origin. This observation was in contrast to the binding behavior of identically prepared and radiolabeled, nonhistone chromosomal proteins purified from rat cell nuclei. This finding may rule out, in eukaryotic cells, a well-described prokarytoic regulatory mechanism for the control of integrated viral gene expression.     

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.     

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.     

Sulfated components of enveloped viruses.

The glycoproteins of several enveloped viruses, grown in a variety of cell types, are labeled with 35SO4(-2), whereas the nonglycosylated proteins are not. This was shown for the HN and F glycoproteins of SV5 and Sendai virus, the E1 and E2 glycoproteins of Sindbis virus, and for the major glycoprotein, gp69, as well as for a minor glycoprotein, gp52, of Rauscher leukemia virus. The minor glycoprotein of Rauscher leukemia virus is more highly sulfated, with a ratio of 35SO4- [3H]glucosamine about threefold greater than that of gp69. The G protein of vesicular stomatitis virus was labeled when virions were grown in the MDBK line of bovine kidney cells, although no significant incorporation of 35SO4(-2) into this protein was observed in virions grown in BHK21-F line of baby hamster kidney cells. In addition to the viral glycoproteins, sulfate was also incorporated into a heterogenous component with an electrophoretic mobility lower than that of any labeled with 35SO4(-2) and [3H]leucine, this component had a much greater 35S-3H ratio than any of the viral polypeptides and thus could not represent aggregated viral proteins. This material is believed to be a cell-derived mucopolysaccharide and can be removed from virions by treatment with hyaluronidase without affecting the amount of sulfate present on the glycoproteins.     

Genetic analysis of myeloproliferative leukemia virus, a novel acute leukemogenic replication-defective retrovirus.

The myeloproliferative leukemia virus (MPLV) is a new acute leukemogenic, nonsarcomatogenic retroviral complex that is generated during the in vivo passage of a molecularly cloned Friend ecotropic helper virus. Examination of viral RNA expression in MPLV-producing cells revealed the presence of two distinct molecular species that hybridized with a long terminal repeat or an ecotropic env-specific probe but not with a xenotropic mink cell focus-forming virus env-specific probe derived from a spleen focus-forming virus: an 8.2-kilobase species corresponding to a full-length Friend murine leukemia virus (F-MuLV) and a deleted species with a genomic size of 7.4 kilobases. This deleted virus was biologically cloned by limiting dilutions and single cell cloning in Mus dunni fibroblasts. Three nonproducer clones with normal morphologies and containing one single integrated copy of the deleted virus were superinfected with F-MuLV, Moloney murine leukemia virus, Gross murine leukemia virus, mink cell focus-forming virus (HIX), or the amphotropic 1504 murine leukemia virus. All pseudotypes caused macroscopic and microscopic abnormalities in mice that were similar to those seen in the parental stock. A comparison of the physical maps of F-MuLV and MPLV, which was deduced from the restriction enzyme digests of unintegrated proviral DNAs, indicated that the MPLV-defective genome (i) is probably derived from F-MuLV, (ii) has conserved the F-MuLV gag and pol regions, and (iii) is deleted and rearranged in the env region in a manner that is clearly distinct from that of Friend or Rauscher spleen focus-forming viruses.     

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

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.     

In vitro production of Rauscher murine leukemia virus: influence of culture age on biological properties.

Large-scale production and concentration procedures have been standardized to study the biological properties of Rauscher leukemia virus produced from the high-passaged JLS-V9-H mouse bone marrow cell line. Virus produced early (days 4 to 6) in the harvest and refeed cycle contained higher levels of ribonucleic acid-directed deoxyribonucleic acid polymerase activity and was more infectious than Rauscher leukemia virus produced later (days 7 to 10) in the growth period. The peak of virus production as detected by physical assays (virus particle count, protein, and p30 antigen) was highest at day 6, whereas the optimum biological and ribonucleic acid-directed deoxyribonucleic acid polymerase activity occurred 24 h earlier. When product characterization values of each concentrate were adjusted to a specific activity (i.e., per milligram of protein) basis, virus particle counts averaged 4 x 10(11) through days 5 to 9, and the peak infectivity occurred at day 4, whereas ribonucleic acid-directed deoxyribonucleic acid polymerase activity was highest at day 4 (endogenous) and 5 (exogenous). Sodium dodecyl sulfate-polyacrylamide gel analysis revealed only slight differences in the polypeptide pattern of Rauscher leukemia virus harvested from cultures of varying age, although Rauscher leukemia virus produced between days 3 and 5 contained more glycoprotein than either earlier or later harvests.     

In vitro production of Rauscher murine leukemia virus: influence of culture age on biological properties.

Large-scale production and concentration procedures have been standardized to study the biological properties of Rauscher leukemia virus produced from the high-passaged JLS-V9-H mouse bone marrow cell line. Virus produced early (days 4 to 6) in the harvest and refeed cycle contained higher levels of ribonucleic acid-directed deoxyribonucleic acid polymerase activity and was more infectious than Rauscher leukemia virus produced later (days 7 to 10) in the growth period. The peak of virus production as detected by physical assays (virus particle count, protein, and p30 antigen) was highest at day 6, whereas the optimum biological and ribonucleic acid-directed deoxyribonucleic acid polymerase activity occurred 24 h earlier. When product characterization values of each concentrate were adjusted to a specific activity (i.e., per milligram of protein) basis, virus particle counts averaged 4 x 10(11) through days 5 to 9, and the peak infectivity occurred at day 4, whereas ribonucleic acid-directed deoxyribonucleic acid polymerase activity was highest at day 4 (endogenous) and 5 (exogenous). Sodium dodecyl sulfate-polyacrylamide gel analysis revealed only slight differences in the polypeptide pattern of Rauscher leukemia virus harvested from cultures of varying age, although Rauscher leukemia virus produced between days 3 and 5 contained more glycoprotein than either earlier or later harvests.     

Enhanced production of Rauscher leukemia virus after infection of high-passaged JLS-V9 cells.

JLS-V9, a mouse bone marrow cell line infected with Rauscher leukemia virus at high passage level, produced larger amounts of virus than the standard JLS-V10 cells. The enhanced virus production was attributed to the increased saturation density of JLS-V9 cells.     

Basolateral maturation of retroviruses in polarized epithelial cells.

We have investigated the maturation sites of avian and mammalian C-type retroviruses in polarized epithelial cells. Examination of thin sections of Madin Darby canine kidney cells infected with RD114 or avian reticuloendotheliosis virus revealed that these viruses mature from the basolateral membrane domains. Similar results were obtained with a continuous line of mouse mammary epithelial cells infected with Friend, Moloney, Rauscher, or Kirsten murine leukemia viruses, or Friend virus-related or Moloney virus-related mink cell focus-forming viruses. Immunofluorescence observations indicate that viral glycoproteins are inserted only at the basolateral membranes in these cells. Because of the availability of DNA and protein sequence data, and of molecularly cloned viruses, these virus systems offer advantages for molecular studies on directional transport of plasma membrane glycoproteins.     

Purification of the Moloney and Rauscher Murine Leukemia Viruses by Use of Zonal Ultracentrifuge Systems

The B-IV and B-IX zonal ultracentrifuge rotors were applied to the concentration and purification of the Moloney and Rauscher murine leukemia viruses from large volumes of infected tissue culture fluids and animal materials. Potassium tartrate, potassium citrate and sucrose gradients were used to obtain viral concentrates from the density 1.16 to 1.18 zone. Proteolytic enzyme digestion of tissue culture preparations prior to zonal ultracentrifuge processing was effective in releasing virus from cell debris and producing highly purified, though nonleukemogenic, viral concentrates. Infected Rauscher mouse plasma was processed to give highly purified infectious virus fractions. A single centrifugation of crude Rauscher mouse spleen homogenates resulted in partially purified infectious concentrates with high virus particle counts.     

Expression of the Major Internal Viral Polypeptide in Cells Transformed by Wild-Type and Temperature-Sensitive Murine Sarcoma Virus

Phenotypic expression of the murine intraspecies and interspecies antigenic determinants of the major type C viral structural 30,000-dalton polypeptide, p30, was measured by radioimmunoassay inhibition in cell lines from different species. Uninfected normal rat kidney (NRK) cells did not contain detectable levels of murine intraspecies and interspecies p30 antigen, whereas rat cells transformed by and producing murine sarcoma virus (MSV)-Moloney leukemia virus (M-MSV-MuLV) contained high levels of both murine intraspecies and interspecies p30 antigen. Significant amounts of murine intraspecies and interspecies p30 antigen were detected in wild-type MSV-transformed nonproducer NRK cells. The control of p30 antigen expression was examined in temperature-sensitive MSV-transformed nonproducer cells [NRK(MSV-1b)] which are cold sensitive for maintenance of the transformed phenotype. Both murine intraspecies and interspecies p30 antigens were detected in NRK(MSV-1b) cells when grown at the permissive (39 C) or nonpermissive (33 C) temperature, suggesting that p30 antigen expression is not correlated with maintenance of the transformed phenotype. The results demonstrate that previously undetectable p30 antigens are expressed in MSV-transformed nonproducer NRK cells, and suggest that the expression of p30 antigen may be a useful marker for viral gene expression in mammalian cells.     

Gangliosides of chemically and virally transformed rat embryo cells.

The gangliosides of control rat embryo cells, 3-methylcholanthrene, Rauscher leukemia virus, and combined 3-methylcholanthrene-Rauscher leukemia virus transformants were examined using [14 C]glucosamine as a tracer. All four cell lines exhibited a complex pattern of gangliosides. While N-acetylgalactosaminyl-(N-acetylneuraminyl)-galactosyl-glucosyl-ceramide was the major ganglioside in the control cell line, N-acetylneuraminyl-galactosyl-glucosyl-ceramide was the major ganglioside in the three transformants. The 3-methylcholanthrene transformant possessed a ganglioside pattern different from that of the Rauscher leukemia virus transformant. Hydrolysis of the gangliosides indicated that galactosamine, N-acetyl-and N-glycolylneuraminic acid were the labeled components in all cell lines.     

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.     

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.     

Transcription of 70S RNA by DNA polymerases from mammalian RNA viruses.

DNA polymerases purified by the same procedure from four mammalian RNA viruses, simian sarcoma virus type 1, gibbon ape lymphoma virus, Mason-Pfizer monkey virus, and Rauscher murine leukemia virus are capable of transcribing heteropolymeric regions of viral 70S RNA without any other primer. In this reconstituted system the enzymes from simian sarcoma virus type 1, Mason-Pfizer monkey virus, and Rauscher murine leukemia virus transcribe viral 70S RNA almost as efficiently as the DNA polymerase from the avian myeloblastosis virus, but gibbon ape lymphoma virus DNA polymerase is approximately three-to fivefold less efficient. Although there is a substantial difference among the sizes of these DNA polymerases (160,000 daltons for the avian myeloblastosis virus enzyme, 110,000 daltons for the Mason-Pfizer monkey virus enzyme, and 70,000 daltons for the mammalian type C viral polymerases), the ability to transcribe viral 70S RNA is a characteristic common to these enzymes.