JLS-V9 Cells Research Articles

The induction of myeloid leukaemias by Rauscher murine leukaemia virus.

A number of cloned viral preparations isolated from Rauscher virus-producing JLS-V5 cells were compared in their competence to induce different types of leukaemias. All preparations were able to induce myeloid leukaemias, but the induction of lymphatic or erythroid leukaemias was also observed. Serial infection of newborn mice with either cell-free extracts or serum from animals suffering from a myeloid leukaemia did not result in the occurrence of relatively more myeloid leukaemias nor did the infection with virus harvested from ascites fluid of permanent myeloid cell lines. It appears that the mechanism by which myeloid leukaemias are induced is not virus-specific.

Biological, chemical, and immunological studies of Rauscher ecotropic and mink cell focus-forming viruses from JLS-V9 cells.

Two murine leukemia viruses were isolated from JLS-V9 cells which had been infected with Rauscher plasma virus. One virus was XC positive and failed to grow on mink or cat cells and thus was an ecotropic virus. The other virus formed cytopathic foci on mink cells, was XC negative, and fell into the mink cell focus-forming (MCF) viral interference group and was thus an MCF virus. The glycoproteins of the two viruses could be distinguished immunologically, by peptide mapping, and by size in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The MCF virus produced gp69, and the ecotropic virus produced gp71, explaining the origin of the heterogeneous glycoprotein (gp69 and gp71) of Rauscher leukemia virus. Amino-terminal sequences of gp69 and gp71 were determined. The MCF sequence was distinct from the ecotropic sequence, but retained partial homology to it. The data show that the glycoproteins are encoded by related yet distinct genes. The protein structural data support the proposal that MCF virus gp70 molecules have nonecotropic sequences at the amino terminus, with ecotropic sequences occurring at the 3' end of the gene. The Rauscher MCF virus glycoprotein lacks a glycosylation site found at position 12 of the ecotropic sequence.

Characterization of glycosaminoglycans associated with Rauscher murine leukemia virions.

Host-derived sulfated components that copurify and are physically associated with the envelope of Rauscher murine leukemia virions grown in JLS-V9 cells were characterized by digestion with chondroitinase ABC and chondroitinase AC II, as well as nitrous acid degradation. A dermatan-sulfate-chondroitin-sulfate copolymer and heparin or heparan sulfate wee shown to be associated with the virions. Competitive binding studies indicated a specificity of the virions for association with heparan sulfate. The physiological importance of the association is discussed.

Further Characterization of Virus-like 30S (VL30) RNA of Mice: Initiation of Reverse Transcription and Intracellular Synthesis

We have studied the virus-like 30S (VL30) RNA sequences of mice. Previous work has shown that these sequences are coded in the mouse genome, expressed in some normal cells and released as pseudotypic particles from cells producing murine C-type retroviruses. VL30 sequences have some similarities to standard retrovirus RNA, but differences also exist. To further assess the similarities and differences, several aspects of VL30-specific metabolism were investigated. We studied the initiation of VL30-specific DNA synthesis during an endogenous reverse transcriptase reaction. Short initial VL30-specific cDNA transcripts were covalently attached to RNA as measured by equilibrium banding in caesium sulphate density gradients. Therefore, reverse transcription of VL30-specific cDNA is initiated by an RNA primer. The intracellular synthesis of VL30 RNA was investigated by pulse labelling uninfected JLS-V9 cells with 3H-uridine. Hybridization of the pulse-labelled nuclear RNA indicated that the major VL30-specific RNA evident after a 15 min label was the same size as the mature VL30 RNA. Thus, VL30 RNA is apparently not synthesized via a higher mol. wt. precursor. Both of these results demonstrate similarity of VL30 RNA sequences to standard retroviruses. One unique feature of VL30 RNA was detected. JLS-V9 cells contained both the monomeric VL30 RNA and a hydrogen-bonded 38S form which yielded the monomer when denatured. This contrasts with standard murine leukaemia virus which is only found as a monomer within cells.

Murine leukemia virus proteins expressed on the surface of infected cells in culture.

Infection of JLS-V9 cells in culture with Rauscher murine leukemia virus induced the appearance on the cell surface of two classes of viral proteins: Rauscher murine leukemia virus gp70, and glycoproteins related to the viral core (gag) proteins with apparent molecular weights in sodium dodecyl sulfate polyacrylamide gels of 80 x 10(3) and 95 x 10(3). The latter proteins were identified by lactoperoxidase-catalyzed iodination of the cell surface and by metabolic labeling with [(3)H]mannose followed by immunoprecipitation with an antiserum directed against the major viral core protein, p30. Tryptic peptide maps of chloramine T-iodinated proteins indicated that 80 x 10(3) - and 95 x 10(3)-molecular-weight proteins were closely related. The 95 x 10(3)-molecular-weight protein from Rauscher murine leukemia virus-infected cells had a tyrosine fingerprint which was identical to that of the 95 x 10(3)-molecular-weight gag surface polyprotein of endogenous virus-producing AKR-A cells, suggesting that expression on the cell surface of glycosylated forms of gag precursor polyproteins may not be an exclusive property of leukemic thymocytes, but a more general phenomenon in murine leukemia virus infection. Tryptic fingerprint analysis of iodinated viral and cell-bound gp70's before and after desialylation indicated a lower level of glycosylation in the cell-bound gp70 population than in virions. Analysis of only surface-iodinated gp70 showed a simple pattern of exposed tryptic peptides which was very similar in Rauscher murine leukemia virus-infected cells and in AKR-A cells.

Aminopeptidase activity associated with purified murine leukaemia viruses.

Rauscher leukaemia virus (RLV), extensively purified by rate and density gradient centrifugation procedures, exhibited aminopeptidase (AP) activity. The amount of virion activity was about 0.005 times the specific activity of purified hog kidney aminopeptidase M (EC 3.4.11.2). The activity was also found in purified Moloney and Gross leukaemia viruses, but not in comparable gradient fractions of uninfected JLSV-9 cells. Furthermore replacement of serum by bovine serum albumin in the growth medium of virus producing cells did not affect the AP activity. These results indicate that murine leukaemia viruses (MuLV) possess a tightly bound AP activity which is present as a minor component of the virion preparation and is probably not due to host cell membrane or serum contamination. Characterization of the pH optimum and substrate specificity show the MuLV aminopeptidase activity is similar to but different from both hog kidney, leucine aminopeptidase (EC 3.4.11.1) and aminopeptidase M (EC 3.4.11.2).

Activation of the Endogenous Proviral Genes in Mouse Cells Is Not Followed by Increased Sensitivity to Deoxyribonuclease I Digestion

The disposition of chromosome proteins about the endogenous proviral DNA of BALB-c mouse has been studied. The sensitivity of the endogenous proviral DNA sequences to deoxyribonuclease I (DNaseI) was analysed in BALB-c mouse tissues (liver and spleen) and in the cell line JLS-V9 which does not produce virus. On all of these preparations the endogenous proviral DNA was as sensitive to DNase I digestion as total chromatin. Since the proviral genes in JLS-V9 cells were silent, it was of interest to study possible changes in the chromatin structure following virus induction by iododeoxyuridine. We could not detect any increase in the sensitivity of the endogenous proviral DNA to DNase I digestion following induction. The induction was very efficient, however, since 60% of the cells responded to produce intracellular virus antigens.

Physical map of biologically active Harvey sarcoma virus unintegrated linear DNA

BALB/c JLS V9 cells recently infected with Harvey sarcoma virus-murine leukemia virus (HSV-MuLV) complex contained unintegrated HSV linear DNA of 6.0-kilobase pair mass. The cells also contained two HSV closed circular DNA species along with MuLV-encoded linear and closed circular DNA species. HSV 6.0-kilobase pair linear DNA induced focal transformation upon transfection of NIH 3T3 mouse fibroblasts, and the biological activity of HSV DNA did not require helper MuLV functions. A physical map of restriction endonuclease cleavage sites along HSV 6.0-kilobase pair linear DNA was derived. Comparison of this map with one for Moloney MuLV DNA showed that the HSV and Moloney MuLV genomes are identical near their viral RNA 3' ends.

Glycopeptides of Murine Leukemia Viruses I. Comparison of Two Ecotropic Viruses

The glycopeptides obtained by pronase digestion of two ecotropic strains of murine leukemia virus (MuLV) were compared by gel filtration. Four different glycopeptide size classes, designated G(1), G(2), G(3), and G(4), with molecular weights of approximately 5,100, 2,900, 2,200, and 1,500, respectively, were shown to be associated with Rauscher MuLV virions grown in JLS-V9 cells. Various sugar precursors, including glucosamine, galactose, fucose, and mannose were incorporated into G(1) and G(2), suggesting that these are complex (type I) glycopeptides. The two smaller glycopeptide size classes, G(3) and G(4), were shown to be mannoserich (type II) glycopeptides. G(4) was more sensitive to digestion with endo-beta-N-acetylglucosaminidase H than G(3), suggesting that the core of G(3) may contain fewer mannose residues. Glycopeptides of the same size class as G(1) and G(2) were associated with both Rauscher MuLV and AKR-MuLV grown in III6A (mouse embryo) cells. Previous studies have shown that gp52, a proteolytic cleavage product of gp70, possessed primarily G(1) glycopeptides and that gp52 was more highly sulfated than gp70. We observed that G(1) is approximately twofold more highly sulfated than G(2), explaining the observed difference in sulfation of gp52. The unusually large size of G(1) suggested that infection with MuLV may alter the host cell glycosylation pattern. To test this possibility, glycopeptides from Sindbis virions grown in uninfected and Rauscher MuLV-infected JLS-V9 cells were compared, and no differences were observed. G(1) was not detected in Sindbis virions, indicating that acquisition of G(1) depends on properties of the virus-coded polypeptide backbone of the gp70 molecule.

Interferon-induced synthesis of a 63,000 dalton protein in mouse cells

Cultures of mouse JLSV5 cells (a cell line chronically infected with Rauscher murine leukemia virus) and of fresh uninfected NMRI mouse embryo fibroblasts were treated with interferon and labelled with ( 35S)-methionine. Newly synthesized proteins were then examined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and two-dimensional electrophoresis of the cell lysates. In both cell types interferon treatment resulted in the synthesis of a radioactive 63,000 dalton protein, which was undetectable in a radioactive form in the control cells. The possibility is considered that this protein is a mediator of the biological activities of interferon on cells.

Post-translational modification of Rauscher leukemia virus precursor polyproteins encoded by the gag gene.

Post-translational modifications of retrovirus gag gene-encoded polyproteins include proteolytic cleavage, phosphorylation, and glycosylation. To study the sequence of these events, we labeled JLS-V9 cells chronically infected with Rauscher murine leukemia virus in pulse-chase experiments with the radioactive precursors [35S]methionine, [14C]mannose, [3H]glucosamine, and [32P]phosphate. Newly synthesized gag polyproteins which incorporated label, and the modified products derived from them, were identified by immunoprecipitation of cell lysates with anti-p30 rabbit serum, followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography. Pulse-chase experiments were carried out in the presence as well as in the absence of tunicamycin, an inhibitor of glycosylation. Among the three major polyproteins synthesized in the absence of tunicamycin, two were found to be glycosylated but not phosphorylated. These were designated gPr80gag and gP94gag. Both shared identical [35S]methionine peptides with Pr65gag and p30. Of the two nonglycosylated precursors, Pr65gag and Pr75gag, only Pr65gag was found to be detectably phosphorylated, and Pr75gag could be readily identified only when glycosylation was inhibited. On the basis of these results, a scheme for the post-translational modification of gag polyproteins is proposed. According to this scheme the gag gene-encoded polyproteins are processed from a common precursor, Pr75gag, by two divergent pathways: one leading through the intermediate Pr65gag to internal virion components via cleavage and phosphorylation and the other via tunicamycin-sensitive mannosylation to the intermediate gPr80gag, which is further glycosylated to yield cell surface polyprotein gP94gag.

Virus-Like 30S RNA in Mouse Cells

Uninfected JLS-V9 mouse cells are known to express high levels of viral sequences that hybridize to complementary DNA made by the BrdU-induced virus of JLS-V9 cells. The genome in the BrdU-induced virus has been found to consist mainly of an RNA species that migrates as 30S RNA material during electrophoresis through agarose gels. This virus-like 30S RNA, designated VL30 RNA, apparently represents a new class of endogenous defective retroviruses that are not generally evident because of their defectiveness and lack of biological function. Fingerprint analysis and hybridization studies show that VL30 RNA does not have homology with the standard nondefective murine leukemia viruses. Upon superinfection with a nondefective murine leukemia virus, or upon induction of endogenous virus with BrdU, VL30 RNA is rescued into virions by phenotypic mixing. When VL30 RNA is rescued by BrdU induction, the VL30 RNA is mainly organized as a 50S complex, but when VL30 is rescued by superinfection, VL30 is also found in 70S RNA. Rescued VL30 RNA sequences can be reverse transcribed by the virion-associated DNA polymerase in an endogenous reaction. Many mouse cells express the sequences, whereas heterologous cells such as rat or rabbit cells do not contain them. By using hybridization of a complementary DNA probe to cellular RNA immobilized on paper, no subgenomic RNA related to the VL30 RNA could be found in cells expressing the VL30 sequences. From 20 to 50 copies of these sequences were found to be contained in the mouse genome. VL30 RNA is probably present in most stocks of leukemia and sarcoma viruses made in mouse cells.

Prolonged survival time of allogeneic skin grafts in host pretreated with somatic cell hybrids and prednisolone.

By applying somatic cell hybrids to transplantation immunity, the prolongation of survival time of mouse skin allografts was investigated. The hybrid cells (LJ) were made by cell fusion between L cells derived from C3H/He mouse and JLS-V9 cells derived from BALB/c mouse with UV-irradiated Sendai viruses. Skin grafts from female (C3H/He . JmsX BALB/c . Slc) F1 donors were transplanted to male BALB/c . Slc. The survival of the transplant in BALB/c pretreated with the LJ cells and prednisolone showed a significant prolongation, compared with that in animals pretreated with only prednisolone or the LJ cells. The mean survival time and standard deviation of the grafts in mice injected with 1 X 10(5) LJ cells i.v. 4 days before the grafting, and subsequently treated with prednisolone 50 mg/kg i.p. 1 day before and at the time of the grafting were 17.3 +/- 2.2 days. Controls were 9.2 +/- 0.4 days. The mechanisms of prolongation of skin grafts were discussed in terms of cell-mediated immunity and humoral factors. Somatic cell hybrids are useful, not only for inducing immunological enhancement, but also for studying the function of major histocompatibility antigens in cellular immunity and can be applied to clinical organ transplantation.

Interferon inhibits C-type virus at a posttranscriptional, prerelease step.

The mode of action of interferon in JLSV 5-cells, chronically infected with Rauscher murine leukemia virus (MLV), was studied by examining the fate of preexisting labelled viral RNA in interferon-treated cells and by determining the infectivity/physical particle ratio of cell-associated and extracellular virus. Interferon added together with 3H-uridine inhibited the production of labelled virus particles even when it was only allowed to act after all viral RNA synthesis had been stopped by actinomycin D. This indicated that the interferon-induced antiviral state primarily functions at a posttranscriptional step. When interferon was given after a 3H-uridine pulse label and arrest of label incorporation by glucosamine and unlabelled uridine, it prevented a portion of the preexisting radioactive RNA from occurring in extracellular particles. However, part of the labelled viral RNA had reached a stage beyond which interferon could not prevent it from occurring in extracellular virus particles. The notion that interferon primarily affects release of fully assembled and enveloped MLV particles may be eliminated: interferon-treatment did not affect the release of particle-bound reverse transcriptase in cells treated with cycloheximide after the antiviral state had been established. It was confirmed that interferon-treated JLSV 5-cells contained an increased number of virus particles associated with the cell membrane. However, these particles were found to have a reduced infectivity compared to those associated with control cells, thus confirming the view that virions produced by interferon-treated cells are defective; perhaps lacking in certain components.

Rescue of endogenous 30S retroviral sequences from mouse cells by baboon type C virus

Mus musculus SC-1 cells were infected with M7 baboon type C virus. The progeny of this infection included viral pseudotypes that contained M7 helper virus and endogenous 30S retrovirus-associated sequences derived from SC-1 cells (RAS). The RAS sequences are unrelated by nucleic acid hybridization criteria to previously described types of murine retroviruses and do not code for known murine viral structural proteins. The RAS genome is present in multiple copies in the DNA of laboratory (M. musculus) and Asian (M. caroli and M. cervicolor) mice, is expressed in the RNA of uninfected mouse cells, and can be efficiently rescued by type C, but not type B, viruses. RAS is closely related to 30S virus-associated RNA in NIH/3T3 and BALB/c JLSV-9 cells and may be analogous to the defective 30S RNA sequences found in rats.

Effect of impaired glycosylation on the synthesis of envelope proteins of rauscher murine leukemia virus

The effects of 2-deoxy- d-glucose (deoxyglucose) and cytochalasin B on the synthesis of Rauscher murine leukemia viral envelope proteins were investigated. JLS-V9 cells infected with and producing Rauscher murine leukemia virus (R-MuLV) were cultured in the presence of deoxyglucose (15 m M) or of varying concentrations of cytochalasin B (0.1–25 μg/ml) and the synthesis of virus-specific polypeptides was examined in pulse-chase experiments. Overall protein synthesis was inhibited to some extent by cytochalasin B; at a concentration of 25 μg/ml an inhibition of about 70% was observed. Incorporation of d-[1- 3H]glucosamine, however, was almost completely inhibited (97%) at the same concentration of the drug. Newly formed virus-specific polypeptides were analyzed by sodium dodecyl sulfate-polyacrylamide-gel electrophoresis after immuno-precipitation with polyvalent and monospecific antisera against R-MuLV proteins. No specific effect on the synthesis of the gag gene product p30 was observed. The synthesis of the precursor of the envelope polypeptides, env-pr82, however, was prevented in the presence of cytochalasin B. Instead, the synthesis of a new glucosamine-deficient 70,000-molecular weight polypeptide ( env-pr70 ∗) was observed. Env-pr70 ∗ could be immunoprecipitated with anti-gp69/71 serum and anti-p15(E),p12(E) serum and, therefore, probably represents the protein moiety of env-pr82. In pulse-chase experiments in the presence of cytochalasin B, env-pr70 ∗ was converted to a polypeptide with a molecular weight of about 75,000 which was slowly lost during the chase period. Production of virus-specific gp69/71, p15(E), and p12(E) was inhibited under these conditions. Similar results were obtained with deoxyglucose, except that the inhibition of glycosylation by the latter compound is irreversible.

Virus-specific precursor polypeptides in cells infected with Rauscher leukemia virus: Synthesis, identification, and processing

The synthesis of virus-specific polypeptides in JLS-V9 and JLS-V5 cells infected with Rauscher leukemia virus (R-MuLV) was studied in pulse-chase experiments, followed by radioimmunoprecipitation analysis with polyvalent and monospecific antisera against R-MuLV proteins. Two glycosylated polypeptides with molecular weights of about 8 ( env-pr82) were identified as precursors of the virion envelope polypeptides gp69/71 and p15(E). On the other hand, virion polypeptides p30 and pl5 are derived from a 75,000-( gag-pr75) and a 65,000-dalton ( gag-pr65) precursor polypeptide. These precursor-product relations were confirmed by analysis of chymotryptic digests of virion polypeptides and their precursors. In the presence of the arginine analog canavanine two polypeptides with molecular weights of 82,000 and 72,000 ( gag-pr82 and gag-pr72, respectively) were synthesized instead of gag-pr75 and gag-pr65. Processing of precursor polypeptides is reduced in the presence of canavanine. From these results, we conclude that gag-pr82 is possibly the primary gag-gene product and is cleaved into gag-pr75. These studies provided the following additional information: First, we established that immediately after cleavage of their precursors, gp69/71 is found on the outer surface of the cell and p30, p15, and p12 leave the cell as components of budding virions. Therefore, these polypeptides were detected intracellularly in very small amounts only. Polypeptide p15(E) was present within the cell as well as on its outer surface. Second, despite a great similarity in virus-specific (precursor) polypeptides detected in JLS-V9 and JLS-V5 cells, small differences in molecular weights of some of these polypeptides were observed after SDS-PAGE.

RNA metabolism of murine leukemia virus. III. Identification and quantitation of endogenous virus-specific mRNA in the uninfected BALB/c cell line JLS-V9

mRNA containing type C endogenous virus-specific sequences was indentified in JLS-V9 cells (an uninfected BALB/c-derived cell line) by annealing extracted RNA with 3H-labeled virus-specific DNA. The criterion for virus-specific RNA being mRNA was that it co-sedimented with polyribosomes in a sucrose gradient and that it changed to lower sedimentation value if polyribosomes were disagregated prior to centrifugation. It was not possible to identify virus-specific mRNA in unfractionated cytoplasm from JLS-V9 cells since large amounts of virus-specific ribonucleoprotein which was not mRNA had sedimentation values similar to polyribosomes and obscured the analysis. Virus-specific mRNA could be readily identified in polyribosomes which had been purified through a step gradient of 1 and 2 M sucrose, and consisted of two species with sedimentation values of 38S and 27S. The amount of virus-specific RNA in different JLS-V9 cell fractions was quantitated in comparison to cell fractions obtained from M-MuLV clone no. 1 cells (a line of NIH 3T3 cells producing Moloney murine leukemia virus). Approximately 40% of the total virus-specific mRNA was recovered in the purified polyribosomes in M-MuLV no. 1 cells. The amount of virus-specific RNA on polyribosomes appeared to be quite similar for JLS-V9 cells and M-MuLV clone no.1 cells . In contrast, the level of virus-specific protein in JLS-V9 cells (as monitored by radioimmunoassay of the internal structural protein p30) was less than 2% the level in the M-MuLV clone no. 1 cells.

Translation of Rauscher leukemia virus RNA in heterologous cell-free systems

Rauscher leukemia virus RNA (RLV RNA) is translated in mammalian cell-free systems into distinct polypeptides which are immunoprecipitable by an antiserum directed against RLV proteins. These polypeptides partially comigrate electrophoretically with native viral proteins synthesized in vivo in JLS-V9 cells. Besides 72 000-, 65 000- and 50 000-dalton polypeptides a 15 000-dalton polypeptide is also synthesized in vitro. Analysis of incubations of RLV RNA in different cell-free systems reveals that no virus-specific factors are required in the translation of RLV RNA in vitro.

Method for reproducible large-volume production and purification of Rauscher murine leukemia virus.

Rauscher murine leukemia virus was produced in roller-bottle cultures of chronically infected JLS-V9 cells. Virus from this culture fluid was concentrated and purified by two semi-isopycnic bandings in sucrose gradients. Virus material obtained from young, nonconfluent cultures (early-harvest virus) yielded products characteristically containing endogenous ribonucleic acid-dependent deoxyribonucleic acid polymerase with high specific activity (400 to 1,000 pmol of [3H]thymidine 5'-triphosphate incorporated per milligram of protein per hour). Fluids obtained from older confluent cultures (late-harvest virus) yielded products with endogenous ribonucleic acid-dependent deoxyribonucleic acid polymerase with little or no specific activity (200 pmol or less of [3H]thymidine 5'-triphosphate incorporated per milligram of protein per hour), but with higher virus particle counts and greater amounts of protein and gs antigen than the early-harvest products.