Transformation of Rat Liver Cells with Chicken Sarcoma Virus B77 and Murine Sarcoma Virus

Rat liver cells in vitro were transformed with chicken sarcoma virus B77, giving RL(B77) cells, and with murine sarcoma virus (Harvey), giving RL(MSV) cells. Rat liver cells transformed spontaneously in vitro were designated RL cells. In addition, the RL(MSV) cell line was adapted for growth in culture fluid containing 25 mug of 5-bromodeoxyuridine per ml. All cell lines were tumorigenic in 1-wk-old rats. The number of cells needed for induction of tumor growth was 1,000-fold higher in the case of RL(B77) cells in comparison with RL(MSV) cells and RL cells. No production of viral particles from any of the cell lines investigated was detected by plating concentrated supernatant fluid of the cultures on different secondary embryo cells with and without fusion by Sendai virus, by labeling with uridine-5-(3)H, or by assay for deoxyribonucleic acid polymerase activity. The viral genome was rescued by fusion of RL(B77) cells with chicken cells. Chicken sarcoma virus rescued from (RL(B77) cells differed in plating efficiency on duck cells from B77 virus rescued from transformed rat embryo cells. No virus was rescued after fusion of RL(MSV) and RL cells with mouse, rat, or chicken embryo cells. Infectious murine sarcoma virus can be induced by 5-bromodeoxyuridine from RL(MSV) cells.     

Expression of a New Complement-fixing Antigen reactive with Murine Sarcoma Virus Rat Antiserum in Rat Cells transformed by Polyoma Virus

WE reported accelerated transformation by DNA viruses (SV40 and polyoma) in rat embryo (RE) cells chronically infected with a C-type RNA virus^1,2. Recently we found in RE cells transformed by polyoma virus a new complement-fixing (CF) antigen detectable by rat antisera having broad reactivity with the various intraspecies and interspecies antigens of the RNA tumour viruses^3–8; this antigen, however, was distinct from the murine intraspecies and interspecies group-specific (gs) antigens both immunologically and by virtue of other properties. It is also distinct from the polyoma virion (capsid) and tumour (“T”) antigens.     

Translational Products of Moloney Murine Sarcoma Virus RNA: Identification of Proteins Encoded by the Murine Sarcoma Virus src Gene

In vitro translation of virion RNA of Moloney murine sarcoma virus (MSV) strain 124 yielded major products having molecular weights of 63,000 (63K), 43K, 40K, 31K, and 24K daltons. A molecularly cloned subgenomic fragment of Moloney MSV comprised of the cellular insertion (src) region was utilized in hybridization arrest translation as a means of identifying products of the MSV src gene. MSV src DNA specifically inhibited synthesis of the 43K, 40K, 31K, and 24K proteins, implying that each of these proteins was coded within the MSV src gene. The MSV src-specific nature of this family of proteins was further confirmed by partial purification of MSV src-containing RNAs from MSV non-producer cells. In vitro translation of enriched cellular RNAs yielded products with molecular weights identical to those of the 43K family of proteins synthesized from virion RNA. Nucleotide sequence analysis of the MSV transforming region has revealed a long open reading frame which includes five methionine codons (Reddy et al., Proc. Natl. Acad. Sci. U.S.A. 77:5234-5238, 1980). The molecular weights of the four largest proteins that could be synthesized within this open reading frame corresponded closely to the molecular weights of the 43K family of proteins. Partial cyanogen bromide cleavage of each of the three largest proteins resulted in an uncleaved fragment having a molecular weight equal to that of the smallest (24K) protein. These findings provide direct biochemical evidence that the 43K, 40K, 31K, and 24K proteins are related in their carboxy-terminal regions, as well as information concerning the MSV src gene coding sequences from which each protein originates:     

Structure and Functions of the Kirsten Murine Sarcoma Virus Genome: Molecular Cloning of Biologically Active Kirsten Murine Sarcoma Virus DNA

The unintegrated closed circular form of viral DNA prepared from NIH3T3 cells infected with Kirsten murine sarcoma virus was cloned into bacterial plasmid pBR322. The closed circular DNA, which consisted of two different-sized populations, was enriched from the virus-infected cells, linearized with BamHI, and inserted into pBR322 DNA. Four different recombinant DNAs (clones 2, 4, 6, and 7) were obtained, and a physical map of each was constructed by using various restriction enzymes. Clone 4 DNA had the largest insertion, corresponding to a complete copy of the linear DNA. This suggested that this insertion contained two copies of the 0.55-kilobase pair long terminal redundant sequence. Clone 2 and clone 6 insertion DNAs had deletions of 0.2 and 0.5 kilobase pair, respectively, which mapped near the right end (3' side of viral RNA) of the linear DNA. Clone 7 DNA appeared to have a deletion of a single copy of the large terminal redundant sequence. Transfection of BALB3T3 cells with the clone 4 DNA insertion showed that this DNA had transforming activity. The efficiency of transfection with clone 4 Kirsten murine sarcoma virus DNA was enhanced eightfold by inserting EcoRI-cleaved viral DNA into the EcoRI site of pBR322. The EcoRI-inserted DNA produced foci with single-hit kinetics, suggesting that a single molecule of Kirsten murine sarcoma virus DNA can induce transformation. Results of transfections with EcoRI-inserted Kirsten murine sarcoma virus DNA cleaved with various restriction enzymes suggested that the first 3.3-kilobase pair region at the left end of the linear DNA is important for the initiation of transformation or maintenance of transformation or both.     

Co-Infection of Mouse Spleen Cells with Murine Sarcoma Virus and Guaroa Virus

Enhancement of tumor induction by oncornaviruses through a dual viral infection has been described by several investigators. The mechanism(s) of this enhancement has not as yet been determined. By using a murine sarcoma virus and a Bunyawera group arbovirus (Guaroa virus) in an in vitro system, evidence was obtained for enhancement of the oncogenic potential of the viruses by genetic interaction with the nononcogenic virus as well as the production of increased amounts of the oncogenic virus. These results confirm and extend similar responses obtained in in vivo systems.     

Isolation of Temperature-Sensitive Mutants of Murine Sarcoma Virus

Three separate murine sarcoma virus nonproducer cell lines have been isolated which are temperature sensitive for the maintenance of transformation. In each case, a viral rather than a cellular genetic mutation is the reason for the temperature-sensitive effect. Superinfection of one of the mutants with murine leukemia virus overcomes the temperature-sensitive change in the transformed state.     

Immunological Cross-Reactions Between Two Low-Molecular-Weight Polypeptides from A Murine Type C Virus

Two low-molecular-weight type-specific virion polypeptides from the Kirsten strain of Murine leukemia virus, polypeptides p10 and p12, are immunologically related by radioimmunoassay competition techniques.     

Activation of the Murine Sarcoma Virus Genome After Infection with the Murine Leukemia Virus as Determined by Cell Agglutination

Non-virus-producing NIH/3T3 cells transformed by the murine sarcoma virus are agglutinated by conconavalin A to the same low level as normal NIH/3T3 cells. Infection with the murine leukemia virus greatly increases the agglutination of transformed cells but not that of normal cells. These data suggest that the morphological expression of cell transformation and the surface alterations associated with increased cell agglutination are controlled by the expressions of different sarcoma virus genes.     

Relationship Between Moloney Murine Leukemia and Sarcoma Virus RNAs: Purification and Hybridization Map of Complementary DNAs from Defined Regions of Moloney Murine Sarcoma Virus 124

Complementary DNAs (cDNA's) specific for various regions of the Moloney murine sarcoma virus (MSV) 124 RNA genome were prepared by cross-hybridization techniques. A cDNA specific for the first 1,000 nucleotides adjacent to the RNA 3' end (cDNA 3') was prepared and shown to also be complementary to the 3'-terminal 1,000 nucleotides of a related Moloney murine leukemia virus (MLV) genome. A cDNA complementary to the "MSV-specific" portion of the MSV 124 genome was prepared. This cDNA was shown not to anneal to Moloney MLV RNA and to anneal to a portion of the viral RNA of about 1,500 to 1,800 nucleotides in length, located 1,000 nucleotides from the 3' end of MSV RNA. A cDNA common to the genome of MSV and MLV was also obtained and shown to anneal to the 5'-terminal two-thirds, as well as to the 3'-terminal 1,000 nucleotides, of the MSV RNA genome. This cDNA also annealed to the RNA from MLV and mainly to the 5'-terminal half of the MLV genome. It is concluded that the 6-kilobase Moloney MSV 124 RNA genome has a sequence arrangement that includes (i) a 3' portion of about 1,000 nucleotides, which is also present at the 3' terminus of MLV; (ii) an MSV-specific region, not shared with MLV, which extends between 1,000 and 2,500 nucleotides from the 3' terminus; and (iii) a second "common" region, again shared with MLV, which extends from 2,500 nucleotides to the 5' terminus. This second common region appears to be located in the 5' half of the 10-kilobase MLV genome as well. Experiments in which a large excess of cold MLV cDNA was annealed to (3)H-labeled polyadenylic acid-containing fragments of MSV RNA gave results consistent with this arrangement of the MSV genome.     

Production of Virus by Mammalian Cells Transformed by Rous Sarcoma and Murine Sarcoma Viruses

Cultured cells of mammalian tumors induced by ribonucleic acid (RNA)-containing oncogenic viruses were examined for production of virus. The cell lines were established from tumors induced in rats and hamsters with either Rous sarcoma virus (Schmidt-Ruppin or Bryan strains) or murine sarcoma virus (Moloney strain). When culture fluids from each of the cell lines were examined for transforming activity or production of progeny virus, none of the cell lines was found to be infectious. However, electron microscopic examination of the various cell lines revealed the presence of particles in the rat cells transformed by either Rous sarcoma virus or murine sarcoma virus. These particles, morphologically similar to those associated with murine leukemias, were found both in the extracellular fluid concentrates and in whole-cell preparations. In the latter, they were seen budding from the cell membranes or lying in the intercellular spaces. No viruslike particles were seen in preparations from hamster tumors. Exposure of the rat cells to (3)H-uridine resulted in the appearance of labeled particles with densities in sucrose gradients typical of virus (1.16 g/ml.). RNA of high molecular weight was extracted from these particles, and double-labeling experiments showed that this RNA sedimented at the same rate as RNA extracted from Rous sarcoma virus. None of the hamster cell lines gave radioactive peaks in the virus density range, and no extractable high molecular weight RNA was found. These studies suggest that the murine sarcoma virus produces an infection analogous to certain "defective" strains of Rous sarcoma virus, in that particles produced by infected cells have a low efficiency of infection. The control of the host cell over the production and properties of the RNA-containing tumorigenic viruses is discussed.     

Rat C-Type Virus induced in Rat Sarcoma Cells by 5-Bromodeoxyuridine

HALOGENATED derivatives of uridine are highly effective inducers of latent C-type RNA viruses^1,2 and have been successfully used to induce viruses identical to, or similar to, the C-type RNA tumour viruses in mouse, rat and human cells^3–6. In previous experiments we used 5-bromodeoxyuridine (BrUdR) for induction of focus-forming virus in non-productive rat cells that have been transformed by mouse sarcoma virus². We describe here the induction of a C-type RNA virus in the cells of the rat tumour cell line XC, which contains the Rous sarcoma virus genome⁷. The induced virus possesses the group specific (gs) antigens of rat C-type viruses but not those of chicken C-type viruses.     

Enhancement of Murine Leukemia Virus Replication in Rat Tumor Cells Containing Rat C-Type Virus

The yield of Maloney leukemia virus (MLV) from MLV-infected rat cells was shown to be enhanced in rat cells containing rat C-type virus. The MLV produced in these cells was shown to be identical to murine-derived MLV and devoid of properties related to rat C-type virus.     

Isolation of a Transformation-Defective Deletion Mutant of Moloney Murine Sarcoma Virus

A transformation-defective (td) deletion mutant of Moloney murine sarcoma virus (td Mo-MSV) and a transforming component termed Mo-MSV 3 were cloned from a stock of clone 3 Mo-MSV. To define the defect of the transforming function, the RNA of td Mo-MSV was compared with those of Mo-MSV 3 and of another transforming variant termed Mo-MSV 124 and with helper Moloney murine leukemia virus (Mo-MuLV). The RNA monomers of td Mo-MSV and Mo-MSV 3 comigrated on polyacrylamide gels and were estimated to be 4.8 kilobases (kb) in length. In agreement with previous analyses, the RNA of Mo-MSV 124 measured 5.5 kb and that of Mo-MuLV measured 8.5 kb. The interrelationships among the viral RNAs were studied by fingerprinting and mapping of RNase T₁-resistant oligonucleotides (T₁-oligonucleotides) and by identification of T₁-oligonucleotides present in hybrids formed by a given viral RNA with cDNA's made from another virus. The nontransforming td Mo-MSV RNA lacked most of the Mo-MSV-specific sequence, i.e., the four 3′-proximal T₁-oligonucleotides of the six T₁-oligonucleotides that are shared by the Mo-MSV-specific sequences of Mo-MSV 3 and Mo-MSV 124. The remaining two Mo-MSV-specific oligonucleotides identified td Mo-MSV as a deletion mutant of MSV rather than a deletion mutant of Mo-MuLV. td Mo-MSV and Mo-MSV 124 exhibited similar deletions of gag, pol, and env sequences which were less extensive than those of Mo-MSV 3. Hence, td Mo-MSV is not simply a deletion mutant of Mo-MSV 3. In addition to their MSV-specific sequences, all three MSV variants, including td Mo-MSV, shared the terminal sequences probably encoding the proviral long terminal repeat, which differed from their counterpart in Mo-MuLV. This may indirectly contribute to the oncogenic potential of MSV. A comparison of td Mo-MSV sequences with either Mo-MSV 124 or Mo-MSV 3 indicated directly, in a fashion similar to the deletion analyses which defined the src gene of avian sarcoma viruses, that Mo-MuLV-unrelated sequences of Mo-MSV are necessary for transformation. A definition of transformation-specific sequences of Mo-MSV by deletion analysis confirmed and extended previous analyses which have identified Mo-MuLV-unrelated sequences in Mo-MSV RNA and other studies which have described transformation of mouse 3T3 fibroblasts upon transfection with DNAs containing the Mo-MSV-specific sequence.     

Rescue of Murine Sarcoma Virus from a Sarcoma-Positive Leukemia-Negative Cell Line: Requirement for Replicating Leukemia Virus

The nature of murine sarcoma virus (MSV) "defectiveness" was investigated by employing an MSV-transformed mouse 3T3 cell line which releases noninfectious virus-like particles. Rescue kinetics of MSV, observed after murine leukemia virus (MuLV) superinfection of these "sarcoma-positive leukemia-negative (S + L -)" mouse 3T3 cells, consisted of a 9- to 12-hr eclipse period followed by simultaneous release of both MSV and MuLV with no evidence for release of infectious MSV prior to the production of progeny MuLV. Addition of thymidine to the growth medium of MuLV-superinfected S + L - cells at a concentration suppressing deoxyribonucleic acid synthesis inhibited the replication of MuLV and the rescue of MSV. MSV production closely paralleled MuLV replication under a variety of experimental conditions. These results suggest that replication of MuLV is required for the rescue of infectious MSV from S + L - cells and that one (or more) factor, produced late in the MuLV replicative cycle, is utilized by both viruses during virion assembly. During the course of these experiments, virus stocks were recovered which contained infectious MSV in apparent excess over MuLV. These stocks were used for generating new S + L - cell lines by simple end point dilution procedures.     

Genetic Determinants of Rous Sarcoma Virus Particle Size

The Gag proteins of retroviruses are the only viral products required for the release of membrane-enclosed particles by budding from the host cell. Particles released when these proteins are expressed alone are identical to authentic virions in their rates of budding, proteolytic processing, and core morphology, as well as density and size. We have previously mapped three very small, modular regions of the Rous sarcoma virus (RSV) Gag protein that are necessary for budding. These assembly domains constitute only 20% of RSV Gag, and alterations within them block or severely impair particle formation. Regions outside of these domains can be deleted without any effect on the density of the particles that are released. However, since density and size are independent parameters for retroviral particles, we employed rate-zonal gradients and electron microscopy in an exhaustive study of mutants lacking the various dispensable segments of Gag to determine which regions would be required to constrain or define the particle dimensions. The only sequence found to be absolutely critical for determining particle size was that of the initial capsid cleavage product, CA-SP, which contains all of the CA sequence plus the spacer peptides located between CA and NC. Some regions of CA-SP appear to be more important than others. In particular, the major homology region does not contribute to defining particle size. Further evidence for interactions among CA-SP domains was obtained from genetic complementation experiments using mutant ΔNC, which lacks the RNA interaction domains in the NC sequence but retains a complete CA-SP sequence. This mutant produces low-density particles heterogeneous in size. It was rescued into particles of normal size and density, but only when the complementing Gag molecules contained the complete CA-SP sequence. We conclude that CA-SP functions during budding in a manner that is independent of the other assembly domains.     

Effectiveness of Therapy with a Multistranded Synthetic Polynucleotide Complex Against a Murine Sarcoma Virus

A known interferon stimulator, polyriboinosinic acid:polyribocytidylic acid (rI:rC), was shown to have a protective effect against a highly tumorigenic variant of the murine sarcoma virus (Moloney) designated as (MSV-PV). Mice treated intraperitoneally with different regimens of rI:rC survived significantly longer than untreated controls. The treatment was effective whether the infection with MSV-PV was intraperitoneal or intracerebral. The antiviral effectiveness of rI:rC was demonstrated by marked reduction of viral replication. The kinetic study of interferon induction by rI:rC showed a peak titer of interferon occurring at 18 hr after injection, followed by a rapid decrease in titer by 24 hr.     

Suppression of Murine Retrovirus Polypeptide Termination: Effect of Amber Suppressor tRNA on the Cell-Free Translation of Rauscher Murine Leukemia Virus, Moloney Murine Leukemia Virus, and Moloney Murine Sarcoma Virus 124 RNA

The effect of suppressor tRNA's on the cell-free translation of several leukemia and sarcoma virus RNAs was examined. Yeast amber suppressor tRNA (amber tRNA) enhanced the synthesis of the Rauscher murine leukemia virus and clone 1 Moloney murine leukemia virus Pr200^gag-pol polypeptides by 10- to 45-fold, but at the same time depressed the synthesis of Rauscher murine leukemia virus Pr65^gag and Moloney murine leukemia virus Pr63^gag. Under suppressor-minus conditions, Moloney murine leukemia virus Pr70^gag was present as a closely spaced doublet. Amber tRNA stimulated the synthesis of the “upper” Moloney murine leukemia virus Pr70^gag polypeptide. Yeast ochre suppressor tRNA appeared to be ineffective. Quantitative analyses of the kinetics of viral precursor polypeptide accumulation in the presence of amber tRNA showed that during linear protein synthesis, the increase in accumulated Moloney murine leukemia virus Pr200^gag-pol coincided closely with the molar loss of Pr63^gag. Enhancement of Pr200^gag-pol and Pr70^gag by amber tRNA persisted in the presence of pactamycin, a drug which blocks the initiation of protein synthesis, thus arguing for the addition of amino acids to the C terminus of Pr63^gag as the mechanism behind the amber tRNA effect. Moloney murine sarcoma virus 124 30S RNA was translated into four major polypeptides, Pr63^gag, P42, P38, and P23. In the presence of amber tRNA, a new polypeptide, Pr67^gag, appeared, whereas Pr63^gag synthesis was decreased. Quantitative estimates indicated that for every 1 mol of Pr67^gag which appeared, 1 mol of Pr63^gag was lost.     

Transformation by Murine Sarcoma Virus: Fixation (Deoxyribonucleic Acid Synthesis) and Development

Transformation of rat embryo cells by murine sarcoma virus (MSV) was contingent upon synthesis of deoxyribonucleic acid (DNA) during the first 12 hr of infection. Inhibition of DNA synthesis by thymidine (20 mm) or cytosine arabinoside (0.1 mm) resulted in the protection of cells from transformation by MSV. Transient suppression of DNA synthesis prior to infection or after a 12-hr delay had little effect on subsequent transformation, emphasizing the critical time period in in which DNA synthesis was necessary for intracellular fixation of the viral genome. These results are similar to those previously described for Rous sarcoma virus. Development of transformed cells after viral fixation was shown to be influenced by cellular density. Under conditions which allowed fixation of virus in confluent cellular monolayers, less than 20% of these cells developed into transformed foci.     

Biological and Serological Properties of Viral Particles from a Nonproducer Rat Neoplasm Induced by Murine Sarcoma Virus (Moloney)

The viral particles present in a nonproducer rat neoplasm induced by murine sarcoma virus (MSV) Moloney isolate, as detected by electron microscopy, were found to be biologically active on normal kidney cells of random-bred Osborne-Mendel rats. The virus is designated here as MSV (0). MSV (0) differs from other pseudotypes of MSV in its host range, antigenicity, and interference pattern.