Rescue of Rous Sarcoma Virus from Rous Sarcoma Virus-Transformed Mammalian Cells

Rat cells transformed by the B77 strain of avian sarcoma virus produce no virus-like particles, yet B77 virus was rescued from these cells by Sendai virus-mediated fusion with chicken cells. This virus rescue was not affected by treatment of the chicken cells with agents that rendered the cells incapable of dividing, although such treatment greatly reduced the ability of the chicken cells to plate as infectious centers after infection with B77 virus. Fusion of R(B77) cells with chicken erythrocytes also led to virus rescue, although with less efficiency than fusion with chicken fibroblasts. Therefore, virus rescue was probably due to a factor or factors contributed by chicken cells which aid in virus production.     

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.     

Ribonuclease-Sensitive Deoxyribonucleic Acid Polymerase Activity in Uninfected Rat Cells and Rat Cells Infected with Rous Sarcoma Virus

Rat cells infected with the B77 strain of avian sarcoma virus [R(B77) cells] produced no virus-like particles but contained information for the production of infectious B77 virus. (3)H-labeled deoxyribonucleic acid (DNA) product of the B77 virus endogenous DNA polymerase system was used to determine the relative amounts of B77 virus-specific ribonucleic acid (RNA) in B77 virus-infected chicken and R(B77) cells. R(B77) cells were found to contain much less B77 virus RNA than did B77 virus-infected chicken cells. Ribonuclease-sensitive DNA polymerase activity was present in high-speed pellet fractions from Nonidet extracts of B77 virus-infected rat cells. Similar preparations from some uninfected rat cells contained lesser amounts of a similar ribonuclease-sensitive DNA polymerase activity. The endogenous template for the DNA polymerase activity in high-speed pellet fractions from R(B77) cells was not related to B77 virus RNA or to RNA of a rat C-type virus. The DNA product of the endogenous DNA polymerase in high-speed pellet fractions of R(B77) cells hybridized to a small extent with RNA from the same fraction and to a similar extent with RNA from uninfected rat cells.     

Intracellular Viral RNA Species in Mouse Cells Nonproductively Transformed by the Murine Sarcoma Virus

The size and quantity of virus-specific RNA in five non-virus-producing mouse cells transformed by the Moloney isolate of murine sarcoma virus (MSV) was determined. Hybridization of RNA from transformed cells with the [(3)H]DNA product of the RNA-directed DNA polymerase of the murine sarcoma-leukemia virus was used to detect and quantitate virus-specific RNA. The amount of virus-specific RNA in non-virus-producing cells was less than one-sixth of that found in virus-producing cells. A striking correlation was found between the amount of intracellular virus-specific RNA and the degree of agglutination by conconavalin A previously reported for the four non-virus-producing NIH/3T3 cell lines (Salzberg and Green, 1974). A major RNA subunit sedimenting at 26 to 28S was detected in all five MSV-transformed non-virus-producing cells. This could represent the RNA genome of defective MSV.     

Transformation and Productive Infection of Human Osteosarcoma Cells by a Feline Sarcoma Virus

FELINE sarcoma virus (FSV) transforms human embryo cells in vitro¹; it therefore seemed interesting to determine whether this virus could transform human osteosarcoma cells. Defective Moloney sarcoma virus genome can be rescued from non-producer hamster tumour cells by feline leukaemia virus (FeLV)² and because FSV stocks also contain excess FeLV (ref. 1 and unpublished observations of R. V. G.), it was hoped that human osteosarcoma cells transformed by FSV and co-infected with FeLV might yield a human sarcoma virus.     

Spontaneous Conversion of Nontransformed Avian Sarcoma Virus-Infected Rat Cells to the Transformed Phenotype

Normal rat kidney (NRK) fibroblasts were infected with the Schmidt-Ruppin strain (SR-D) of avian sarcoma virus (ASV) and cloned 20 h after infection without selection for the transformed phenotype. Most infected clones initially exhibited the flat, nontransformed morphology that is characteristic of uninfected NRK cells. In long-term culture, however, the majority of the SR-D NRK clones began segregating typical ASV-transformed cells. Transforming ASV could be rescued by fusion with chicken embryo fibroblasts from most of the infected clones tested. Three predominantly flat, independently infected clones were further analyzed by subcloning 8 to 10 weeks after infection. Most flat progeny subclones derived at random from two of these "parental" SR-D NRK clonal lines did not yield virus upon fusion with chicken embryo fibroblasts, although a nondefective transforming ASV was repeatedly recovered from the parental clones. This observation suggested that most, but not all, daughter cells in these SR-D NRK clones lost the ASV provirus after cloning. The progeny of the third independent parental cell clone, c17, gave rise to both flat and transformed subclones that carried ASV. In this case, ASV recovery by fusion and transfection from the progeny subclones was equally efficient regardless of the transformation phenotype of the cells. The 60,000-dalton phosphoprotein product of the ASV src gene was, however, expressed at high level only in the transformed variants. The results of a Luria-Delbruck fluctuation analysis and of Newcombe's respreading test indicated that the event leading to the spontaneous conversion to the transformed state occurred at random in dividing cultures of these flat ASV NRK cells at a rate predicted for somatic mutation.     

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.     

Baboon Virus Isolate M-7 with Properties Similar to Feline Virus RD-114

A virus (M-7) isolated from baboon placental tissue demonstrates many similarities to endogenous feline virus RD-114. Immunodiffusion analysis shows a group-specific antigen (gs-1) line of identity between M-7 and RD-114. Anti-RD-114 DNA polymerase IgG inhibits M-7 polymerase by 57% compared to 97% for RD-114. M-7 virus has helper activity as demonstrated by rescue of murine sarcoma virus (MSV) from sarcoma-positive leukemia-negative human amnion cells. The host range of the rescued M-7 pseudotype of MSV, MSV (M-7), is similar to that of RD-114 virus. MSV (M-7) is also able to transform baboon cells and causes no detectable transformation of feline cells without addition of helper feline leukemia virus. Interference properties of M-7 and RD-114 virus are identical. Virus-specific neutralizing antisera, although partially cross-reacting, can distinguish MSV (M-7) from MSV (RD-114). These similarities and differences between RD-114 and M-7 viruses are best explained as type-specific differences between two viruses within the same strain.     

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.     

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.     

Isolation and Characterization of Revertant Cell Lines VI. Susceptibility of Revertants to Retransformation by Simian Virus 40 and Murine Sarcoma Virus

The susceptibility of two classes of revertants of Simian virus 40 (SV40)-transformed 3T3 cells to retransformation by SV40 or murine sarcoma virus (MSV) was studied. Both serum-sensitive and density-sensitive revertants are not retransformable by SV40. MSV can transform both types of revertants. The MSV-transformed revertants grow to high cell densities and form colonies when suspended in semi-solid methylcellulose medium, but are unable to grow in 1% calf serum. The MSV-transformed revertants produce infectious MSV and murine leukemia virus and possess the same number of chromosomes as the untransformed revertants.     

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.     

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:     

Murine Sarcoma and Leukemia Viruses: Assay Using Clonal Lines of Contact-Inhibited Mouse Cells

A continuous cell line of highly contact-inhibited cells (NIH/3T3) has been developed from NIH Swiss mouse embryo cultures. Its growth properties are similar to those of 3T3 and BALB/3T3. Although 3T3 is relatively insensitive to focus formation by murine sarcoma viruses, cloned lines of both NIH/3T3 and BALB/3T3 have been isolated that are highly sensitive to sarcoma virus focus formation and leukemia virus growth. The sensitivity and specificity are comparable to those found with primary embryo cells. MSV-transformed lines of NIH/3T3 have been obtained.     

Retention of Antigen Specificity of Murine Sarcoma Viruses grown in Hamster Cells <Emphasis Type="Italic">in vitro</Emphasis>

TUMOURS can be induced in hamsters by the various strains of murine sarcoma virus (MSV)^1–6. Tumours differ, however, in the antigens which are expressed. Whereas the cell line HT-1, derived from early passages of a hamster tumour induced by the Moloney strain of MSV (M-MSV), contains no trace of infectious virus or virion antigen^2,7, tumours induced by the Harvey (H), Kirsten (Ki) and later passages of the M-MSV-(GLV) viruses have yielded sarcoma viruses with a hamster-specific host range^3–6,8 which do not share envelope^4–6,9 or group specific¹⁰ antigens with murine viruses. The HT-1 cell does retain the MSV genome which can be rescued by murine leukaemia viruses². Such rescued viruses are termed pseudo-types and contain the envelope and group-specific antigens of the rescuing virus. The virus preparation from tumours induced by M-MSV(GLV) differed from the other hamster-specific viruses in that a non-sarcomagenic C-type virus could be isolated from cultures infected beyond the cell transformation end point⁶. This virus was also hamster-specific in host range and antigenic properties and specifically interfered with cell transformation by the various hamster-specific virus strains⁹. This virus also shared an ether-stable virion-antigen with a C-type virus found in a lymphoma which occurred spontaneously in a hamster¹⁰. This shared antigen seems to be the principal structural polypeptide of hamster C-type viruses and is structurally similar but antigenically distinct from its mouse homologue (unpublished work of S. O., C. Foreman, G. K. and R. V. G.). These findings led us to propose that the hamster-specific non-sarcomagenic C-type virus was a hamster leukaemia virus (in the generic but not necessarily the pathological sense) and the virus is therefore designated HaLV^9,10. The hamster-specific sarcoma viruses are considered to be pseudotypes of MSV rescued in vivo by HaLV and are abbreviated accordingly; for example, M-MSV(HaLV) represents the hamster-specific sarcoma virus rescued from M-MSV induced tumours. This is plausible because HaLV is able to rescue the MSV genome from HT-1 cells⁶. (This change in the nomenclature has been made in order to reflect the antigenic composition of the hamster-specific virus more accurately. In addition, to indicate the virus rescued from M-MSV(GLV)-induced hamster tumours, a terminal G is added after the parentheses. This has been done only to distinguish it from the virus obtained from M-MSV induced hamster tumours, for there is no evidence of residual activity from GLV.)     

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.     

Arrangement of Integrated Avian Sarcoma Virus DNA Sequences Within the Cellular Genomes of Transformed and Revertant Mammalian Cells

We have examined the arrangement of integrated avian sarcoma virus (ASV) DNA sequences in several different avian sarcoma virus transformed mammalian cell lines, in independently isolated clones of avian sarcoma virus transformed rat liver cells, and in morphologically normal revertants of avian sarcoma virus transformed rat embryo cells. By using restriction endonuclease digestion, agarose gel electrophoresis, Southern blotting, and hybridization with labeled avian sarcoma virus complementary DNA probes, we have compared the restriction enzyme cleavage maps of integrated viral DNA and adjacent cellular DNA sequences in four different mouse and rat cell lines transformed with either Bratislava 77 or Schmidt-Ruppin strains of avian sarcoma virus. The results of these experiments indicated that the integrated viral DNA resided at a different site within the host cell genome in each transformed cell line. A similar analysis of several independently derived clones of Schmidt-Ruppin transformed rat liver cells also revealed that each clone contained a unique cellular site for the integration of proviral DNA. Examination of several morphologically normal revertants and spontaneous retransformants of Schmidt-Ruppin transformed rat embryo cells revealed that the internal arrangement and cellular integration site of viral DNA sequences was identical with that of the transformed parent cell line. The loss of the transformed phenotype in these revertant cell lines, therefore, does not appear to be the result of rearrangement or deletions either within the viral genome or in adjacent cellular DNA sequences. The data presented support a model for ASV proviral DNA integration in which recombination can occur at multiple sites within the mammalian cell genome. The integration and maintenance of at least one complete copy of the viral genome appear to be required for continuous expression of the transformed phenotype in mammalian cells.