Revertants of an ASV-transformed rat cell line have lost the complete provius or sustained mutations in src

We have isolated and characterized 12 revertants of a clonal line (B31) of avian sarcoma virus (ASV)-transformed rat-1 cells. The B31 cells contain a single normal ASV provirus, display the classical features of virally transformed cells, and revert to normal phenotype at low frequency. Revertants isolated after selective killing of transformed cells resemble uninfected rat-1 cells morphologically, fail to grow in suspension, and are at least 100-fold less tumorigenic than B31 cells. Two mechanisms of reversion have been identified in these cells. (i) Three of the revertant lines have lost the entire provirus, including both copies of the sequences repeated at the ends of the provirus; the manner in which the provirus is lost is not known. (ii) The other nine revertants retain a provirus of normal size and unaltered flanking cellular DNA: contain the same species of viral RNA at the same concentrations as in the parental line, B31; are susceptible to retransformation by wild-type ASV; and yield transformation-defective (td) virus after fusion with chicken cells. In one case, the rescued virus transforms chicken cells, but produces fusiform rather than normal foci and does not retransform rat cells morphologically. Hence these revertants arise as a consequence of nonconditional mutations (base substitutions or small deletions) in the viral transforming gene,src. In several cases, the revertant cells retransform spontaneously, or transforming virus appears in stocks of rescued td virus after passage through chicken cells, indicating back mutations to wild-type. Several of the rescued td viruses can also recombine to restore a wild-type phenotype. Analysis of the structure and enzymatic activity of products ofsrc confirms that the revertant cells bear various mutations insrc.     

Specific restriction of avian sarcoma viruses by a line of transformed lymphoid cells.

MSB-1 cells are a line of transformed chicken lymphoid cells derived from tumors induced by Marek's disease viruses and free of exogenous avian leukosis viruses (ALV). They can be infected by ALV of subgroups A and C including transformation-defective (td) deletion mutants of avian sarcoma viruses (ASV). In terms of virus titers in supernatant culture medium, proportion of virus-producing cells, and levels of viral RNA detected by hybridization with a cDNA probe, infection by td ASV of MSB-1 cells was indistinguishable from infection of chicken embryo fibroblasts. In contrast, wild type ASV was restricted in its growth on MSB-1 cells. Different clones of ASV varied in their restriction by all these parameters of viral growth by factors of 10^?1 to 10^?4 Studies of a severely restricted viral clone showed equal quantities of hybridizable viral DNA in Hirt supernatant fractions of both fibroblasts and MSB-1 cells at 10 hr after high multiplicity infection, and transfection assays indicated infectious viral DNA in both cell types. Viral DNA largely disappeared from Hirt supernatant fractions of MSB-1 cells by 48 hr after infection, and sarcoma virus-specific DNA was not detected in Hirt pellet fractions from MSB-1 cells at levels found in comparably infected fibroblasts. Infectious ASV DNA, while easily detected in fibroblasts, could not be detected on MSB-1 cells at 48 hr or later times after infection. Because replication of td ASV does not appear restricted in MSB-1 cells, the failure of ASV DNA to integrate normally in these cells seems to be related to the presence of src sequences in the viral genome.     

Mutants of Rous sarcoma virus with extensive deletions of the viral genome.

Deletion mutants of Rous sarcoma virus (RSV) have been isolated from a stock of Prague RSV which had been irradiated with ultraviolet light. Quail fibroblasts were infected with irradiated virus and transformed clones isolated by agar suspension culture. Three clones were obtained which did not release any virus particles. Analysis of DNA from these non-producer clones with restriction endonucleases and the Southern DNA transfer technique indicated that the clones carry defective proviruses with deletions of approximately 4 × 10⁶ daltons of proviral DNA. The defective proviruses, which retain the viral transformation (src) gene, contain only 1.7–2.0 × 10⁶ daltons of DNA. Multiple species of viral RNA containing the sequences of the src gene were detected in these clones; some of these RNAs may contain both viral and cellular sequences. The protein product of the src gene, p60^src (Brugge and Erikson, 1977), was also synthesized in the nonproducer clones. However these clones did not contain the products of the group-specific antigen (gag), DNA polymerase (pol), or envelope glycoprotein (env) genes, nor did they contain the 35 and 28 S RNA species which are believed to represent the messengers for these viral gene-products. The properties of these mutants indicate that expression of the src gene is sufficient to induce transformation. These clones may represent useful tools for the study of the expression of this region of the genome.     

Infection of rat cells by avian sarcoma virus: factors affecting transformation and subsequent reversion

We have investigated factors that influence (1) the initial incidence of transformation in rat cells infected by avian sarcoma virus B77 and (2) the transformed phenotype of the cell and its stability. Transformation is not limited to a genetically highly competent subpopulation of either viruses or cells but can potentially involve any of the viruses or hosts. The kinetics of transformation are one-hit, even at input virus multiplicities of infection (m.o.i.) of 1 to 10, suggesting that a single virus particle is sufficient to induce transformation and that there is not a physiologically highly susceptible cell population whose response reaches a plateau at high m.o.i. However, as judged by levels of virus recovery and morphological reversion, transformed clones obtained after infection at high m.o.i. show a more uniform virus expression than those obtained at low m.o.i. Moreover, analysis of integrated proviral DNA in cloned transformed cells indicated that seven clones transformed at low (0.01) m.o.i. contain only single proviruses whereas five clones derived from high (1 or 7.5) m.o.i. infections each bear a minimum of three integrated proviruses. These findings suggest that, in the high m.o.i. transformants, multiple viral genome copies contribute to the transformed cell phenotype, making it unlikely that transformation is due simply to randomly occurring virus-cell interactions. Studies on transformants obtained at low m.o.i. show that heterogeneity in virus recovery and morphological reversion can be stable but is not, in most cases, due to virus mutants. Since levels of virus recovery and morphological reversion in these clones did not show a uniform inverse relationship, it is also unlikely that their phenotypic variability is due simply to modulations in overall levels of virus expression. Most revertants isolated from clones transformed at low m.o.i. show decreased but detectable rescuability of transforming virus, suggesting that virus expression is reduced, but reversion by other mechanisms has also been detected in these clones (Varmus et al., 1980).     

Growth stimulation of chicl embryo neuroretinal cells infected with Rous sarcoma virus: relationship to viral replication and morphological transformation.

Neuroretinal (NR) cells from 7-day-old chick embryos infected with Rous sarcoma virus (RSV) are morphologically transformed, synthesize virus, and are induced to proliferate for several generations. By contrast, uninfected cells have a limited growth capacity and cannot be propagated in vitro. The relationship of induction of cell multiplication to viral replication and morphological transformation was analyzed by infecting NR cells with conditional and nonconditional transformation defective viruses.NR cells infected with a temperature-sensitive mutant of RSV defective for cell transformation, but not for virus replication, were induced to multiply at both nonpermissive and permissive temperatures, although expression of the transformed phenotype, as tested by several parameters, was suppressed in these cells at nonpermissive temperature.Nonconditional, nontransforming viruses replicated normally in NR cells, but failed to induce their multiplication. These results indicate that only transforming viruses can induce NR cell multiplication and that viral replication alone does not account for the growth changes in infected NR cells. These data also suggest that expression of the transformed phenotype and induction of NR cell proliferation may depend on distinct viral informations.     

Expression of viral envelope glycoprotein and transformation genes in cells transformed by a defective Kirsten murine sarcoma virus.

Concurrent expression of transformation properties and virion envelope glycoproteins has been observed as a property of several clones of normal rat kidney cells transformed by, but not producing, Kirsten murine sarcoma virus. The present studies were carried out to determine whether a genetic linkage exists between the viral sarcoma and envelope genes in these cells. Several alternative models for the possible structure and origin of the sarcoma and envelope genes were considered. One possibility, that the viral envelope gene was derived from an endogenous rat virus, was studied by characterization of the interference properties of the transformed cells. The sarcoma virus genome of envelope-positive clones was efficiently rescued by woolly monkey and murine xenotropic but not by murine ecotropic viruses. Thus, the interference properties of cells producing the envelope glycoprotein are analogous to those of a cell producing murine ecotropic virus, indicating that the envelope was of murine viral origin. In these experiments it was also found that sarcoma viruses rescued from envelope-positive cells upon superinfection with primate and xenotropic murine viruses could transform host cells for both xenotropic and ecotropic viruses, indicating that these superinfecting viruses became phenotypically mixed with the ecotropic envelope expressed in transformed, envelope-positive cells. Possible linkage between the envelope and transformation genes was analyzed by the frequency of concurrent rescue of sarcoma and envelope genes. Transfer of the Kirsten sarcoma viral genome to uninfected cells upon rescue by superinfection with woolly monkey virus showed a high frequency of apparent segregation of the transformation and envelope genes [from 29 to 57% for (KSV env⁺)NRK-6]. The model supported by the present data is that the transformed, envelope-positive cells were infected with a virus which contained both the envelope and the sarcoma genes.     

Critical role for SV40 small-t antigen in human cell transformation

Defining the ability of simian virus 40 (SV40) to transform human cells has become of even greater importance with the increased understanding that this virus may play a role in some human malignancies. This report documents the requirement for viral small-t (ST) antigen in large-T (LT)-driven transformation of primary fibroblasts, a requirement that cannot be met by a well-known oncogene, c-Ha-ras (EJ-ras), which can cooperate with LT in rodent systems. The cellular gene telomerase is not essential for transformation, although transformed clones are not immortal without it. Similarly, an immortal mesothelial cell line has been developed using LT and telomerase. Immortalized mesothelial cells are morphologically normal, but can be transformed by introduction of ST, or ST + ras, but not by ras alone. It is likely that ST will be required along with LT for transformation of most human cell types.     

Evidence for helper independent murine sarcoma virus. I. Segregation of replication-defective and transformation-defective viruses

In the conventional focus assay for murine sarcoma virus (Hartley and Rowe, 1966), the formation of a focus involves repeated rounds of infection, and, as is shown in this report, the possibility of alterations in the genome of the virus is thereby increased. Multiple rounds of infection were avoided by infecting cells in suspension, plating them sparsely, and allowing them to grow into colonies. XC cells were added to detect which colonies were producing leukemia virus. When cells were infected with the Moloney sarcoma-leukemia virus (M-MuSV(MuLV)), four types of colonies were seen: (1) morphologically normal with syncytia (XC⁺) or (2) without syncytia (XC^?), (3) morphologically transformed with no syncytia, (4) transformed with syncytia. The proportions infected by MuSV (transformed cells) or by MuLV (XC⁺) conformed to Poisson's distribution, and this allowed the calculation of the titers of MuSV and MuLV. Clones of chronically infected cells could readily be isolated.A clone of transformed cells called G8 was derived from JLS-V9 cells infected with M-MuSV(MuLV). The cells produced no MuLV detectable by cocultivation with XC cells, but they did produce sarcoma virus detected by the production of sarcomas in mice and morphological transformation of several lines of mouse cells in culture. The virus had a density of 1.16 g/cm³.The kinetics of focus formation were one-hit when assayed by the conventional assay. Virus picked from most ($\text{32}\text{38}$) of these foci consisted of a mixture of sarcoma virus and leukemia virus but some ($\text{4}\text{38}$) foci were found that produced sarcoma virus alone (presumably “competent” sarcoma virus, i.e., helper-independent). The presumed “competent” sarcoma virus was carried through 4 successive passages and each time, most of the foci were found to contain both MuSV and MuLV, but some produced MuSV only. In contrast, the original, chronically infected G8 cells did not release detectable MuLV through more than 30 passages. Leukemia virus or defective sarcoma virus segregated from the competent MuSV with low and equal frequencies only when new mouse cells were infected.Examination of the individual cells within foci formed by spread of viral infection showed that some cells produced only competent virus; other cells from the foci produced MuSV and MuLV; others were transformed nonproducers containing defective MuSV that could be rescued by superinfection with MuLV; and still others were transformed but MuSV could not be rescued from them.No evidence was found for the presence of a helper virus in excess of the concentration of sarcoma virus and competence appears to be a property of the virion itself. The data suggest that the competent MuSV is similar to the helper-independent strain of the Rous avian sarcoma virus. It is not known why this virus is negative in the XC assay, although the MuLV that segregates from it is positive in this assay.In the search for a helper virus, a form of MuSV was found that did not morphologically transform the cells it infected, nor was it produced by them, but both transformation and release of MuSV appeared on superinfection with MuLV.     

Transformation parameters induced in chick cells by incubation in media of altered NaCl concentration

Lowering the NaCl concentration of the medium in which normal chick cell cultures are incubated causes them to enlarge, vaculate, and appear morphologically similar to cells transformed by the Bryan strain of Rous sarcoma virus (RSV). Raising the NaCl concentration of the medium causes cells to become round or spindle shaped and to appear morphologically similar to cells transformed by the Schmidt-Ruppin strain of RSV. Uninfected chick cell cultures incubated in either low or high NaCl medium also express many characteristics of transformed cells. They lose contact inhibition of growth and movement, grow to higher saturation densities, synthesize reduced amounts of fibronectin, exhibit increased lectin agglutinability, transport increased amounts of hexoses, have reduced levels of succinic dehydrogenase activity, produce increased amounts of lactate and pyruvate, and can be serially passaged in culture significantly longer than normal chick fibroblasts. We were not able to induce normal chick cells to exhibit all transformation parameters by incubation an altered NaCl media: the cells did not grow in soft agar or in low serum medium. Cells transformed by the Bryan and Schmidt-Ruppin strains of RSV exhibit strain-specific changes in their intracellular Na⁺ and K⁺ concentrations. Cells incubated in altered NaCl media exhibit altered intracellular monovalent cation concentrations similar to those seen in the cells they resemble morphologically. Results presented in this publication identify a possible new transformation parameter, alteration of the intracellular Na⁺ and K⁺ concentrations, and suggest that many, but not all transformation parameters may be consequences of the altered intracellular monovalent cation concentrations.     

The persistence and expression of virus-specific DNA in revertants of Rous sarcoma virus-transformed BHK-21 cells

Clones of Rous sarcoma virus-transformed baby hamster kidney cells and subclones reverted to normal phenotype were found to contain one copy of integrated virus-specific DNA per diploid cell. In the transformed clones, virus-specific DNA is transcribed into RNA at low levels similar to those observed in other nonpermissive RSV-transformed cells; in the revertant clones, virus-specific RNA is detected, but at a still lower concentration. The amount of virus-specific RNA in these clones correlates with the frequency with which RSV can be rescued after fusion with chick embryo cells. Treatment of a revertant clone with 5-bromodeoxyuridine increased the concentration of viral RNA and the frequency of rescue.     

Virus-specific DNA sequences in mouse and rat cells transformed by the Harvey and Moloney murine sarcoma viruses detected by in situ hybridization.

The [³H]DNA product of the murine sarcoma-leukemia virus (MSV(MLV)) and viral 60–70 S [³H]RNA was annealed with cytological preparations of mouse and rat cells transformed by the Harvey and Moloney strains of MSV. With viral [³H]DNA as cytological probe, these in situ hybridization measurements detected from 25 to 30 autoradiographic grains per interphase nucleus of transformed cells and 1 to 3 grains per nucleus of uninfected rat and mouse cells. With viral 60–70 S [³H]RNA of lower specific radioactivity as cytological probe, 12 grains per transformed cell nucleus were detected. These findings indicate that transformation of cells with MSV(MLV) produces a several-fold increase in the content of some virus-specific DNA sequences. Virus-specific sequences in transformed mouse cells were localized in the chromocenters of interphase nuclei.     

Comparisons of two early gene functions essential for transformation in polyoma virus and SV-40.

We have compared ts-a mutants of polyoma virus with ts-A mutants of SV-40, and hr-t mutants of polyoma virus with the viable deletion mutants of SV-40 mapping between 0.54 and 0.59 map unit (referred to as dl). All four groups of mutants are either totally or partially defective in inducing stable transformation as assayed by anchorage-independent growth. In each virus system, two classes of mutants—hr-t and ts-a of polyoma virus and dl and tsA of SV-40—complement to induce stable transformation. Two distinct functions essential for transformation are therefore encoded within the early regions of these papova viruses. Two approaches have been taken in attempts to define the roles of these early viral genes in cell transformation. In the first approach, a clonal analysis was made of cells transformed at the permissive temperature by ts-a/A mutants. Selections were carried out either for anchorage-independent growth or for focus formation. Although the variation in expression of the selected parameter of transformation among multiple clones derived with the same virus and cell line is often high, the majority of clones show no temperature dependence of either selected or unselected properties when compared to wild-type virus-transformed clones. In some instances, temperature-sensitive clones are observed. No correlation is seen between the appearance of a temperature-sensitive phenotype in individual clones and the expression of T-antigen species at permissive and nonpermissive temperatures determined by immunofluorescence or immunoprecipitation of [³⁵Slmethio-nine-labeled proteins. In the second approach, mutants of all four groups were tested for their ability to induce abortive transformation measured as the transient loss of anchorage-dependent growth. This assay circumvents the problem of clonal variation and gives a clearcut result. ts-a/A mutants retain the ability to induce abortive transformation, behaving like wild-type virus at the nonpermissive temperature. hr-t mutants are virtually negative, while the dl mutants show a reduced ability to induce abortive transformation. The simplest explanation which is adequate for the majority of the results is that the ts-a/A function is required only transiently to carry out an initiation event which stabilizes transformation, while the hr-t/dl function acts to induce parameters of the transformed phenotype in the manner of a maintenance function. Additional interpretations are put forward to explain the results with temperature-sensitive clones transformed by ts-a/A mutants.     

A novel cell transformation with DNA-containing cytoplasmic yaba tumor poxvirus

Monkey kidney cells were morphologically transformed in vitro with uv-irradiated Yaba tumor poxvirus. Cell lines established were shown to be virus nonproducers and exhibited biological characteristics typical of transformed cells. These characteristics included increased saturation density, reduced serum requirements for growth, and ability to grow in soft agar. The morphological alterations of transformed cells were similar to Yaba virus-induced tumor cells and were characterized by loss of contact inhibition, multinucleated cells, and cytoplasmic lipid droplets. Southern blot hybridization revealed that sequences homologous to low-molecular-weight viral DNA (5.1, 4.8, 3.9 kbp) were present in the transformed cells. Yaba virus-specific antigens detected by immunofluorescence assays were found to be in the cytoplasm of transformed cells. Four virus-specific proteins, with molecular weights of 160,000, 140,000 107,000, and 74,000 daltons, were detected in transformed cells immunoprecipitated with sera from tumor-bearing monkeys. These results indicate that DNA-containing Yaba tumor virus, which replicates exclusively in the cytoplasm, is capable of inducing cell transformation in vitro.     

Hematopoietic cell transformation by reticuloendotheliosis virus: Characterization of the genetic defect

A quantitative transformation assay has been developed for reticuloendotheliosis virus (REV) using cells of hematopoietic origin. The number of transformed colonies produced in soft agar was shown to be linearly related to the concentration of REV in the inoculum. Many of the in vitro transformed clones produce infectious REV and its helper virus, reticuloendotheliosis associated virus (REV-A). An analysis of the RNA monomers from particles released from virus-producing REV-transformed clones on denaturing methyl-mercuric hydroxide gels indicated that two distinct RNA species were present. The larger RNA species of REV-A had a molecular lenght of 8.7 kb and the smaller RNA monomer of REV had a molecular lenght of approximately 5.9 kb. Therefore, the defectiveness of REV is due to deletion of sequences essential for replication. Various clones of transformed cells produce different relative amounts of particles containing the RNA monomer of REV and REV-A. REV-transformed non-virus-producing cells were isolated which did not release infectious or noninfectious particles. Superinfection of REV-transformed non-virus-producing cells with the nontransforming members of the reticuloendotheliosis virus group resulted in pseudotype formation. Leukemogenesis and lymphoid cell transformation by REV pseudotypes were helper independent. In REV-transformed non-virus-producing cells the precursor polypeptides synthesized in virus-producing cells were not detected.     

Evolution and properties of Aedes albopictus cell cultures persistently infected with sindbis virus

Replicate cultures of Aedes albopictus cells were infected with Sindbis virus and then maintained for long periods of time by weekly subculture. During the first week the viral titers ranged between 10⁸ and 10⁹ PFU/ml, but then gradually fell and within a few weeks stabilized at about 10⁵ to 10⁸ PFU/ml. Such cultures were followed for the appearance of temperature-sensitive (ts) virus, small-plaque virus, and for the appearance within the cells of small (12–15 S) double-stranded viral RNA (dsRNA). Cloning experiments carried out 6 months or more after the initial infection showed that persistently infected cultures gave rise to both virus-yielding and nonyielding clones. Similar results were obtained when virus-positive clones were recloned. Prolonged treatment of persistently infected cultures with anti-Sindbis virus serum resulted in curing of the virus infection. Cured cultures behaved in every way tested as normal uninfected A. albopictus cells. The ability to cure with anti-viral serum suggests that in this system extracellular virus is needed to perpetuate the long-term infection. After persistently infected cultures were subcultured, viral RNA synthesis and viral yields were maximal during the first 3–4 days. Thereafter, although the cell number continued to increase for several days, viral RNA synthesis and viral yields both decreased sharply. This result strongly suggests that A. albopictus cells have efficient means for the regulation of viral biosynthesis. Although the resistance of persistently infected cultures to superinfection could be accounted for by interfering temperature-sensitive nondefective virus, the presence in these cells of 12 S dsRNA suggests that defective viral genomes are also present.     

RNA in mammalian sarcoma virus transformed nonproducer cells homologous to murine leukemia virus RNA

Mammalian sarcoma virus transformed nonproducer clones of mouse and rat cells contain RNA which hybridizes to the DNA product made from virus preparations containing leukemia virus, or sarcoma and leukemia virus. The hybridization of this virus-specific [³H]thymidine-labeled DNA to either total cellular or polysomal RNA extracted from these cells was detected with an enzymatic assay using a nuclease preparation either from mung beans or from Aspergillus oryzae, or by centrifugation in Cs₂SO₄ density gradients. The enzymatic assays were found to be more sensitive than Cs₂SO₄ for detection of such hybrids.     

Simple system for isolation of cellular and viral mutants for transformation by retrovirus.

To investigate the cellular mechanism of transformation by retroviruses, we established a system for isolation of cellular and viral mutants for transformation of a rat cell line. Mutagenized untransformed cells of this line were infected with recombinant murine retrovirus containing the src gene of Rous sarcoma virus and the selective marker gene, neo. After reaching confluence, cells transformed by the src gene tend to overgrow and die. Utilizing this property of src transformed rat cells and the selective marker gene, we could easily select untransformed cell clones containing the retrovirus genome. Expression of the src gene product in the flat clones selected was examined by in vitro assay of src kinase activity. To determine whether the mutations of these flat clones were viral or cellular, the susceptibilities of the clones to transformation were examined after superinfection with the wild-type virus and also characterized the retroviruses recovered from these clones. With this system, two novel clones were isolated. One had a defect in viral information affecting the transformed phenotype, but still retained src kinase activity like fully transformed cells. The other showed low src kinase activity but retained wild-type transforming virus, suggesting that a cellular gene involved in viral gene expression was mutated.     

Isolation of three new avian sarcoma viruses: ASV 9, ASV 17, and ASV 25

The newly isolated avian sarcoma viruses, ASV 9, 17, and 25, cause fibrosarcomas in young chickens and induce foci of transformed cells in chick embryo fibroblast cultures. They are defective in replication and belong to envelope subgroup A. The sizes of their genomes are 6 kb (ASV 9), 5 kb (ASV 17), and 6 kb (ASV 25), respectively. All three contain long terminal repeat (LTR) and gag sequences but lack pol. env is absent from ASV 9 and ASV 25, but some env sequences are detectable in ASV 17. None of the defective viral genomes hybridized to selected onc probes representing src, fps, yes, myc, myb, and erb A. erb B appears absent from ASV 9 and ASV 17, but some hybridization between the erb B probe and the RNA of ASV 25 was detected. ASV 9 codes for a transformation-specific gag-linked protein of 130kDa. Multiple gag-linked transformation-specific proteins are seen in ASV 17 and 25; they require further study.