Biological properties and translational products of three independent isolates of feline sarcoma virus

Three transforming retroviruses have been isolated from naturally occurring tumors of outbred cats. In the present report, the biological properties of the SM, GA, and ST strains of feline sarcoma virus (FeSV) have been characterized in tissue culture. Like previous mammalian transforming viruses, each FeSV strain was demonstrated to be replication defective, requiring a type-C RNA virus as a helper. Reproducible differences were demonstrated in the morphology of transformed foci induced by each FeSV strain. These findings were independent of helper virus or assay cell utilized, suggesting that the transforming activities of the three FeSV strains are distinguishable. Nonproducer transformants of SM-FeSV expressed feline leukemia virus (FeLV) gag gene products, p15, p12, and p30. In contrast, the GA-and ST-FeSV genomes coded only for p15 and p12 proteins, further distinguishing SM-FeSV from the other FeSV strains. Each remained stable with respect to viral protein expression upon transmission to new cells, demonstrating the stable association of FeLV gag gene information with each FeSV genome. Analysis of the FeLV structural proteins translated in nonproducer transformants of the three FeSV strains revealed the presence of precursor molecules, whose sizes could not be accounted for solely by the number of FeLV gag proteins present in them.     

Isolation of a new feline sarcoma virus (HZ1-FeSV): Biochemical and immunological characterization of its translation product

A new strain of feline sarcoma virus, designated HZ1-FeSV, was isolated from a 4-year-old domestic cat with multicentric fibrosarcoma. A primary tumor cell line was established and virus produced from that line was found to induce foci in feline embryonic lung fibroblasts (FLF3) and mink lung fibroblasts (CCL64) in tissue culture and fibro-sarcomas in inoculated 10-week-old kittens. The derivation of transformed nonproducer clones of FLF3 and CCL64 cells containing helper virus-rescuable, focus-forming activity indicated that HZ1-FeSV was defective for replication. The only discernible translation product of the HZ1-FeSV genome in cultured cells was a 100,000-Da polyprotein (P100) which contained amino-terminal sequences of the FeLV gag gene precursor protein covalently linked to a sarcoma virus-specific domain. Immunoprecipitates containing P100 exhibited a protein kinase actvity capable of phosphorylating tyrosine residues of P100. Immunologically, P100 was highly cross-reactive with gag-fes polyproteins encoded by two previously characterized strains of FeSV, the GA- and the ST-FeSV. By comparison of methionine-containing tryptic peptides, the HZ1-FeSV protein was shown to be more closely related to the GA-FeSV protein than to the ST-FeSV protein, but to be distinguishable from both other proteins.     

Avian sarcoma virus PRCII: Conditional mutants temperature sensitive in the maintenance of fibroblast transformation

Avian sarcoma virus PRCII was mutagenized with 5-azacytidine, and from single foci of transformed cells induced by this virus preparation three conditional mutants were isolated that are temperature sensitive for oncogenic transformation of fibroblasts. Two of these, LA42 and LA47, are not affected in their replication potential at the nonpermissive temperature (41.5°) nor are the virions heat labile. The third mutant, LA46, is coordinately temperature sensitive in replication and transformation. A shift of the incubation temperature from 36° to 41.5° at any time after infection causes transformed cells by any one of the three mutants to revert to normal morphology.     

Transformation with subgenomic fragments of feline sarcoma virus proviral DNA

Murine fibroblasts can be transformed by subgenomic fragments of feline sarcoma virus (FeSV) proviral DNA generated by BamH-I digestion of DNA of Snyder-Theilen FeSV and FeLV-infected mink cells. The efficiency of transformation of NIH 3T3 cells by BamH-I-treated FeSV proviral DNA was the same as that of untreated FeSV DNA. Focus-forming virus could not be rescued from the BamH-I-digested FeSV DNA-transformed cells. However, DNA from these transformed cells was able to induce transformation in secondary transfection experiments. Analysis of the DNAs from two BamH-I-digested FeSV DNA-transformed clones showed that the transformants contained only the 5′ two-thirds of the FeSV provirus. Cells transformed by subgenomic fragments of FeSV DNA produce the 85,000-dalton gag-x protein, but do not express FOCMA. These experiments support the notion that the gag-x protein plays an active role in maintenance of the transformed phenotype.     

Feline sarcoma virus-specific transformation-related proteins and protein kinase activity in tumor cells

Polyproteins (gag-fes) encoded by the Synder-Theilen (ST) and the Gardner-Arnstein (GA) strains of feline sarcoma virus (FeSV) were previously shown to be associated with mink or rat cells that were nonproductively transformed in vitro. In the present study we demonstrated that the same gag-fes proteins were found in cat cells transformed in vitro. Of greater importance, these transformation-related proteins were also in cells taken from fresh biopsies of FeSV-induced tumors. Cells from fibrosarcomas induced with ST-FeSV had gag-fes proteins that were characteristic of this strain. Fibrosarcomas and melanomas were induced with GA-FeSV and both types of tumors contained the protein that is characteristic of cells transformed in vitro with this virus. Expression of these proteins in cultured tumor cells appeared to be independent of the passage level. Based on two-dimensional tryptic peptide analysis, the gag-fes proteins of cat tumor cells appeared to be indistinguishable from those found in cells transformed in vitro. The polyproteins of the cat tumor cells have a closely associated protein kinase activity, as demonstrated in the in vitro assay, and phosphorylated tyrosine residues. Gag-fes proteins of either the ST or GA class were not present in cell cultures initiated from five spontaneous cat tumors.     

Similar transforming growth factors (TGFs) produced by cells transformed by different isolates of feline sarcoma virus

Fisher rat embryo cells transformed by each of three independent isolates of feline sarcoma virus (FeSV) are shown to release transforming growth factors (TGFs) into cell culture medium. These acid- and heat-stable peptides compete for binding to, and stimulate phosphorylation of, EGF membrane receptors and promote anchorage-independent cell growth. Cells transformed by the Gardner and Snyder-Theilen strains of FeSV produce high titers of TGF (60-200 ng eq EGF/liter) while cells transformed by McDonough FeSV produce TGF at only low levels (<10 ng eq EGF/liter). Growth factors produced by cells transformed by each of the three FeSV isolates functionally and biochemically resemble each other, mouse sarcoma growth factor (SGF), and TGFs produced by human tumor cells.     

Genetic analysis of the defectiveness in strain MC29 avian leukosis virus.

Avian myelocytomatosis virus MC29 is defective in replication. The extent of this defectiveness was analyzed in complementation experiments. Continuous nonproducer quail cell lines transformed by MC29 were superinfected with different helper viruses. Infectious MC29 pseudotypes were formed only with helper viruses which belonged to the avian leukosis-sarcoma virus complex, e.g., Rous-associated virus type 1 (RAV-1) or ring-necked pheasant virus (RPV). Helper viruses of other retroviral species, such as reticuloendotheliosis virus (REV), golden pheasant virus (GPV), or an amphotrophic murine leukemia virus (MuLV-1313), could not rescue MC29 from these nonproducer cells but complemented the envelope-defective Bryan high titer strain of Rous sarcoma virus. Host range and interference patterns of MC29 rescued from nonproducer cells indicated that the envelope specificity of the pseudotypes was determined exclusively by the activating helper virus. Cocultivation or Sendai virus-induced fusion of MC29 nonproducer cells with quail or chicken cells transformed by the defective Bryan high titer strain of Rous sarcoma virus did not result in complementation of the defects in either of the two viruses. Fluorescent antibody staining failed to detect virus-specific antigens at the surface of MC29 nonproducer cells. Temperature-sensitive mutants of Rous sarcoma virus with lesions in the genes coding for the RNA-directed DNA polymerase (pol gene) or the nonglycosylated structural proteins (gag gene) did not acquire wild-type characteristics when grown in MC29 nonproducer cells. It is concluded that MC29 is defective in all three genes essential for the replication of retroviruses, namely, env, pol, and gag.     

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.     

A transformation defective mutant of Rous sarcoma virus inducing chick embryo neuroretinal cell proliferation.

Infection of chick embryo neuroretinal (NR) cells by Rous sarcoma virus (RSV) results in neoplastic transformation and induction of sustained cell proliferation. Both cell transformation and multiplication depend on genetic information(s) only present in sarcoma viruses. However, the observation that NR cells infected with T-class temperature sensitive mutants of RSV are induced to multiply at nonpermissive temperature, in the absence of transformation, also suggests that “growth stimulation” and “transformation” may represent distinct information expressed by sarcoma viruses in NR cells.Using induction of cell multiplication as a genetic marker, it has been possible to isolate a mutant of RSV, designated td PA-101, in which the mitogenic and transforming properties are permanently dissociated in NR cells. This mutant is defective in transforming NR cells and chick embryo fibroblasts and does not produce sarcomas in chicks. Therefore, td PA-101 represents a novel class of nonconditional mutants of RSV which differ from other transformation defective viruses in fully retaining the capacity to induce NR cell proliferation.     

Transforming genes of avian (v-fps) and mammalian (v-fes) retroviruses correspond to a common cellular locus

The Gardner (GA) and Snyder-Theilen (ST) isolates of feline sarcoma virus (FeSV) represent genetic recombinants between feline leukemia virus (FeLV) and transformation-specific sequences (v-fes gene) of cat cellular origin. A related transforming gene (v-fps), common to the Fujinami, PRC II, and UR 1 strains of avian sarcoma virus has also been described. Translational products of each of these recombinant virus isolates are expressed in the form of polyproteins exhibiting protein kinase activities with specificity for tyrosine residues. In the present study, v-fes and v-fps homologous sequences of GA-FeSV, ST-FeSV, and Fujinami sarcoma virus (FSV) are defined and these independently derived transforming genes are shown to correspond to a common cellular genetic locus which has remained highly conserved throughout vertebrate evolution.     

Restitution of fibroblast-transforming ability in src deletion mutants of avian sarcoma virus during animal passage.

The Schmidt-Ruppin strain of Rous sarcoma virus subgroup D (SR-D) gives rise to transformation defective (td) mutants which have lost either all or almost all of the src gene (standard td or std viruses) or have only a partial deletion of src. These partial deletion mutants, designated ptd viruses, contain genomic RNA slightly larger than std isolates, and heteroduplex analyses suggest that ptd viruses retain approximately 25% of src from the 5′ end of that gene [Lai et al. (1977) Proc. Natl. Acad. Sci. USA74, 4781–4785]. Several ptd isolates of SR-D were injected into newly hatched chickens and after prolonged latent periods caused sarcomas in about 30% of the birds. The tumors occurred in internal organs away from the site of injection. Infectious sarcoma viruses isolated from these growths show the envelope markers of subgroup D are nondefective for replication and induce a transformation in vitro which is morphologically distinct from that of SR-D. Electrophoresis of 35 S genomic RNA from these recovered sarcoma viruses shows it to be of the size characteristic for nondefective sarcoma viruses. Fingerprint analysis of ³²P-labeled RNA from one of the new sarcoma viruses detected all oligonucleotides present in ptd viruses, the src-specific oligonucleotides of SR-D, and one new oligonucleotide not present in SR-D. This new RNase T₁-resistant oligonucleotide and the src-specific oligonucleotides identical to those of SR-D map close to the 3′ end in the genome of the recovered sarcoma virus, which is the position expected for the src gene. These studies suggest that recovered avian sarcoma viruses have acquired cellular sequences which are closely related in structure and function to the viral src gene.     

Transformation parameters of chick embryo fibroblasts transformed by Fujinami, PRCII, PRCII-p, and Y73 avian sarcoma viruses

The transformation parameters of chicken embryo fibroblasts transformed by replication defective avian sarcoma viruses were compared with those of cells transformed by Rous sarcoma virus (RSV). Two classes of avian sarcoma virus with transforming genes distinct from the src gene of RSV were examined: Fujinami sarcoma virus (FSV), PRCII, and PRCII-p were used as representatives of one class, and Y73 as a representative of the other class. The transformation parameters examined included morphological markers: cell surface morphology, the disappearance of microfilament bundles, and the loss of cell surface fibronectin; alterations in growth properties: growth to high densities and growth in suspension; and metabolic changes: increased uptake of hexose, release of plasminogen activator, and increased synthesis of hyaluronic acid. In general, all of the viruses induced a phenotype which was qualitatively similar to that of RSV-transformed cells, although the extent of each change was less than that observed with RSV. One strain of FSV was found to be coordinately temperature sensitive for the induction of all phenotypic markers examined, whereas another strain of FSV and the other viruses examined were temperature resistant. PRCII-transformed cells had a lower efficiency of colony formation in agar suspension and showed only partial disruption of microfilament bundles; this partial transformation phenotype may be correlated with the low oncogenicity of this virus. These findings in conjunction with previous studies on protein phosphorylation in ASV-transformed cells are consistent with the idea that the replication-defective avian sarcoma viruses transform cells by a mechanism similar to that involved in transformation by RSV.     

Tyrosine phosphorylation sites common to transforming proteins encoded by Gardner and Snyder-Theilen FeSV

The Gardner and Snyder-Theilen strains of feline sarcoma virus (FeSV) encode 110,000 molecular weight (M_r) polyproteins as their primary translational products. Cells transformed by a variant of Snyder-Theilen FeSV express an 85,000 M_r polyprotein. Single tyrosine phosphorylation sites identified within each of these viral gene products are shown to represent major in vitro acceptors phosphorylated by the polyprotein associated protein kinases. By two-dimensional tryptic peptide analysis, these acceptor sites are highly related although exhibit minor differences in map position, while by analytical high-performance liquid chromatography, all three elute at approximately the same acetonitrile concentration (21–23%). The tyrosine acceptor sites in these FeSV-encoded polyproteins have been localized by sequential Edman degradation at a position seven amino acid residues distal to their trypsin cleavage site. The major tyrosine acceptors, two serine phosphorylation acceptors common to the p12 structural components of all three polyproteins, a single major phosphothreonine site, and several minor less well-resolved acceptor sites are shown to be phosphorylated in vivo. In addition to the major tyrosine acceptor site initially phosphorylated in vitro, phosphorylation of secondary tyrosine acceptors is shown to occur to proportionately greater extents when in vitro phosphotransferase reactions are carried out for longer times or at higher temperatures.     

Sarcoma growth in 15l5×72 chickens infected with avian sarcoma viruses of subgroup B or G

A comparative study was made of sarcoma growth in 15I₅×7₂ chickens infected in the wing web at 4 weeks of age with strains of subgroup B or G avian sarcoma viruses. Infection with sarcoma viruses of either subgroup B or G resulted in the formation of progressive wing web sarcomas at the site of inoculation. The survival times of the subgroup G virus-infected chickens were generally at least twice as great as the survival times of the subgroup B virus-infected chickens, which averaged 6–9 weeks postinoculation. At 5 weeks postinfection, a significantly higher titer of virus neutralizing antibody was detected in the subgroup G virus-infected chickens. Necropsy indicated that a high percentage of subgroup B virus-infected chickens exhibited fibrosarcomas at sites distal to the primary wing web sarcomas, whereas only a small percentage of subgroup G virus-infected chickens exhibited distal sarcomas. The results further indicated that the viral env gene is a determinant of the pattern of distal sarcoma formation.     

RNA species obtained from clonal lines of avian sarcoma and from avian leukosis virus

Gel electrophoresis of the dissociated 60–70 S RNA prepared from cloned avian sarcoma viruses showed a single major peak corresponding to size class a. Class b RNA was not detectable in these preparations. Class a RNA derived from the Schmidt-Ruppin strain of Rous sarcoma virus had a slightly but reproducibly lower electrophoretic mobility than class a RNA of Prague strain Rous sarcoma virus. The presence of avian tumor virus group specific (gs) antigens in chicken cells before infection did not detectably influence the electropherograms of the dissociated avian sarcoma virus RNA: cloned sarcoma virus from gs positive and gs negative cells showed identical gel patterns. However, two avian sarcoma virus clones derived from a clonal colony of transformed cells contained a new RNA component which migrated slower than class a RNA. This unusual component was found consistently in the original clonal cultures but was not seen if new cells were infected with the same virus, suggesting a cell mediated modification of viral RNA.Additional evidence was obtained for the absence of class a RNA from avian tumor viruses which cannot form foci in fibroblast cultures. Several leukosis and transformation defective viruses which had not been analyzed previously showed class b RNA only. No significant differences between the class b RNA preparations obtained from several independently isolated transformation defective viruses were detected.     

Differential expression of transformation in rat and chicken cells infected with an avian sarcoma virus ts mutant

Cells of the NRK line were infected with ts 339, a mutant of avian sarcoma virus B77, and clones were isolated which displayed the characteristics of transformed cells at 33 C. These cells reverted to a normal phenotype at 37 C. In comparison, chicken cells infected with the same mutant remained transformed at 37 C and acquired properties of normal cells only after temperature shift to 41 C.     

Esh avian sarcoma virus codes for a gag-linked transformation-specific protein with an associated protein kinase activity

Esh sarcoma virus, initially isolated from a spontaneous tumor of a chicken, transforms fibroblasts in vitro and induces fibrosarcomas in vivo. It is defective for replication, and infectious viral stocks consist of a mixture of a sarcomagenic virus (ESV) and an a avian leukosis virus of subgroup A (EAV) which serves as helper. Cloned stocks of infectious ESV contain two RNA components of M_r, 3 and 1.5 × 10⁶, respectively, as determined by electrophoresis in sodium dodecyl sulfate-polyacrylamide gels. The component of M_r 1.5 × 10⁶ appears to be the genome of defective ESV, since it is not detected in preparations of the helper virus EAV. The size of the ESV genome suggests major deletions of replicative genes, and ESV-transformed nonproducer cells fail to express functional translation products of the gag, pol, and env genes. ESV-transformed producer and nonproducer clones also do not express pp60^src but contain a gag-related protein of M_r 80,000 (p80). Two-dimensional analyses of the [³⁵S]methionine-labeled tryptic peptides of p80 indicate that this protein contains part of the sequences of gag-p19 covalently linked to additional sequences unrelated to gag, pol, and env gene products. These ESV-specific sequences are also unrelated to pp60^src and to gag-linked polyproteins found in cells transformed by defective avian sarcoma viruses PRCII and Fujinami or defective leukemia viruses AEV, MC29, and MH2. P80 is phosphorylated in vivo at two major sites, one involving phosphoserine and the other phosphotyrosine residues. Immunoprecipitates containing ESV-p80 are associated with a protein kinase activity that is specific for tyrosine residues of several acceptor molecules including p80 itself, rabbit immunoglobulin H chain of the immune complex and exogenously added α-casein. p80 is phosphorylated in vitro at the same tyrosine site as in vivo suggesting that the enzyme activity detected in vitro is of physiological significance. The p80-associated protein kinase activity is strictly dependent on the presence of Mg²⁺ or Mn²⁺ but was found independent of known effectors of cellular protein kinases Ca²⁺, cAMP, or cGMP.     

The transformation-specific proteins of avian (Fujinami and PRC-II) and feline (Synder--Theilen and Gardner--Arnstein) sarcoma viruses are immunologically related

The detection of protein kinase activity associated with transformation-specific gene products of Rous sarcoma virus (RSV), Abelson murine leukemia virus (Ab-MuLV), the Snyder-Theilen (ST), and Gardner-Arnstein (GA) strains of feline sarcoma virus (FeSV) and the recently characterized Fujinami (FSV) and PRCII avian sarcoma viruses suggests that these viruses may induce oncogenic transformation by similar mechanisms. In spite of their similar enzymatic properties, the transformation-specific proteins of RSV or Ab-MuLV were not found to share common antigenic determinants with those of the ST or the GA strains of FeSV. These results suggest that vertebrates contain multiple unrelated genes that code for proteins with associated tyrosine-specific protein kinase activity and oncogenic properties when incorporated into the genome of a retrovirus. In contrast, the transformation-specific proteins of ST and GA-FeSV were shown to be immunologically related to those of the FSV and PRCII avian sarcoma viruses. The antigenic determinants shared by these viral proteins have been mapped within their respective sarcoma virus-specific regions, suggesting that the cellular insertions present in these avian and feline sarcoma viruses are related. These observations indicate that potentially oncogenic sequences have been conserved during the evolution of feline and avian genomes and have been independently acquired by two sets of sarcoma viruses.