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Transforming growth factors (TGFs) were purified from serum-free medium conditioned by retrovirus-transformed Fisher rat embryo fibroblasts, mouse 3T3 cells, and two human melanoma cell lines. The purification of each TGF was monitored in a radioreceptor assay based on receptor crossreactivity with mouse submaxillary gland epidermal growth factor (mEGF) and was achieved by gel permeation chromatography of the acid-soluble TGF-containing activity, followed by reverse-phase high-pressure liquid chromatography with sequential use of acetonitrile and 1-propanol in the presence of aqueous trifluoroacetic acid. The amino-terminal sequences of rat, mouse, and human TGFs were determined. Extensive sequence homology was found among TGF polypeptides from different species and cell types. Alignment of the amino acid sequences of rat TGF, mEGF, and human urogastrone (hEGF) reveals statistically significant sequence homology. The reported results suggest that TGFs that compete for binding to the cellular EGF receptor and EGF may have evolved from a common progenitor.

Endogenous xenotropic cat type C virus (RD-114)- and infectious feline leukemia virus (FeLV)-specific gene expressions were measured in spontaneous sarcomas carcinomas, and nonmalignant cat tissues by molecular hybridization for virus-specific RNA and competition radio-immunoassays for the major internal protein (p30) of these two viruses. The results indicate that RD-114 gene expression in sarcomas and carcinomas at both RNA and p30 levels is significantly higher than histologically normal tissues from cats free of cancer. In contrast, the levels of FeLV viral RNA and p30 are fount to be low or undetectable in the majority of these tumored and normal tissues examined. Whereas variability in the amounts of RD-114 OR FeLV RNA and p30 expressed is found in tissues from different cats, their expression is fairly uniform in multiple malignant tissues of the same cat. The finding of widespread occurrence of elevated RD-114 gene expression in sarcomas and carcinomas is consistent with our similar observation with natural lymphomas of domestic cats and suggests that expression of certain functions of this endogenous virus may be etiologically involved in the development of many different spontaneous neoplasms of cats.

Type C RNA viruses have been isolated from several Old World vertebrates, and an even larger number of Old World species have been shown to contain endogenous viral genetic sequences. The present report describes the first isolation of type C virus endogenous to a species originating in the New World. This virus, isolated from cells of the Columbian black-tailed deer, Odocoileus hemionus, is shown to possess genetic sequences in common with DNA of its species of origin. While it shares biochemical and immunologic characteristics with other mammalian type C viruses, its immunologic properties readily distinguish it from known endogenous viruses.

A new acute transforming type C retrovirus was isolated from mice inoculated with a virus stock obtained by iododeoxyuridine induction of methylcholanthrene-transformed C3H/10T1/2 mouse cells. This virus, designated 3611-MSV, transforms embryo fibroblasts and epithelial cells in culture and induces fibrosarcomas in vivo. 3611-MSV is replication defective, requiring a type C helper virus for propagation both in vitro and in vivo. By using endpoint transmission of 3611-MSV to MMCE C17 mouse and FRE 3A rat cells, several nonproductively transformed clonal cell lines have been derived. Pseudotype virus stocks obtained from such clones transform cells in vitro, are highly oncogenic in vivo, and exhibit host range and serological properties that are characteristic of their helper virus component. Analysis of viral antigen expression in 3611-MSV-transformed cells has led to the demonstration of a 90,000-molecular-weight (Mr) polyprotein and a 75,000-Mr probable cleavage product, both containing the amino-terminal murine leukemia virus gag gene proteins p15 and p12. In contrast to gene products of many previously described mammalian transforming viruses, 3611-MSV-encoded polyproteins lack detectable protein kinase activity, and 3611-MSV-transformed cells resemble chemically transformed cell line C3H/MCA-5, from which 3611-MuLV was originally derived, in that they do not exhibit elevated levels of phosphotyrosine. By using molecular hybridization the 3611-MSV transforming gene was found to be distinct from previously described mammalian cellular oncogenic sequences, including c-ras, c-abl, c-fes, c-fms, c-sis, and c-mos.

The previously described high-molecular-weight polyprotein major translational product of the Snyder-Theilen strain of feline sarcoma virus (FeSV) was shown to possess protein kinase activity with specificity for tyrosine acceptor sites. Cells transformed by Snyder-Theilen FeSV exhibited constitutively elevated levels of phosphotyrosine and a concomitant reduction in epidermal growth factor (EGF) binding sites. By endpoint cloning in microtiter plates, a number of transformation-defective (tf) mutants of the Snyder-Theilen strain of FeSV were isolated. Mink cells nonproductively infected by such mutants were morphologically nontransformed, failed to grow in soft agar, bound EGF as efficiently as control mink cells, and lacked rescuable transforming virus. Although the level of expression of the major viral polyprotein translational product in td mutant-infected clones was comparable to that of wild-type (wt) transformants, the polyprotein in mutant clones lacked detectable protein kinase activity and total cellular phosphotyrosine levels were not elevated significantly above control values. Of a large number of wt Snyder-Theilen FeSV-transformed mink cell clones isolated, the majority were found to revert to a nontransformed morphology upon continuous passage in cell culture. Such nontransformed variants, as well as a Gardner FeSV-transformed mink cell revertant, lacked detectable polyprotein expression and exhibited levels of phosphotyrosine and EGF binding similar to those of control mink cells. These findings provide strong evidence favoring the involvement of the Snyder-Theilen FeSV-encoded high-molecular-weight polyprotein and its associated tyrosine-specific protein kinase activity in transformation.

The translation product of the gag gene of mammalian type-c- RNA viruses is a 65,000-68,000 molecular weight precursor polypeptide (Pr65) whose cleavage leads to the formation of four virion proteins, p30, p15, p12 and p10. An immunological approach has been used to establish the arrangement of the sequences coding for these proteins within the viral genome as (5') p15-p12-p30-p10 (3').

Polyproteins encoded by several independent isolates of feline sarcoma virus (FeSV) were analyzed with respect to molecular weight, extent of phosphorylation, and tryptic peptide composition. As previously reported, cells nonproductively transformed by the Gardner strain of FeSV express a polyprotein which has a molecular weight of approximately 115,000 and contains feline leukemia virus p15, p12, and minor portion of p30. In addition, a major 72,000-dalton possible cleavage product can be identified. Snyder-Theilen FeSV-transformed cells express a major polyprotein of approximately 115,000 daltons and a second highly related 80,000-dalton protein. The p12 structural component of Gardner FeSV P115, but not Snyder-Theilen FeSV 115, corresponds to feline leukemia virus subgroup A with respect to immunological type specificity, a finding consistent with the independent origin of these viruses. Tryptic peptide analysis revealed five methionine-containing peptides specific to the nonstructural portion of Gardner FeSV 115, three of which were also represented in Snyder-Theilen FeSV P115, three of which were also represented in Snyder-Theilen FeSV P115. None of these [35S]methionine-labeled tryptic peptides were present in translational products representative of the complete feline leukemia virus subgroup A genome, including Pr180gag-pol, Pr65gag, and Pr82env. Similarly phosphorylated tryptic peptides within the structural (p12) and nonstructural components of Gardner FeSV P115 and Snyder-Theilen FeSV P115 Are highly related. These findings support the possibility that acquired sequences of two independently derived isolates of FeSV encode structurally related proteins.

The amino acid composition, the COOH-terminal amino acid, and the NH2-terminal amino acid sequence of the first 55 residues of the major internal structural protein, p24, of bovine leukemia virus (BLV) were determined. The compositional data and the results of end-group analysis revealed that, although BLV p24 is chemically distinct, it more closely resembles the p30 structural proteins than the other gag gene products of mammalian retroviruses. It was found that BLV p24 shares the common NH2-terminal proline and COOH-terminal leucine but lacks the common prolylleucylarginine tripeptide and the larger conserved region found near the NH2 terminus of all mammalian type C viral p30s. Alignment of the amino acid sequence of BLV p24 with the previously determined sequence of feline leukemia virus p27 revealed a statistically significant sequence homology. A more distant relationship was found between BLV p24 and other mammalian p30s. The finding of a definite sequence homology between BLV p24 and mammalian type C virus p30s clearly establishes the origin of these contemporary viral proteins from common progenitor genes.

Healthy cats, including those exposed to feline leukemia virus in leukemia cluster households, lack evidence of humoral immunity to virion surface antigens of the endogenous RD-114 virus.

Type-C viral polypeptides are expressed at very low levels in C57BL/10 liver and embryo cells as compared to cells of other mouse strains. The 12,000-mol. wt. polypeptide in C57BL/10 cells was purified and shown to possess type-specific antigenic determinants indistinguishable from those of a previously described class of endogenous virus. The very low levels of endogenous virus-specific RNA in cells of C57BL/10 as compared to other strains suggests control of this endogenous virus at the level of transcription. Analysis of viral antigen expression F1 hybrids of C57BL/10 with strains characterized by intermediate and high levels of viral antigen expression indicates that the restriction of this endogenous virus by C57BL/10 cells is not due to simple dominance by a repressor of virus expression.

Mink cells morphologically transformed by either Snyder-Theilen feline sarcoma virus (ST-FeSV) or Abelson murine leukemia virus (Abelson-MuLV) exhibit relatively high rates of reversion to the nontransformed phenotype. The proviral DNAs are conserved within the revertant lines and have not undergone changes in integration sites due to translocations or other genomic rearrangements. In contrast, expression of well-defined viral-encoded transforming proteins is blocked and elevated levels of phosphotyrosine characteristic of the parental transformed cells are reduced to control levels. Loss of the transformed phenotype is associated with increased cytosine methylation of proviral DNA sequences while levels of methylation resume control levels upon spontaneous retransformation of revertant clones. Following molecular cloning, and transfection to Rat-2 cells, ST-FeSV proviral DNAs from revertant and transformed cells induced similar numbers of transformed foci. Cytosine methylation sites involved in regulation of expression of the major ST-FeSV encoded transforming protein have been localized within the proviral DNA itself rather than in adjacent cellular flanking sequences. In contrast to the v-fes proviral DNA, c-fes, the cellular homolog of the ST-FeSV acquired transforming sequences, is highly methylated in cytosine residues in both transformed and revertant clones. These findings demonstrate regulation of viral oncogene-mediated transformation by cytosine methylation and suggest that expression of cellular homologs of viral oncogenes, such as c-fes, are also subject to regulation at this level.

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.

Mouse cells contain several biologically distinguishable endogenous type-C viruses. In the present studies, the immunologic response of different strains of mice to the natural expression of these viruses has been examined. High titers of neutralizing antibodies have been detected to a class of virus that lacks the ability to exogenously infect mouse cells. The expression of this virus, in vivo as well as in vitro, is shown to be vertically transmitted as a dominant genetic trait. The findings indicate the widespread occurrence of this endogenous virus and the ability of the mouse to immunologically respond to a class of type-C virus whose mode of transmission is similar to that of cellular genes.

The immunological responses of inbred mice to structural components of one class of endogenous virus were investigated by means of radioimmunoassays utilizing highly purified viral proteins. Naturally occurring antiviral antibodies were demonstrated only in those strains possessing information for induction of a mouse cell-tropic endogenous virus. Moreover, these antibodies invariably appeared subsequent to the detection of spontaneous replication of this virus in the same animal. The immune responses elicited were much stronger against endogenous viral gp70 than p30, consistent with previous findings of tolerance in the mouse to the major structural antigen of its endogenous virus. However, the demonstration of an immune response to p30 under conditions of both natural and experimental immunization establishes that tolerance to this viral antigen can be overcome.

Cells of mouse and rat origin contain endogenous type C viruses. There are intrinsic regulatory factors which restrict the ability of different classes of these inducible viruses to persist in cells after chemical activation. The cellular block to certain viruses is shown to be overcome under conditions in which the virus-activated cells are allowed continued exponential growth. These findings may help to explain the high frequency at which replicating type C viruses are detectable in naturally occurring tumors of some species.

Cell lines transformed by woolly monkey sarcoma virus (WSV) in the absence of infectious virus production were analyzed for the expression of woolly monkey helper viral p30, p12, and gp70 antigens. Several lines produced high levels of both p30 and p12, whereas gp70 was not detectable. One transformed clone expressed only p12, and in another cell line, none of the helper viral antigens were detected. The properties of each sarcoma virus bred true upon transmission, indicating that each variant represents a distinct genotype. The different cell lines were examined with respect to properties characteristic of the transformed state. The in vitro growth properties and oncogenicity of each WSV-transformed clone were indistinguishable, indicating that transformation by WSV occurs independently of the expression of at least three helper viral polypeptides.

The major nonglycosylated structure proteins of mammalian type C RNA tumor viruses are synthesized in the form of a high molecular weight precursor coded for by a viral gene disignated "gag". Previous genetic analysis of a prototype virus isolated of mouse origin has led to a determination of the internal arragnement of the regions within the gag gene coding for individual structural proteins. In the present study, the biochemical properties of structural proteins of type C virus isolates of additional mammalian species were analyzed. The results obtained indicate that the biochemical properties of immunologically cross-reactive proteins have been highly conserved throughout the evolution of this group of viruses. Moreover, these findings provide a means of mapping the gag genes of a broad range of mammalian type C viruses. In view of the results obtained, a new nomenclature system for type C viral gag gene-coded translational products is proposed.

A highly sensitive and specific radioimmunoassay has been developed for the major structural protein of an oncornavirus etiologically associated with bovine lymphosarcoma. This test can be used to identify cattle which have been exposed to the bovine leukemia virus and may thus develop or transmit the disease. Analysis of randomly obtained serums indicates that infection that infection with this virus is widespread among cattle.

Several dual-tropic isolates derived from the thymuses of preleukemic or leukemic AKR mice and a more recrnt group of viruses generated by in vitro or in vivo passage of a poorly infectious endogenous virus of C3H mouse cells have been shown to be highly oncogenic. By analysis of the immunological properties of their gag gene-coded structural proteins, each of the AKR-derived isolates and two dual-tropic C3H-derived isolates were found to closely resemble AKR murine leukemia virus. In contrast, gag gene-coded proteins of two other leukemogenic isolates of C3H origin, including one ecotropic and one dual-tropic virus, were indistinguishable from those of Moloney murine leukemia virus. All of the oncogenic isolates, including those of AKR and C3H origin, were found to possess common envelope glycoprotein determinants of a unique class not shared by the nononcogenic ecotropic viruses from which they were derived. These findings support the possibility that oncogenic variants of endogenous ecotropic mouse type C viruses are derived by genetic recombination. This recombinational event appears to involve the acquisition, by different ecotropic viruses, of a common class of endogenous virus-coded envelope glycoprotein determinants which are presumably required, but not necessarily sufficient, for oncogenicity.

To define the human homolog (or homologs) of transforming sequences (v-fes gene) common to Gardner (GA) and Snyder Theilen (ST) isolates of feline sarcoma virus (FeSV), a representative library of human lung carcinoma DNA in a cosmid vector system was constructed. Three cosmid clones were isolated containing GA/ST FeSV v-fes homologous cellular sequences, within 32- to 42-kilobase cellular inserts representing 56 kilobases of contiguous human cellular DNA. Sequences both homologous to, and colinear with, GA or ST FeSV v-fes are distributed discontinuously over a region of up to 9.5 kilobases and contain a minimum of three regions of nonhomology representing probable introns. A thymidine kinase selection system was used to show that, upon transfection to RAT-2 cells, the human c-fes sequence lacked detectable transforming activity.

Cells nonproductively transformed by a variant of the Snyder-Theilen strain of feline sarcoma virus (FeSV) expressed an 85,000-dalton polyprotein (P85) with associated tyrosine-specific protein kinase activity. We identified within this polyprotein a single tyrosine acceptor site for its enzyme activity. This acceptor site, as well as two serine phosphorylation sites localized with the p12 structural component of Snyder-Theilen FeSv P85, was phosphorylated in cells nonproductively transformed by Snyder-Theilen FeSv. In contrast, infection by Snyder-Theilen FeSV transformation-defective mutants resulted in phosphorylation only of the two serine acceptor sites, indicating phosphorylation of the tyrosine acceptor site to be transformation specific. In addition, we describe in vitro labeling conditions, using unfractionated cell extracts, which resulted in preferential phosphorylation of the single Snyder-Theilen FeSV tyrosine-specific acceptor site.

Sensitive radioimmunoassays were developed for avian type C viral gag gene-coded proteins. These assays were used to examine the restriction to virus production by avian embryo cells and mammalian cells transformed by avian sarcoma viruses. The results indicate that although a high-molecular-weight primary translational product of the gag gene is expressed, its cleavage and processing are incomplete. Furthermore, analysis of intermediate cleavage products provided information regarding the order of sequences coding for the individual viral proteins within the avian type C viral gag gene.

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 Synder-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 (less than 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.

RNA C-type viruses are known to exist in an unexpressed form in all mouse cells. These endogenous viruses can be activated from cells in tissue culture under certain experimental conditions. The biologic activity in vivo of one class of chemically-activated C-type virus has been studied. This virus is shown to induce a specific tumor, lymphatic leukemia, in mice of a low leukemic incidence strain.

The Gardner strain of feline sarcoma virus (FeSV) has previously been shown to encode as its major translational product, a phosphoprotein of molecular weight (Mr) = 115,000 (P115) with an associated protein kinase activity and probable transforming function. In the present study, we demonstrate that this enzyme specifically phosphorylates tyrosine and that Gardner FeSV-transformed mink and rat cells exhibit constitutively higher levels of phosphotyrosine than nontransformed control cells. In addition, we identify a Mr = 150,000 cellular substrate for the FeSV P115-associated protein kinase activity. This latter protein, designated P150, is expressed in cells of a number of mammalian species including cat, mink, dog, rat, mouse and human, possesses broadly reactive interspecies antigenic determinants, and exhibits binding affinity for FeSV P115. Although P150 exhibits an associated protein kinase activity, this activity differs from the FeSV P115-associated enzyme in that it phosphorylates serine and threonine rather than tyrosine. On the basis of immunologic criteria and tryptic peptide analysis, the possibility that P150 is structurally related to Gardner FeSV P115 is excluded. As a consequence of its affinity for FeSV P115, P150 is efficiently incorporated into FeSV pseudotype virions, providing a source for its purification and preparation of antisera for study of its expression in mammalian cells.

The major envelope glycoprotein of bovine leukemia virus was isolated by lectin-bound Sepharose and DEAE-cellulose column chromatography. This protein was shown to have a molecular weight of about 41,000 and to lack detectable immunological cross-reactivity with glycoproteins of other oncornaviruses. Sera obtained from 100% of cattle examined with clinically diagnosed lymphosarcoma contained high-titered antibody to 125I-labeled bovine leukemia virus glycoprotein, whereas sera from animals in a disease-free herd were antibody negative.

The feline oncornavirus-associated cell membrane antigen (FOCMA) acts as a target for natural immuno-surveillance against tumor development in the cat. In the present study, mink and rat cells nonproductively transformed by feline sarcoma virus (FeSV) were shown to express FOCMA as well as 5'-terminal feline leukemia virus (FeLV) gag gene proteins, p15 and p12. In contrast, such cells lack detectable levels of other FeLV gag gene-coded proteins or the env gene product, gp70. FOCMA, p15, and p12 antigen expression is initially in the form of an 80,000-100,000 molecular weight precursor which, upon post-translational cleavage, gives rise to a 65,000 molecular weight component that contains FOCMA and a 25,000 molecular weight component containing p15 and p12. Feline lymphoma cells, including those from several tumors that lacked detectable levels of FeLV structural protein expression, were shown to be FOCMA-positive. These findings strongly suggest that FOCMA represents an FeSV-coded transformation specific protein and provide preliminary information regarding the position within the FeSV genome coding for its synthesis.

The integrated form of McDonough FeSV proviral DNA, including cellular flanking sequences, was molecularly cloned from nonproductively transformed Fisher rat cells. Acquired cellular-derived (v-fms) sequences within the cloned proviral DNA were mapped from between 2.6 and 5.5 kb from the 5'LTR. Upon transfection, the cloned proviral DNA was biologically active; it caused induction of the transformed phenotype and the resulting transformed cells expressed the major McDonough FeSV translational product, P170gag-fms at high level. Using a series of molecular probes representing subgenomic regions of the viral v-fms gene, a cosmid library of human lung carcinoma DNA was screened for v-fms homologous sequences. Three cosmid clones containing overlapping v-fms homologous cellular DNA inserts, representing a contiguous region of cellular DNA sequence of approximately 64 kb in length, were isolated. Within this region of human genomic DNA, v-fms homologous sequences are dispersed over a total region of around 32 kb. These represent the entire human cellular homolog of v-fms, are colinear with the viral v-fms transforming gene, and contain a minimum of four intervening sequences. At least 12 regions of highly repetitive DNA sequences have been mapped in close proximity to c-fms coding sequences.