Gardner-Arnstein Research Articles

Structure of the feline c-fes/fps proto-oncogene: genesis of a retroviral oncogene

The nucleotide sequence of the feline c-fes/fps proto-oncogene was analyzed. Comparison with v-fes and v-fps revealed that all v-fes/fps homologous sequences were dispersed over 11 kilobase pairs in 19 interspersed segments. All segments, numbered exon 1 to exon 19 as in the chicken and human loci, were flanked by consensus splice junctions. The putative promoter region contained a CATT sequence and three CCGCCC motifs which were also found in the human locus at similar positions. About 200 nucleotides downstream of a translational stop codon in exon 19, a putative poly(A) addition signal was identified. Using the putative translation initiation codon in exon 2, a 93,000-molecular-weight protein could be deduced. This protein resembled very well the putative protein of the human c-fes/fps proto-oncogene (94% overall homology) and, although less well, the putative protein of the chicken c-fes/fps proto-oncogene (70% overall homology). As far as the feline c-fes/fps proto-oncogene sequences transduced to the Gardner-Arnstein (GA) and Snyder-Theilen (ST) strains of feline sarcoma virus (FeSV) are concerned, homology in deduced amino acid sequences between the GA- and ST-v-fes viral oncogenes and the proto-oncogene was 99%. Analysis of the recombination junctions between feline leukemia virus and v-fes sequences in GA- and ST-FeSV proviral DNA revealed for the left-hand junction the involvement of homologous recombination, presumably at the DNA level. The right-hand junction, which appeared identical in the GA-FeSV and ST-FeSV genomes, could have been the result of a site-specific recombination at the RNA level.

Monoclonal antibodies to the v-fes product and to feline leukemia: Virus P27 interspecies-specific determinants encoded by feline sarcoma viruses

Monoclonal antibodies to p27 gag and v- fes specific determinants on the gag-onc polyprotein encoded by Snyder-Theilen feline sarcoma virus (ST-FeSV) were prepared. In order to obtain hybridoma clones specific to the antigenic determinants encoded by the FeSV genome, Lou rats were immunized with ST-FeSV-transformed, virus-nonproducing syngeneic cells, and boosted with either the same cells or affinity-purified feline leukemia virus (FeLV) p27. Three distinct clones reactive to both FeLV p27 and p85 gag-fes , and one clone specific for a p85 fes determinant were established. The anti-p27 monoclonal antibodies also reacted with the polyproteins p959 gag-fes and p839 gag-fgr from Gardner-Arnstein (GA) and Theilen-Pedersen (TP1) FeSV, respectively. The anti-p27 monoclonal antibodies reacted with MuLV p30 and RD114 p28 but not with RSV, MMTV, or BLV. These results indicated that the part of the p27 gag gene that is preserved in ST-, GA, and TPI-FeSV encodes interspecies-specific p27 determinants.

Molecular cloning of the feline c-fes proto-oncogene and construction of a chimeric transforming gene

The feline c-fes proto-oncogene, different parts of which were captured in feline leukemia virus (FeLV) to generate the transforming genes (v-fes) of the Gardner-Arnstein (GA) strain of feline sarcoma virus (FeSV) and the Snyder-Theilen strain (ST) of FeSV, was cloned and its genetic organization determined. Southern blot analysis revealed that the c-fes genetic sequences were distributed discontinuously and colinearly with the v-fes transforming gene over a DNA region of around 12.0 kb. Using cloned c-fes sequences, complementation of GA-FeSV transforming activity was studied. Upon replacement of the 3' half of v-fesGA with homologous feline c-fes sequences and transfection of the chimeric gene, morphological transformation was observed. Immunoprecipitation analysis of these transformed cells revealed expression of high Mr fusion proteins. Phosphorylation of these proteins was observed in an in vitro protein kinase assay, and tyrosine residues appeared to be involved as acceptor amino acid.

The Hardy–Zuckerman 2-FeSV, a new feline retrovirus with oncogene homology to Abelson-MuLV

There is substantial evidence that oncogenes (v-onc) of acute transforming retroviruses have been acquired by transduction of cellular genes (c-onc) with retroviruses. Feline leukaemia virus (FeLV)-associated feline fibrosarcomas have proven to be extremely useful for the isolation of acute transforming retroviruses of a mammalian species. Three different v-onc genes have been identified in five acute transforming feline retroviruses. The Susan McDonough feline sarcoma virus (SMFeSV) contains the oncogene fms (ref. 4). The Snyder-Theilen (ST) and Gardner-Arnstein (GA) FeSVs contain the oncogene fes (ref. 4), which is homologous to the oncogene fps of the avian sarcoma viruses FSV, RRCII, PRCIV and 16L (refs 7, 8). The v-onc sequences of the Parodi-Irgens (PI) FeSV have recently been found to be homologous with the v-sis sequences of the simian sarcoma virus. We report here the isolation of another acute transforming feline retrovirus from a naturally occurring feline fibrosarcoma, designated the Hardy-Zuckerman 2 feline sarcoma virus (HZ2-FeSV) and demonstrate that the HZ2-FeSV and Abelson murine leukaemia virus (A-MuLV) have homologous oncogenes.

Mutant feline sarcoma proviruses containing the viral oncogene (v-fes) and either feline or murine control elements.

The sequences required for transformation by the Gardner-Arnstein (GA) strain of feline sarcoma virus (GA-FeSV) were defined by site-directed, in vitro mutagenesis of molecularly cloned proviral DNA. Portions of the Ga-FeSV provirus, subcloned in the plasmid pBR322, were mutagenized by deletion or frameshift at XhoI restriction sites flanking the nucleotide sequences presumed to encode the GA-FeSV transforming polyprotein (P108(gag-fes)). The biological activity of subgenomic and reconstructed full-genome-length molecules was assayed by transfection and focus induction in NIH 3T3 cells. Both mutant and wild-type molecules containing the intact P108(gag-fes) coding region induced foci of transformed cells at efficiencies between 10(4) and 10(5) focus-forming units per pmol of DNA; a deletion mutant lacking 3'-terminal v-fes sequences was completely nontransforming in parallel assays. Representative subcloned foci of transformed NIH 3T3 cells synthesized P108(gag-fes) with associated in vitro protein kinase activity. Focus-forming viruses could be rescued from transformed subclones induced by full-length proviral DNA, but not from cells transformed by subgenomic DNA lacking a 3' long terminal repeat (LTR). It was concluded that: (i) nucleotide sequences encoding P108(gag-fes) and its associated kinase activity are responsible for transformation, (ii) the GA-FeSV 3' env and LTR sequences are not required for focus induction, and (iii) the 3' LTR is necessary for rescue of infectious FeSV RNA. A chimeric DNA containing the 5' LTR and P108(gag-fes) coding region of GA-FeSV joined to the 3' LTR of Moloney murine sarcoma virus was both transforming and rescuable at high efficiency. Restriction analysis showed that passaged stocks of rescued transforming virus contained Moloney murine sarcoma virus U3 sequences at both proviral DNA termini, consistent with generally accepted models for LTR formation during reverse transcription. Wild-type GA-FeSV and the chimeric virus (here designated as GAHT), each rescued from NIH 3T3 cells with the same amphotropic murine leukemia virus, yielded approximately equal numbers of foci when titrated on CCL 64 mink cells. By contrast, on mouse NIH 3T3 cells, the focus-forming titer of GAHT was 1 to 2 log higher than that of FeSV. The foci induced on NIH 3T3 cells by GAHT appeared earlier and were reproducibly larger than those induced by GA-FeSV. Differences in transforming activity on NIH 3T3 cells were also found using colony formation in agar, showing that the more rapid appearance and larger size of foci formed in liquid media were not due to virus spread. These data suggest that transcriptional control signals within the viral LTR regulate the levels of the transforming gene product in a species-specific manner.

Antibodies of predetermined specificity detect two retroviral oncogene products and inhibit their kinase activities.

Oligopeptides predicted from the nucleotide sequence of the oncogene v-fes of feline sarcoma virus (FeSV) were synthesized chemically and used to generate specific antibodies. Antisera against a 12-amino-acid-long oligopeptide (12-mer) located 42 residues from the carboxyl terminus of the v-fes coding sequence efficiently recognized the transforming proteins encoded by Snyder-Theilen (ST) and Gardner-Arnstein (GA) strains of FeSV. This 12-mer also contains 10 amino acid residues homologous in order and position to those predicted from the nucleotide sequence of the oncogene v-fps of avian Fujinami sarcoma virus (FSV). The anti-12-mer immunoprecipitated the FSV-specific transforming protein molecules from FSV-transformed cells. Binding of these antipeptide antibody molecules to the v-fes and the v-fps gene products inhibited their associated tyrosine-specific protein kinase (EC 2.7.1.37) activities. The ability to generate such site-specific antisera to the products of related oncogenes will be valuable in the molecular characterization of retroviral transforming proteins and their normal cellular homologs.

Association of the transforming proteins of the ST and GA strains of feline sarcoma virus and their in vitro associated protein kinase activities with cellular membranes.

The translation products of the Snyder-Theilen (ST) and Gardner-Arnstein (GA) strains of feline sarcoma virus (FeSV), termed gag-fes proteins, are high molecular weight polyproteins containing different amounts of the amino terminus of the feline leukemia virus (FeLV) gag gene-coded precursor protein linked to a similar sarcoma virus-specific polypeptide. Both polyproteins are phosphoproteins with indistinguishable in vitro associated tyrosine-specific protein kinase activities. The polyproteins are extremely hydrophobic proteins which are intimately associated with the plasma membrane fraction of transformed cells. Approximately 10% of the proteins are modified by glycosylation and expressed on the cell surface where they are accessible to lactoperoxidase-mediated radio-iodination and trypsinization. Cell surface localization of the polyproteins does not appear to be necessary for transformation. However, preliminary evidence suggests that the amount of FeLV p30 sequences at the amino end of the proteins may have some effect on the intracellular distribution of the gag-fes polyproteins and on the phenotype of the transformed cell.

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

Polyproteins (gag-fes) encoded by the Snyder-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.

Biochemical characterization of protein kinase activities associated with transforming gene products of the Snyder-Theilen and Gardner-Arnstein strains of feline sarcoma virus

Translation products of the genomes of the Snyder-Theilen (ST) and Gardner-Arnstein (GA) feline sarcoma viruses (FeSV) have been shown to possess associated protein kinase activities. These activities are similar to those associated with a number of rapidly transforming retroviruses in that they appear to mediate transfer of radiolable from [γ- 32P]ATP to selected tyrosine residues in substrates and to be independent of the effects of cAMP and cGMP in vitro. The FeSV activities, when detected in specific immune complexes bound to Staphylococcus aureus, result predominantly in autophosphorylation of FeSV polyproteins. Partially purified polyprotein also induces phosphorylation of an exogenous acceptor (casein) in a soluble protein kinase assay system. The FeSV enzymes display marked preference for Mn +2 and Co +2 over Mg +2 while Ca +2 fails to initiate activity in vitro. The optimal pH for the reactions is near 7.0. There is a strict requirement for ATP as phosphoryl donor. High concentrations of ADP and dADP are inhibitory to the reaction but low concentrations of dADP are stimulatory. A comparison of these properties with those of other retroviral-associated protein kinases suggests that these activities fall into three, and possibly four, classes.

The transformation-specific proteins of avian (Fujinami and PRC-II) and feline (Snyder-Theilen and Gardner-Arnstein) sarcoma virsuses 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.

Three sizes of subunits in RNAs from feline sarcoma-leukaemia virus mixtures.

RNA from the Snyder-Theilen feline sarcoma-leukaemia virus complex (ST-FeSV-FeLV) sedimented in a double-peaked band between 50 and 70S, but Gardner-Arnstein (GA) FeSV-FeLV RNA sedimented in a single 70S peak. FeLV isolated from the ST virus mixture contained RNA which sedimented in a 70S band like GA-FeSV-FeLV RNA, but F422 FeLV RNA sedimented more slowly, at 50 to 60S. After thermal denaturation, resedimentation revealed three classes of RNA subunits in ST-FeSV-FeLV RNA: the first class, 35 to 37S, was also found in ST-FeLV and other FeLVs (except F422 FeLV), in the endogenous feline virus, RD114 and in GA-FeSV-FeLV; the second class, 32 to 34S, was similar to subunits in F422 FeLV and minor components of GA-FeSV-FeLV and ST-FeLV; the third class, 25S, was detected only in ST-FeSV-FeLV RNA. Electrophoresis of RNA species in buffered formamide provided evidence that the three classes of RNA subunits distinguishable on the basis of sedimentation rates actually represent three size classes of subunits. The ST virus mixture was shown to contain about equal titres of infectious FeLV and transforming FeSV whereas GA-FeSV-FeLV had at least a 10-fold excess fo FeLV over FeSV. These observations are discussed in terms of possible origins of the three sizes of FeSV-FeLV RNA subunits and their relationships to three species of FeSV-FeLV proviral DNA described recently (Sherr et al. 1979).

Transformation of baboon cells with feline sarcoma virus.

Testes cells from two baboons have been transformed by the Snyder-Theilen (ST) and Gardner-Arnstein (GA) strains of feline sarcoma virus (FeSV). The resulting cell cultures formed tumors in immunosuppressed mice, but not in the autologous baboons. The cells transformed by GA FeSV contained more feline gs antigen than those transformed by ST FeSV. Conversely, the cells transformed by ST FeSV produced more type-C virus. Unlike FeSV, which has a predilection for feline cells, the virus from baboon cells transformed by ST FeSV replicated in baboon and beagle embryo cells, but not in feline embryo cells, suggesting that this virus is different from FeSV.