Localization of temperature-sensitive transformation mutations and back mutations in the Rous sarcoma virus src gene.

Cloning and sequencing of two temperature-sensitive transforming mutations of Rous sarcoma virus reveal that their lesions are due to distinct but close single amino acid changes near the carboxy terminus of the v-src gene product. Back mutations to wild type result from second mutations at either nearby or distant sites.     

Regulation of cellular morphology by the Rous sarcoma virus src gene: analysis of fusiform mutants.

We have been interested in how Rous sarcoma virus (RSV) influences transformed cell morphology and compared the molecular properties of chicken embryo cells (CEC) infected with mutants of RSV that induce the fusiform transformed cell morphology with those of CEC infected by wild-type RSV, which induces the more normal round transformed cell morphology. We looked for properties shared by all fusiform mutant-infected cells, because these may be responsible for maintaining the fusiform morphology. Five different fusiform mutants, two wild-type RSVs, and one wild-type back revertant of a fusiform mutant were studied. In the fusiform mutant-infected cells, the localization and myristylation of pp60src were determined and the extent of expression of the extracellular matrix protein fibronectin was examined at both the mRNA and protein levels. The phosphorylation of vinculin on tyrosine also was examined in the same CEC. Within all fusiform mutant-transformed CEC, pp60src was dramatically absent from the adhesion plaque sites normally seen in cells transformed with wild-type RSV, and these transformed CEC all expressed more fibronectin mRNA and protein in the extracellular matrix than did the wild-type RSV-transformed CEC. The absence of pp60src from the adhesion plaques was not due to lack of myristylation of the src protein, and tyrosine phosphorylation of vinculin was not related to fibronectin expression. These results suggest that the inverse relationship between pp60src in the adhesion plaques and fibronectin expression in the extracellular matrix may be interconnected phenomena and could be related to the maintenance of the fusiform transformed morphology.     

Rous sarcoma virus variants that encode src proteins with an altered carboxy terminus are defective for cellular transformation.

The src gene of Rous sarcoma virus (v-src) and its cellular homolog, the c-src gene, share extensive sequence homology. The most notable differences between these genes reside in the region encoding the carboxy terminus of the src proteins. We constructed mutations within the 3' end of the v-src gene to determine the significance of this region to the transforming potential of the v-src protein, pp60v-src. The mutants CHdl300 and CHis1511 contain mutations that alter the last 23 amino acids of pp60v-src, whereas the mutant CHis1545-C contains a linker insertion that alters the last 11 amino acids of pp60v-src, and the mutant CHis1545-H contains a linker insertion that results in a 9-amino-acid insertion at position 415. Plasmids bearing each of these mutations were unable to transform chicken cells when introduced into these cells by DNA transfection. In addition, the structurally altered src proteins encoded by the mutants had much-reduced levels of tyrosine protein kinase activity in vivo, as measured by autophosphorylation and phosphorylation of the 34,000-Mr cellular protein, and in vitro, as determined by measuring the level of pp60src autophosphorylation. These data indicate that the carboxy-terminal amino acid sequences play an important role in maintaining the structure of the catalytic domain of pp60v-src. In contrast, the transfection of chicken cells with plasmid DNA containing a chimeric v-c-src gene resulted in morphological cell transformation and the synthesis of an enzymatically active hybrid protein. Therefore, the carboxy-terminal sequence alterations observed in the c-src protein do not alone serve to alter the functional activity of a hybrid v-c-src protein appreciably.     

Characterization of Rous sarcoma virus src gene products synthesized in vitro.

The cell-free synthesis of three major proteins from virion RNA of nondefective Rous sarcoma virus (RSV), but not from RNA of transformation-defective deletion mutants, has been observed. The apparent molecular weights of these transformation-specific proteins are approximately 60,000 (60K), 25K, and 17K. Tryptic maps of methionine-containing peptides revealed the 17K, 25K, and 60K proteins to be overlapping in sequence. However, only partial homology was observed between the 17K, 25K and 60K proteins synthesized from Schmidt-Ruppin strain, subgroup D, RSV RNA and those synthesized from Prague strain, subgroup B, RSV, RNA. About half of the methionine peptides in the Schmidt-Ruppin strain, subgroup D, 60K protein were shared with the Prague strain, subgroup D, 60K protein, and the rest were distinct to each. The virion RNAs coding for the 60K, 25K, and 17K proteins were found to be polyadenylated and to sediment with maximal mRNA activity at about 23, 19 to 20, and 18S, respectively. In addition, transformation-specific proteins with molecular weights of 39K and 33K were observed by in vitro synthesis. These proteins are also related to the 60K, 25K, and 17K proteins and were synthesized from polyadenylated RSV RNA of approximately 21 to 22S. RNase T1-resistant oligonucleotides were analyzed in parallel, and the src-specific oligonucleotides were found to be first present in equimolar amounts in those gradient fractions sedimenting at 21 to 22S. Our data suggest that synthesis of the 60K protein is initiated near the 5' terminus of the src gene, whereas the 39K, 33K, 25K, and 17K proteins are initiated internally in the src gene. All of these proteins appear to be initiated independently, but they may have a common termination site.     

Site-directed mutagenesis of the src gene of Rous sarcoma virus: construction and characterization of a deletion mutant temperature sensitive for transformation

Transformation of cells by Rous sarcoma virus results from the expression of the viral src gene product, pp60src. Site-directed mutagenesis techniques have been used to construct defined deletion mutations within the src gene of Prague A strain of Rous sarcoma virus. The deletion of DNA sequences at the Bg/II restriction site in the src gene yielded both transformation-defective mutants (tdCH4, 64, and 146) and a mutant temperature sensitive for morphological transformation (tsCH119). The genome of tsCH119 contains an in-phase deletion of approximately 160 base pairs, which mapped to the immediate 3' side of the Bg/II restriction site. Upon infection of chicken cells, tsCH119 encoded a structurally altered src protein, pp53src, containing a deletion of amino acid residues 202 to 255. Immune complexes containing pp53src isolated from tsCH119-infected cells grown at 41 degrees C exhibited only 50% less tyrosine-specific kinase activity than immune complexes isolated from cells grown at 35 degrees C. pp53src immunoprecipitated from tsCH119-infected cells grown at either 35 or 41 degrees C contained phosphoserine and phosphotyrosine. We suggest that tsCH119 represents a class of mutants containing mutations mapping within a functionally important domain of the src protein, distinct from the domain specifying the protein kinase activity.     

Evidence the pp60src, the product of the Rous sarcoma virus src gene, undergoes autophosphorylation.

F/St mice are unique in producing high levels of both ecotropic and xenotropic murine leukemia virus. The high ecotropic virus phenotype is determined by three or more V (virus-inducing) loci. A single locus for inducibility of xenotropic murine leukemia virus was mapped to chromosome 1 close to, but possibly not allelic to, Bxv-1. Although the high ecotropic virus phenotype is phenotypically dominant, the high xenotropic virus phenotype was recessive in all crosses tested. Suppression of xenotropic murine leukemia virus is governed by a single gene which is not linked to the xenotropic V locus.     

Evidence that the phosphorylation of tyrosine is essential for cellular transformation by Rous sarcoma virus

All cells transformed by Rous sarcoma virus contain levels of phosphotyrosine in protein which are 6–10 fold greater than the very low levels present in uninfected cells. The increase is due largely to modification of cellular polypeptides. The abundance of phosphorylated tyrosines in protein in cells infected with tsLA29, a mutant of Rous sarcoma virus which is temperature-sensitive for cellular transformation, increases to 60% of maximum within 60 min of a shift to the permissive temperature and drops to a level close to that in uninfected cells within 60 min of a shift to the restrictive temperature. In light of the fact that pp60^src phosphorylates tyrosine in vitro, these results suggest strongly that the modification of one or more cellular polypeptides by way of pp60^src is critical for cellular transformation by Rous sarcoma virus. There is, however, no increase in the abundance of phosphotyrosine in protein in mouse cells transformed by Kirsten sarcoma virus, Moloney sarcoma virus, or SV40 virus, in chick embryo cells infected with avian myelocytomatosis virus MC29, and in rat and hamster cells transformed by polyoma virus. Thus increased phosphorylation of tyrosine is neither a universal mechanism of transformation nor an inevitable secondary cellular response to transformation.     

Transformation-defective mutants of Rous sarcoma virus with src gene deletions of varying length.

The RNAs of transformation-defective (td) deletion mutants of the Schmidt-Ruppin strain of Rous sarcoma virus were found to vary in size when compared by polyacrylamide gel electrophoresis. Three of seven td mutants appeared to recombine with a mutant of Rous sarcoma virus (Schmidt-Ruppin), which has a temperature-sensitive sarcoma (src) gene and is termed ts68, to give rise to recombinants with a reduced temperature sensitivity. The results suggested that different clones of td mutants exist: some in which the src gene appears to be deleted, and others in which the src gene is only partially deleted. A direct correlation between RNA size and the extent of src gene deletion measured by recombination was not obtained, possibly because the recombination assay could only detect src sequences homologous to the lesion(s) of ts68, whereas the electrophoretic analysis of the RNA measured src deletions as well as other possible alterations of the RNA.     

Construction and isolation of a transforming murine retrovirus containing the src gene of Rous sarcoma virus.

Recombinant murine retroviruses containing the src gene of the avian retrovirus Rous sarcoma virus were isolated. Such viruses were isolated from cells after transfection with DNAs in which the src gene was inserted into the genome of the amphotropic murine retrovirus 4070A. The isolated viruses had functional gag and pol genes, but they were all env defective since the src gene was inserted in the middle of the env gene coding region. Infectious transforming virus could be isolated only from cells transfected with DNA constructions in which the src gene was in the same polarity as that of a long terminal repeat of the amphotropic viral genome. These recombinant viruses encoded a pp60src protein with a molecular weight similar to that of the Schmidt-Ruppin strain of Rous sarcoma virus. In addition, the src protein(s) of these recombinant viruses was as active as protein kinases in the immune complex protein kinase assay. Intravenous injection of helper-independent Moloney and Friend murine leukemia virus pseudotypes of the src recombinant viruses into 6-week-old NIH Swiss mice resulted in the appearance of splenic foci within 2 weeks, splenomegaly and, later after infection (8 to 10 weeks), anemia. Infectious transforming virus could be recovered from the spleens of diseased animals. Such viruses encoded pp60src but not p21ras or mink cell focus-forming virus-related glycoproteins.     

Tyrosine phosphorylation of a 50K cellular polypeptide associated with the Rous sarcoma virus transforming protein pp60src.

We have examined the phosphorylation of a 50,000-dalton cellular polypeptide associated with the Rous sarcoma virus (FSV) transforming protein pp60-src. It has been shown that pp60src forms a complex with two cellular polypeptides, an 89,000-dalton heat-shock protein (89K) and a 50,000-dalton phosphoprotein (50K). The pp60src-associated protein kinase activity phosphorylates at tyrosine residues, and the 50K polypeptide present in the complex contains phosphotyrosine and phosphoserine. These observations suggest that the 50K polypeptide may be a substrate for the protein kinase activity of pp60src. To examine this possibility, we isolated the 50K polypeptide by two-dimensional polyacrylamide gel electrophoresis from lysates of uninfected or virally infected cells. Tryptic phosphopeptide analysis indicated that the 50K polypeptide isolated by this method was the same polypeptide as that complexed to pp60src. In uninfected cells or cells infected by a transformation-defective mutant, the 50K polypeptide contained phosphoserine but little or no phosphotyrosine. In cells infected by Schmidt-Ruppin or Prague RSV, there was a 40- to 50-fold increase in the quantity of phosphotyrosine in the 50K protein. Thus, the phosphorylation of the 50K polypeptide at tyrosine is dependent on the presence of pp60src. However, the 50K polypeptide isolated from cells infected by temperature-sensitive mutants of RSV was found to be phosphorylated at tyrosine at both permissive and nonpermissive temperatures; this behavior is different from that of other substrates or putative substrates of the pp60src kinase activity. It is possible that the 50K polypeptide is a high-affinity substrate of pp60src.     

Reverse transformation of vole cells transformed by avian sarcoma virus containing the src gene

Vole cells transformed by avian sarcoma virus carrying the src gene lose their fibroblastic morphology, the organized cytoskeletal system of the normal fibroblastic cell, the typical fibronectin deposit around the cell membrane, and the ability to shut off multiplication when suspended in liquid medium. All of these transformation characteristics are reversed by treatment with cAMP derivatives. Moreover, the cAMP treatment does not cause loss of activity of the src gene product. These data imply that cAMP exerts its effect at or after the point in the metabolic pathway affected by the src gene product, pp60src. Presumably, the decision to adopt the transformed or the normal state is determined by the degree to which the src gene or cAMP-mediated kinase activities respectively predominante in the cell. The development of all four transformation characteristics as a result of introduction of the src gene, and their coordinate reversal by cAMP derivatives, supports the previous thesis that in the normal vole or CHO fibroblast all four properties are part of a common regulatory system.     

Product of in vitro translation of the Rous sarcoma virus src gene has protein kinase activity.

In vitro translation of Rous sarcoma virus virion RNA resulted in the synthesis of a protein kinase which, when immunoprecipitated with antitumor serum, phosphorylated the immunoglobulin heavy chain. Even though in vitro translation of virion RNA resulted in the synthesis of a number of polypeptides which were recognized by antitumor serum, control experiments demonstrated that an immunoprecipitable protein kinase activity was found only when an immunoprecipitable p60src, the polypeptide product of the src gene, was synthesized. A protein kinase with similar properties was therefore intimately associated with p60src which was synthesized in vitro in the reticulocyte lysate, just as it is with p60src which is obtained from transformed chick and mammalian cells. It is therefore highly unlikely that this association is artifactual. ts NY68 is a mutant of Rous sarcoma virus which is able to transform cells at 36 but not at 41 degrees C. In vitro translation of ts NY68 virion RNA at 30 degrees C resulted in efficient synthesis of immunoprecipitable p60src, but very inefficient synthesis of an immunoprecipitable protein kinase. The p60src obtained by in vitro translation of wild-type virion RNA was more than 20-fold more active as a protein kinase than was that obtained from ts NY68 RNA. The correlation in the case of ts NY68 of a deficiency in protein kinase activity with an inability to transform cells at high temperature suggests that the protein kinase activity associated with p60src is indeed critical to cellular transformation.     

Monoclonal antibodies to Rous sarcoma virus pp60src react with enzymatically active cellular pp60src of avian and mammalian origin

The derivation and characterization of 22 hybridoma clones producing monoclonal antibodies (Mabs) specific for the transforming protein of Rous sarcoma virus, pp60src, are described. All Mabs reacted with pp60v-src encoded by Prague, Schmidt-Ruppin, and Bratislava 77 strains of Rous sarcoma virus. Of these Mabs, 10 efficiently immunoprecipitated pp60c-src from chicken embryo cells. Of these 10 Mabs, 2 (GD11 and EB8) readily detected pp60c-src from a variety of rodent and human cultured cells and from rat brain tissue in an in vitro immune complex kinase assay. Mapping experiments have tentatively localized the determinant(s) recognized by GD11 and EB8 to a region of the src protein bounded by amino acid residues 82 to 169, whereas the remaining Mabs appeared to recognize determinants residing within residues 1 to 82 or 169 to 173. Most of the Mabs complexed denatured pp60v-src in a Western immunoblot, and several were used to localize pp60v-src in Rous sarcoma virus-transformed chicken embryo cells by indirect immunofluorescence microscopy.     

Comparison of the expression of the src gene of Rous sarcoma virus in vitro and in vivo.

We have compared the polypeptide products of the src gene of several strains of Rous sarcoma virus produced by in vitro translation of heat-denatured 70S virion RNA in the nuclease-treated reticulocyte lysate with those present in chick cells transformed by these viruses. We have done this by immunoprecipitation, using sera from rabbits injected at birth with Schmidt-Ruppin Rous sarcoma virus. In vitro translation results in the synthesis of at least nine polypeptides which appear to be encoded by the src gene. These range in size from 17,000 to 60,000 daltons. The sera from tumor-bearing rabbits precipitated these polypeptides arising from the in vitro translation of RNA from Schmidt-Ruppin Rous sarcoma virus of both subgroup A and subgroup D and from one stock of Prague Rous sarcoma virus of subgroup C. In each case, all of this family of related polypeptides could be precipitated except the smallest, the 17,000-dalton polypeptide. No precipitation of analogous polypeptides resulting from the translation of RNA from other strains of Rous sarcoma virus was observed. Cells transformed by these three strains of Rous sarcoma virus contain easily detectable amounts of a polypeptide, p60src, essentially identical to the 60,000-dalton in vitro product. With one exception, they do not contain significant amounts of polypeptides analogous to the smaller in vitro products which can be precipitated by these sera. Cells transformed by one stock of Schmidt-Ruppin Rous sarcoma virus of subgroup A did contain a 39,000-dalton polypeptide, which was related, by peptide mapping, to the 60,000-dalton polypeptide and was similar in size to a precipitable in vitro product. The 60,000-dalton polypeptide present in transformed cells appeared to be phosphorylated 10 to 25 min after its synthesis, metabolically very stable, and not derived from a precursor polypeptide. All immunoprecipitates from transformed cells which contained p60src also contained an 80,000-dalton phosphoprotein. This polypeptide is unrelated to p60src, as determined by peptide mapping, and may well be a host cell polypeptide which is specifically associated with p60src.     

The specific interaction of the Rous sarcoma virus transforming protein, pp60src, with two cellular proteins

Sera from rabbits bearing tumors induced by Rous sarcoma virus (RSV) were previously found to contain antibody to the RSV transforming protein, pp60^src. Two additional transformation-specific phosphoproteins from RSV-transformed avian cells are immunoprecipitated with these sera. These proteins, having molecular weights of 90,000 (pp90) and 50,000 (pp50), are not precipitated from uninfected or transformation-defective virus-infected cells and are not related to any RSV structural proteins. Neither pp50 nor pp90 shares any partial or complete proteolytic cleavage peptides with pp60^src, suggesting that pp90 and pp50 do not represent either a precursor or a cleavage product of pp60^src. Sedimentation analysis of RSV-transformed cell lysates on glycerol gradients revealed that the RSV pp60^src protein is present as two forms, one of which represents the majority (95%) of pp60^src and sediments as a monomer, 60,000 molecular weight protein and the other of which sediments with pp90 and pp50 as an apparent 200,000 molecular weight complex. Lysates from cells transformed by viruses containing a temperature-sensitive defect in the src gene contain a greater percentage of pp60^src associated with pp90 and pp50 under both permissive (35°C) and nonpermissive (41°C) conditions compared to wild-type virus-infected cell lysates. Phosphoserine and phosphotyrosine were found associated with pp60^src molecules that sedimented as a monomer, whereas pp60^src molecules that are complexed with pp90 and pp50 contain phosphoserine and greatly reduced amounts of phosphotyrosine. Only the monomer form of pp60^src is capable of phosphorylating IgG in the immune complex phosphotransferase reaction. Normal uninfected chicken cells contain a protein that shares identical partial proteolytic cleavage peptides with the pp90 protein immunoprecipitated from RSV-transformed cells. This pp90 protein is one of the major cytoplasmic proteins in uninfected cells. Antibody directed against pp90 also immunoprecipitates pp60^src and pp50 from lysates of RSV-transformed chicken cells.     

Adeno-associated virus gene expression inhibits cellular transformation by heterologous genes.

In this paper we report that adeno-associated virus (AAV) genomes inhibit stable transformation by several dominant selectable marker genes upon cotransfection into mouse tissue culture cells. Cotransfection of AAV genomes also inhibited the expression of pSV2cat in transient assays. In both cases, the inhibitory effect was independent of AAV DNA replication but required the AAV p5 and p19 genes, which encode proteins required for AAV DNA replication and regulation of AAV gene expression. Finally, addition of a cloned E4 gene in the transfection experiments partially blocked the AAV-mediated inhibitory activities.     

Genetic lesions involved in temperature sensitivity of the src gene products of four Rous sarcoma virus mutants.

The src genes of four Rous sarcoma virus (RSV) mutants temperature-sensitive (ts) for cell transformation were analyzed. The mutant src genes were cloned into a replication-competent RSV expression vector, and the contribution of individual mutations to the ts phenotype was assessed by in vitro recombination with wild-type src sequences. Three of the mutants, which were derived from the Schmidt-Ruppin strain of RSV, each encoded two mutations within the conserved kinase domain. In all three cases, one of the two mutations was an identical valine to methionine change at amino acid position 461. Virus encoding recombinant src genes containing each of these mutations alone were not ts for transformation, demonstrating that two mutations are required for temperature sensitivity. The sequence of the src gene of the Bryan high-titer strain of RSV was determined and compared with that of the fourth ts mutant which was derived from it, again revealing two lesions in the kinase domain of the mutant.     

Phosphorylation of the solubilized insulin receptor by the gene product of the Rous sarcoma virus, pp60src

Both the insulin receptor and the gene product of the Rous sarcoma virus, pp60src, are protein kinases which phosphorylate themselves and other proteins on tyrosine residues. Addition of the solubilized insulin receptor to purified pp60src increased the phosphorylation of the beta-subunit of the insulin receptor. Phosphorylation of the insulin receptor by pp60src occurred both in the absence and presence of insulin but did not alter the insulin dose response for autophosphorylation of the receptor. Increasing concentrations of pp60src increased the phosphorylation of the receptor and at high concentrations equaled the maximal effect produced by insulin. Our observations suggest a possible mechanism by which the metabolically regulated insulin receptor tyrosine kinase could be altered by other tyrosine kinases such as that associated with pp60src. Further studies will be required to determine if the insulin receptor is phosphorylated by pp60src in Rous sarcoma virus-infected cells.