Cell-free translation of purified avian sarcoma virus src mRNA

Avian sarcoma virus 21 S RNA, purified by hybridization from virus-infected cells, was translated in a cell-free system. The major product of translation was a protein of 60,000 daltons. This protein was the same as authentic pp60^src, the product of the ASV src gene, when compared by electrophoretic mobility in polyacrylamide gels, immunological reactivity and partial protease digestion. These findings confirm that the 21 S ASV RNA serves as mRNA for pp60^src. Furthermore, pp60^src is the only major product of translation of the src gene and is apparently synthesized without a cleavable signal sequence.     

Temperature-sensitive kinase activity associated with various mutants of avian sarcoma viruses which are temperature sensitive for transformation

Antisera against the transforming gene product of avian sarcoma virus (ASV), called pp60^src, were raised in tumor-bearing rabbits by inoculation of a high dose of the avian sarcoma virus Schmidt-Ruppin strain of subgroup D (SR-D) into newborn animals. Four mutants of ASV with temperature-sensitive defects for transformation have been investigated for temperature sensitivity of the sarcoma gene-associated protein kinase activity in comparison to that of their wild-type parents. The inactivation rate of the protein kinase from the mutants MI 100, OS122, OS538, and NY68 was two-to threefold faster than that of the respective parents which belong to various subgroups of the Schmidt-Ruppin strain, SR-B, SRD, and SR-A. During heat inactivation, dephosphorylation of ³²P-labeled pp60^src immune precipitated from mutant virus-infected cells was not parallel to the inactivation of the kinase. The amount of [³⁵S]methionine-labeled pp60^src precipitated from mutant- and wild type-infected cells was not sensitive to heat treatment. The kinase activity associated with pp60^src of mutant- and wild type-infected cells was protected from inactivation by the presence of sera from tumor-bearing rabbits (TBR) during the heating procedure. Protein kinase activity of MI100 increased up to fourfold during this process, while with other mutant and wild-type kinase activities such a curing by TBR-sera was also observed but not to the same extent. Immune complexes obtained from transformed cells treated with TBR-serum and Staphylococcus aureus were used as a source of kinase(s) to phosphorylate exogenously added cell lysates as targets. Alternatively, [γ-³²P]ATP was added to cell lysates to allow phosphorylation of polypeptides present in the lysates. This procedure resulted in phosphorylation of proteins with molecular weights of 60K, 35K, and 23K which were absent from the normal or leukosis-virus-infected controls. Several proteins of high molecular weight, one of them of 150K, were enhanced. Treatment of these phosphoproteins with TBR-serum resulted in precipitation of the 60K and 23K proteins but not of the 150K and 35K phosphoproteins.     

Novel localization of pp60src in Rous sarcoma virus-transformed rat and goat cells and in chicken cells transformed by viruses rescued from these mammalian cells

We have investigated by indirect immunofluorescence and subcellular fractionation the intracellular location of pp60^src in RSV-transformed mammalian cells and in CEF cells transformed by virus rescued from these cells. Two independently derived cell lines were examined: RR1022 cells isolated from an in vivo sarcoma induced in an Amsterdam rat by infection with SR-RSV-D; and Pcl, cells isolated from a soft agar colony of normal goat skin fibroblasts transformed in vitro by SR-RSV-D. Transforming viruses (RSV-RR and RSV-Pcl) were rescued from RR1022 and Pcl cells by fusion with CEF cells. Immunofluorescence microscopy showed association of pp60^src with the nuclear envelope and the juxtanuclear reticular membranes in the transformed mammalian cells and in CEF cells transformed by the rescued viruses, in contrast to the plasma membrane localization of pp60^src seen in SR-RSV-transformed CEF cells. Results of subcellular fractionation by differential centrifugation and fractionation of particulate fractions by equilibrium centrifugation in discontinuous sucrose gradients were in agreement with the differences in pp60^src distribution observed by immunofluorescence microscopy. Although the mammalian cell lines were independently derived, pp60^srcs isolated from RR1022 and Pcl cells both lacked amino-terminal 21- and 18-kilodalton [³⁵S]methionine S. aureus V8 protease peptides found in SR-RSV-D pp60^src. Proteolytic peptides identical to those of pp60^src from the mammalian cells were obtained from pp60^src proteins isolated from rescued virus-transformed CEF cells, suggesting that the alteration in the amino-terminal half of the src protein represents a stable change, and that an alteration in the primary structure of pp60^src is responsible for the altered intracellular membrane localization of pp60^src in these cells.     

The expression of pp60src and its associated protein kinase activity in cells infected with different transformation-defective temperature-sensitive mutants of Rous sarcoma virus

A set of five transformation-defective temperature-sensitive mutants of Rous sarcoma virus has been used to investigate the relation between pp60^src its associated protein kinase activity, and expression of the transformed phenotype. In radioimmune competition experiments, the levels of pp60^src induced by the mutants did not vary by more than a factor of two, either among the mutants at a given temperature or between nonpermissive and permissive temperatures for a given mutant. The mutants fell into two distinct classes with respect to the temperature conditional expression of pp60^src-associated kinase activity. Three mutants (GI 201, GI 202, and GI 251) induced two- to fivefold higher levels of pp60^src-associated kinase activity at the permissive temperature. The other mutants GI 203 and 253 induced only very low levels of pp60^src-associated kinase at either temperature. The pp60^src-associated kinase activity induced by GI 201, 202, and 251 at the permissive temperature was significantly more heat labile in vitro than that of the wild type. Furthermore, downshift of the mutant-infected cells to the permissive temperature resulted in a rapid increase (within 15 min) in the pp60^src-associated kinase activity only with mutants GI 201, GI 202, and GI 251, i.e., only with those mutants having an elevated activity at the permissive temperature. The results taken as a whole suggest that there is not a simple relationship between pp60^src, pp60^src-associated kinase activity, and transformation and support the idea of multifunctionality of the src gene product.     

An avian sarcoma virus mutant which produces an aberrant transformation affecting cell morphology

ST 529 is a temperature-sensitive mutant of Rous sarcoma virus (RSV) strain SR-A, which causes an unusual pattern of phenotypic changes in cells that it transforms. At 35°, ST 529-transformed cells exhibit an elongated, fusiform morphology (morph^f), and also possess an aberrant and “unlinked” phenotypic pattern of transformation-related properties. ST529-transformed cells, at 35°, resemble classically transformed cells in respect to density-independent growth, sugar uptake, protease levels, and ability to form soft agar colonies. However, they differ from classically transformed cells in respect to cell morphology, fibronectin levels, adhesiveness, and organization of actin stress fibers. At a nonpermissive temperature (42°), ST529-infected cells appear phenotypically normal. pp60^src isolated from ST529-infected cells at 42° possesses little or no associated protein kinase activity. Kinase activity becomes detectable rapidly, however, within 30 min after a shift to 35°, and reaches maximal levels within 3 hr after the shift. It is probable that an unusual, mutant src gene product is responsible for the novel pattern of transformation-related changes observed in ST529-infected cells. Previous studies utilizing mutants or variants of RSV have suggested that there are probably at least two biologically significant targets for pp60^src. The present experiments provide additional evidence for a multifunctional src gene product.     

Two cellular proteins that immunoprecipitate with the transforming protein of Rous sarcoma virus

The transforming gene (src) of Rous sarcoma virus encodes a 60,000-dalton phosphoprotein (pp60^src) with the ability to phosphorylate tyrosine in certain protein substrates. The enzymatic activity of pp60^src is thought to mediate neoplastic transformation by src. It would therefore be useful to identify cellular proteins that interact with pp60^src on the chance that these proteins might be substrates for the kinase activity of the viral protein or be otherwise involved in neoplastic transformation of the host cell. In pursuit of this objective, we characterized the proteins that coprecipitate with pp60^src in immune complexes. These proteins proved to be of two types. (i) Most immune complexes contained a series of proteins (50,000 to 58,000 daltons) that were apparently derived from pp60^src by sequential degradation from the amino terminus. We do not know if this degradation has a physiological purpose in the infected cell, but it has at least two practical implications: it has proved useful in the analysis of the functional topography of pp60^src; and it can give rise to experimental artifacts in the analysis of proteins obtained from cells infected with Rous sarcoma virus. (ii) Two proteins (50,000 and 89,000 daltons) coprecipitated with pp60^src, probably by virtue of their ability to bind to the viral protein. Both proteins are phosphorylated, both are encoded by the cellular genome, and both can be recovered from either avian or mammalian cells transformed by Rous sarcoma virus. The 89,000-dalton protein contains phosphoserine, irrespective of its source, and its structure is otherwise highly conserved among widely diverged vertebrate species. By contrast, the forms of the 50,000-dalton protein recovered from chicken and rat cells can be readily distinguished by their peptide maps and by their phosphoamino acids (the avian form of the protein contains both phosphoserine and phosphotyrosine, whereas the mammalian form contains only phosphoserine). We used temperature-sensitive mutants in src to explore the possibility that the two cellular proteins might be substrates for the protein kinase activity of pp60^src: propagation of infected cells at the nonpermissive temperature failed to affect the phosphorylation of either of the proteins. We conclude that at least two cellular proteins are associated with pp60^src prior to immunoprecipitation with antisera directed against the viral protein. It is possible that neither of these proteins is a substrate for the protein kinase activity of pp60^src, however, and their role in neoplastic transformation by src (if any) remains moot.     

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

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

Evidence that the src gene product of Rous sarcoma virus is membrane associated

We have investigated the intracellular localization of the src phosphotransferase and of pp60^src by cellular fractionation. Fractionation of cell lysates by differential centrifugation showed that both the phosphotransferase activity, measured using an immune complex assay, and pp60^src cosedimented with particulate fractions enriched in cellular membranes. Upon further fractionation of particulate fractions by equilibrium centrifugation in discontinuous sucrose gradients the src phosphotransferase and pp60^src fractionated in parallel with plasma membranes. The phosphotransferase activity remained associated with cellular membranes under conditions of high ionic strength or of high concentrations of divalent cation chelators further supporting a direct membrane association. The src phosphotransferase activity is higher in buffer containing only Triton X-100 detergent as compared to a more complicated detergent mixture, but under these assay conditions the pp60^scr appears to undergo proteolysis to a 52K-dalton polypeptide (pp52^src) which is active as a phosphotransferase. Pyrimidine triphosphates were shown to act as donors in the immune complex assay of src phosphotransferase activity. [γ-³²P]CTP was about one-third as efficient as [γ-³²P]ATP in serving as a donor. Both CTP and UTP were effective inhibitors of src phosphotransferase activity when either radiolabeled ATP or GTP were used as substrates. The ability of src to interact efficiently with both purine and pyrimidine triphosphates contrasts markedly with most well-studied protein kinases which have been shown to use almost exclusively purine triphosphates.     

Four Rous sarcoma virus mutants which affect transformed cell morphology exhibit altered src gene products

Four Rous sarcoma virus Schmidt Ruppin A (RSV SR-A) morph^f mutants (ST529, WO101, W0201, and WO401), independently isolated in the presence of different mutagens, cause an elongated, or fusiform, transformed cell morphology in chicken embryo fibroblasts at 37°. The src gene products isolated from cells transformed by these mutants exhibited an apparent molecular weight (M_r) of approximately 54,000 to 55,000 on SDS-polyacrylamide gels, in contrast to the approximately 59,000 to 60,000 M_r pp60^src species isolated from wt SR-A-transformed cells, using the same extraction conditions. This difference was observed both in extraction buffers containing SDS, sodium deoxycholate, and NP-40, as well as in buffers not containing SDS and sodium deoxycholate. Partial protease digestion experiments indicated that src gene products of all four mutants were lacking peptides present in the N-terminal half of wt pp60P^src The mutant src species isolated from cells transformed at 37° exhibited kinase activity; in the case of the temperature-sensitive mutant ST529, this activity displayed a pronounced in vitro temperature sensitivity. These results strongly suggest that a common, or similar, structural alteration in “pp60^src” species of the mutants is intimately related to, and perhaps the cause of, the morph^f phenotype exhibited by the cells they transform.     

Isolation of an avian sarcoma virus-specific protein kinase from virus particles

Avian sarcoma viruses (ASV) of the Schmidt-Ruppin strain (SR) contain a protein kinase activity which specifically phosphorylates the IgG of sera from tumor-bearing rabbits (TBR). The amount is comparable with that from transformed cells. The activity is thermolabile in two mutants with a temperature-sensitive lesion in the sarcoma (src) gene. Ion-exchange column chromatography on DEAE-cellulose and phosphocellulose allowed a 250-fold purification of enzymatically active protein kinase with a molecular weight of 60,000 (60K). It phosphorylated casein and the heavy chain of IgG of TBR sera but not of control sera. Phosphorylation of casein could be completely inhibited by TBR serum and resulted in phosphorylation of IgG. Purification of the protein kinase from a mutant virus, OS122, and its wild-type SR-D revealed a threefold higher thermolability for the mutant enzyme. Partial proteolytic digest of the [³⁵S]methionine-labeled 60K protein obtained from the phosphocellulose column by immune precipitation was indistinguishable from that of pp60^STC precipitated from SR-D-transformed cells.     

The structure and protein kinase activity of proteins encoded by nonconditional mutants and back mutants in the sec gene of avian sarcoma virus

We have characterizedsrc proteins encoded by approximately 30 nonconditional transformation-defective mutants of avian sarcoma virus (ASV) and by several back mutants which reestablish a transformed phenotype. We used gel electrophoresis of immunoprecipitated proteins labeled with³²PO₄ or [³⁵S]methionine to assess size, stability, and phosphorylation; partial digestion with staphylococcal V8 protease to determine structure; and an immune complex assay to measure protein kinase activity. The mutants were all isolated as phenotypic revertants of the B31 line of B77-ASV transformed rat cells, each revertant cell bearing a single provirus without appreciable deletions, as described in the accompanying report (Varmuset al., 1980). In several instancesm the mutant proteins were examined both in the revertant rat cells and in chicken cells infected with transformation-defective viruses rescued from the nonpermissive rat cells. In addition, secondary mutations to restore a transformed phenotype (back mutations) occurred in some cases, in the original rat cells and/or chicken cells infected with rescued viruses. Three categories of mutants were identified by this survey. The largest group (Class I) encodedsrc proteins of normal size (60,000M_r); these proteins were hypophosphorylated and exhibited little or no protein kinase activity.Class II mutants displayed immunoprecipitablesrc proteins of less than normal size. In three cases, the shortsrc related proteins were mapped to the amino terminus of wild-type pp60^src and may be the result of nonsense mutations; in two cases, the short proteins were mapped to the car?yl terminus. Most of Class II mutants lacked protein kinase activity, but the 45,000M_r protein in line 000 exhibited moderate levels of activity, thereby mapping the enzymatically active site to the car?yl terminal three-fourths of pp60^src. The smallest group of mutants (Class III) did not produce detectablesrc proteins. Some of the mutant proteins behaved differently in permissive and nonpermissive hosts; in particular, the product of mutant L produced fusiform transformation and was highly phosphorylated and associated with wild-type levels of protein kinase activity in chicken cells, but was nontransforming, hypo-phosphorylated, and associated with low levels of protein kinase activity in rat cells. In all cases, back mutation to a transformed phenotype was accompanied by a restoration of wild-type (or near wild type) levels of protein kinase activity, further documenting the functional significance of the enzymatic activity. Some of the back mutants, however, encoded proteins of atypical size, either smaller or larger than pp60^src. The active proteins larger than pp60^src ranged up to 68,000M_r in size and were altered at or near the amino terminus. In one case (a retransformed derivative of the Class II revertant 000), the generation of a functionalsrc protein of 68,000M_r coincided with the appearance of an insert of ca. 200 base pairs into the ASV provirus, within or adjacent to the coding region for the amino terminus ofsrc. The diversity of reagents, both mutants and back mutants, derived from the single provirus in B31 cells indicates that this system will be useful for correlation of functional and structural attributes ofsrc.     

Characterization of a 105,000 molecular weight gag-related phosphoprotein from cells transformed by the defective avian sarcoma virus PRCII

In cells infected with the replication-defective avian sarcoma virus PRCII a single virus-specific product is detectable, a polyprotein of 105,000 molecular weight (p105). P105 can be precipitated with antisera togag proteins of avian leukosis and sarcoma viruses. By two-dimensional tryptic peptide analysis of [³⁵S]methionine-labeled proteins we have shown that p105 contains peptides of helper viriongag proteins p19 and p27, but not of p15. In addition a number of peptides are present in p105 that are not found in any of the helper virus gene products including gPr95^env and Pr180^gag-pol. These p105-specific peptides are not detectable in the p60^src protein of Rous sarcoma virus (RSV) nor in thegag-related polyproteins encoded by avian myelocytoma and carcinoma viruses MC29 and MH2 or avian erythroblastosis virus AEV. P105 is not detectably glycosylated, but is heavily phosphorylated. In this respect it resembles p60^src of RSV rather than the polyproteins of avian leukemia viruses. Since p105 is the only viral gene product detectable in nonproducing cells transformed by PRCII, this protein may be important in the initiation and maintenance of oncogenic transformation. The nonstructural sequences in p105 would then represent a new class of transforming gene in avian oncoviruses.     

Characterization of the avian sarcoma virus protein p60src.

The intracellular distribution of the avian sarcoma virus (ASV) protein, p60^src, has been investigated in transformed chicken and hamster cells by radioimmunoprecipitation of fractionated cells and by immunofluorescent staining of fixed cells with monospecific antiserum to p60^src. The src protein was found exclusively in the cytoplasmic fraction in both a soluble and an insoluble form. Furthermore, we show that the antigenicity of p60^src was extremely heat-labile in vitro relative to Pr76, the precursor to the internal structural proteins of ASV. Antibody directed against p60^src which is present in serum from rabbits bearing tumors induced by the Schmidt-Ruppin (subgroup D) strain of ASV does not recognize p60^src from cells transformed by other ASV strains including Prague C, B77, and Bryan (RAV-50). Re-evaluation of the precipitation of p60^src from NY68-transformed cells has led to the conclusion that there is no reduction in the synthesis or antigenicity of p60^src when cells are cultured under conditions nonpermissive for transformation by this mutant virus.     

Neuronal pp60c-src(+) in the developing chick spinal cord as revealed with anti-hexapeptide antibody

Summary Polyclonal antibody was raised in rabbits against a synthetic hexapeptide R-K-V-D-V-R corresponding to a unique amino acid sequence of the neuron-specific c-src gene product pp60^c–src(+). The antibody was purified by affinity chromatography. A single band with an apparent molecular mass of 60 kDa was recognized when the supernatant of homogenates of brain and spinal cord from chick embryos and chicks was probed with the affinity purified anti-hexapeptide antibody after SDS-polyacrylamide gel electrophoresis followed by Western blotting. Specificity of the antibody was further characterized by autophosphorylation assay of immunoprecipitate in comparison with the monoclonal antibody 327. Immunocytochemical studies by light microscopy revealed that pp60^c–src(+) was localized in flake-like aggregates in neuronal cell bodies of the spinal cord in 7-15-day-incubated chick embryos and newly hatched chicks. Developing spinal ganglia and muscle cells were also immunoreactive at early developmental stages. By electron microscopy, the reaction product was observed mainly in two regions. One region was at polysomes and along the membranes of the rough endoplasmic reticulum. The other region was along the neuronal plasma membrane — at subsurface cisterns and at synapses. At synapses, the postsynaptic density, presynaptic membrane and synaptic vesicle membranes were immunostained. Immunoreactivity at synapses was more frequently observed at earlier stages than at later stages of development. These findings suggest that pp60^c–src(+) is actively produced in developing neurons and has some important roles in synaptogenesis. In mature synapses, pp60^c–src(+) may be involved in the interaction of synaptic vesicles with the presynaptic membrane.     

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.     

Phosphorylation of pp60src and the cycloheximide insensitive activation of the pp60src-associated kinase activity of transformation-defective temperature-sensitive mutants of Rous sarcoma virus

Chick embryo fibroblasts infected with two transformation defective temperature-sensitive mutants of Rous sarcoma virus (GI 202 and GI 251), when shifted from the nonpermissive temperature (42°) to the permissive temperature (35°) show a rapid increase in the detectable pp60^src-associated kinase activity. When such cells are shifted back to the nonpermissive temperature there is an equally rapid loss of demonstrable kinase activity. The rapid increase in detectable kinase activity is not substantially hindered by cycloheximide at concentrations inhibiting all de novo protein synthesis, and is associated with a cycloheximide-insensitive phosphorylation of the mutant pp60^src.     

The 28 S genomic RNA of avian sarcoma virus PRCII codes for the transformation-specific polyprotein P105

We have characterized the genomic RNA of the defective avian sarcoma virus PRCII. Replicating virus which consists of transforming PRCII and a nontransforming but replication-competent helper, PRCII-AV, contains two RNA species. One is identical in size to the 35 S genome of avian leukosis helper viruses. The second component migrates slightly faster than 28 S ribosomal RNA in polyacrylamide gel electrophoresis but co-sediments with this RNA in sucrose gradients. In vitro translation across a gradient of velocity-sedimented poly(A)-containing PRCII virion RNA yielded three major proteins. The virion protein precursors Pr76^gag and Pr180^gag-pol were translated from 35 S RNA, while the transformation-specific polyprotein P105 was translated from 28 S RNA. P105 may be the only protein coded for by the PRCII genome, although this product would not exhaust the coding capacity of 28 S RNA. Whether translated in vitro or immunoprecipitated from transformed cells, P105 was essentially identical as demonstrated by comparative peptide maps.     

Recovered src genes are polymorphic and contain host markers

Analysis of recovered sarcoma viruses (rASV) and their parental sarcoma virus SR-D by oligonucleotide fingerprinting revealed multiple differences in the src region of the viral genomes. This heterogeneity was further investigated by tryptic peptide mapping of the in vitro translated products of rASV and SR-D RNA. No differences were found in the pr76^gag proteins encoded by the various rASVs or SR-D, but the p60^src proteins showed considerable variation. The p60^src proteins of rASV could be distinguished from that of SR-D on the basis of their mobility in SDS-polyacrylamide gels. Furthermore, two peptides which were absent from SR-D but consistently found in rASV p60^src proteins were also demonstrated in a tryptic peptide map of the cellular src-related protein, p60^sarc. These results provide strong support for the hypothesis that rASV arose by recombination of residual viral src sequences with cellular src-related sequences.     

Characterization of avian erythroblastosis virus p75

We have studied the 75,000-dalton gag-related polyprotein (p75) found in cells transformed by avian erythroblastosis virus (AEV). AEV p75 was found to be phosphorylated but not glycosylated. Phosphorylation occurs at a serine residue(s) present in the p19 portion of the gag sequences contained at the N-terminal end of p75. No other phosphorylation sites were observed in vivo. AEV p75 was not incorporated into the pseudotyped virion particle. An antiserum specific for the non-gag portion of p75 was prepared by hyperimmunizing chickens that had undergone regression of AEV ts34-induced erythroleukemia with AEV-transformed bone marrow cells. Following absorption with disrupted virions, this serum was capable of immunoprecipitating only AEV p75 and none of the proteins seen following in vitro translation of 20–22 S AEV RNA nor the transforming proteins of a variety of other RNA and DNA tumor viruses. AEV p75 did not contain associated protein kinase activities capable of utilizing either ATP or GTP as phosphate donors when p75 was immunoprecipitated by any of the following antisera: rabbit anti-p19, rabbit anti-disrupted Rous-associated virus-2 virion, Rous sarcoma virus-induced tumor-bearing rabbit serum, or chicken anti-p75 serum. AEV p75 also did not contain associated ATPase activity.