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

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

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.     

Heteroduplex analysis of cloned rat endogenous replication-defective (30 S) retrovirus and Harvey murine sarcoma virus

DNA representing endogenous rat replication-defective (30 S) retrovirus has been cloned at the SacI sites of the EK2 vector λgtWes.λB. Heteroduplex analysis of this cloned 30 S DNA to cloned Harvey murine sarcoma virus (Ha-MuSV) DNA suggests that the region of Ha-MuSV which codes for the p21 “src” protein is not found in the rat endogenous replication-defective (30 S) virus and that the p21 gene represents a 1.1-kb (kilobase) substitution of genetic material in the 30 S genome prior to or during the formation of Ha-MuSV. Additionally, a small region (0.56 kb) of nonhomology, representing either a deletion or an insertion, has been found near the middle of the 30 S viral genome and a large region of nonhomology (1.02 kb), representing that portion of Ha-MuSV which is mouse in origin, has been observed at the 3′ end of the respective genomes. These results are in good agreement with the localization of the p21 gene as determined by restriction enzyme analysis and transfection studies of both 30 S and Harvey murine and sarcoma virus DNAs (Ellis et al., submitted for publication).     

Continuous production of Rous sarcoma virus free of transformation defective virus in clones of established RSV-transformed quail cells

Quail embryo fibroblasts were infected with a Schmidt-Ruppin strain RSV × chf recombinant virus. Virus-transformed cells were established as a permanent line and then cloned in methyl cellulose. Out of 140 clones isolated four clones were capable of indefinite growth. These clones were examined for (i) production of sarcoma and td virus particles, (ii) number of integrated virus genome equivalents, and (iii) deletions of the src gene in the provirus. We found that the clones yield about 106 focus-forming units of the sarcoma virus per milliliter of the culture medium. No td virus could be detected by plating of the virus at the endpoint dilution and no 35 S td virus RNA but only 38 S sarcoma virus RNA was found in virions. Hybridization kinetic studies indicated that three different clones contain about 2 virus genome equivalents, and one clone contains about 4 virus genome equivalents per diploid cell. Upon transfection the proviruses of different clones generated sarcoma viruses and no td viruses. Finally digestion with EcoRI restriction endonuclease released in all four clones a 1.9 × 106-dalton fragment characteristic of the complete src gene, while no 0.8 × 10⁶-dalton fragment characteristic of a td provirus could be detected. We concluded that the clones of RSV-transformed quail cells contain only nondefective sarcoma proviruses and produce from these proviruses nondefective focus-forming virions in the absence of any segregant td virions.     

Avian erythroblastosis virus: transformation-specific sequences form a contiguous segment of 3.25 kb located in the middle of the 6-kb genome.

Several foci of chicken embryo fibroblasts transformed by avian erythroblastosis virus (AEV) strain ES-4 were found to produce virus progeny containing the RNA of the replication-defective AEV in excess over the RNA of the helper virus. The size of AEV RNA was determined by methylmercury-agarose gel electrophoresis and electron microscopy to be 28 S or 6 kb. About 40 to 45% of this RNA is homologous by RNA-DNA hybridization to the RNA of other chicken leukosis and sarcoma viruses; the rest of the genome is AEV specific. These AEV specific sequences, which presumably contain the genetic information responsible for transformation form a contiguous stretch of 3.25 kb, located by heteroduplex mapping in the center of the 6 kb genome between two segments of 1.06 kb at the 3′ end and 1.64 kb at the 5′ end which are homologous to the genome of avian sarcoma virus. From the length of the region showing homology between AEV and avian sarcoma virus at the 5′ end of the genome and from the known sequence composition of the AEV-specific 75K protein (Hayman et al., 1979), it can be deduced that the initiation point for the N terminus of the gag protein p19 is located about 1.0 kb from the 5′ end of the genome in avian oncoviruses. Nonproducing AEV-transformed chicken embryo fibroblasts were also isolated. Infectious AEV could be rescued from these cells only with chicken leukosis viruses; unrelated avian retroviruses were ineffective, probably because AEV requires complementation in the gag and pol genes, in addition to env.     

Subgroup classification of feline leukemia and sarcoma viruses by viral interference and neutralization tests

Diverse strains of naturally occurring feline leukemia and sarcoma viruses share the species-specific group specific antigen (gs-1), but reveal similarities and differences between individual members of the group with respect to viral envelope antigens. Such antigens can be demonstrated by viral interference and viral neutralization tests.The occurrence of three envelope antigens enabled us to classify the feline type C viruses into three subgroups of A, B and C. The property of initial viral infectivity (viral attachment and penetration into cells) and the induction of type-specific virus neutralizing antibodies appear to be controlled by the same envelope antigen. Thus, the replication of a feline leukemia virus in feline embryo cell cultures was accompanied by the development in these cultures of a viral resistance to the cell-transforming effects of antigenically related but not unrelated feline sarcoma viruses and feline leukemia pseudotypes of murine sarcoma virus. The presently known naturally occurring feline leukemia and sarcoma viruses of B and C subgroups were found to be antigenic mixtures containing an A subgroup virus as one of the components.These studies suggest that feline type C viruses may gain entry into cells at cell receptor sites specific for each major envelope antigen.     

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.     

Mapping of nonconditional and conditional mutants in the src gene of Prague strain Rous sarcoma virus

Restriction endonuclease EcoRI digestion of the viral DNA of 12 nonconditional transformation defective (td) mutants of Prague strain Rous sarcoma virus (PR-RSV) has divided these mutants into two groups. Five mutants possess an EcoRI B (src gene-containing) fragment of the same size as that from wild type PR-RSV and thus these mutants have no detectable diminution in the transforming src gene. The other 7 mutants bear deletions of 1.0 to 1.8 kilobases in the 3.2-kilobase EcoRI B fragment. The extents of these deletions have been mapped using a number of restriction endonucleases and by comparing these results with studies on the nucleotide sequence of src(Czernilovsky et al., Nature (London)287, 198–203, 1980) we conclude that the td mutants have deleted sequences at the 5′ end of src, and in some cases also in regions between src and env, leaving intact at least some 3′ src sequences. These td mutants recombine in differing patterns with 14 temperature-sensitive (ts) src gene mutants. This enables many of the ts mutations to be localized in limited regions of src, 10 of them being clustered in the 3′ 40% of the gene, the remaining four bearing at least one mutation in the 5′ 60% of src. A nonconditional src gene mutant that transforms cells to a fusiform as opposed to round cell morphology (td SF/LO 104) also possesses a lesion that maps in the 5′ 60% of the src gene.     

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.     

Novel nonconditional mutants in the src gene of Rous sarcoma virus: isolation and preliminary characterization

We have isolated a number of nonconditional transformation-defective (td) mutants of Prague strain Rous sarcoma virus, subgroup A (PR-RSV-A). Many of these resembled td mutants reported previously, but 11 isolates from low-passage stocks of PR--A showed unusual properties and were designated partially td (ptd) mutants. In mixed infections with temperature-sensitive (ts) transformation-defective RSV mutants the ptd viruses produced cell transformation at restrictive temperature (41°), probably by genetic recombination to yield wild-type virus. In tests with a panel of 4 ts mutants, we found that different ptd isolates varied in the number and pattern of ts mutants with which they showed this effect. In mixed infections with one another the ptd viruses yielded transforming virus. Again, the pattern shown by different ptd viruses varied, and on the basis of this variation the 11 ptd isolates appear to comprise at least 10 distinct mutants. The possibility of genome deletions in some of the viruses was examined in Southern blots of EcoRI digests of proviral DNA. Two ptd viruses, which recombined with all 4 ts mutants tested, had EcoRI restriction fragments identical to those of wild-type PR-A. Three isolates which recombined with either 3, 2, or none of the ts mutants, showed deletions in the EcoRI fragment containing the src gene. These deletions corresponded to losses of 1.0, 1.5, and 1.6 kilobases, respectively, from the RNA genome. We conclude that these ptd viruses bear either point mutations or deletions of varying size but all retain part of the src gene. These mutants are stable and should be useful for further genetic and physiological studies on the src gene and its product.     

Recombinant avian oncoviruses. II. Alterations in the gag proteins and evidence for intragenic recombination.

The internal structural (gag) proteins of recombinant avian oncoviruses selected for the env gene of RAV-O (an endogenous chicken virus) and the src gene for PR-RSV-C were examined. Eight of ten clones of such recombinants were found to synthesize altered gag proteins. The gag proteins of one recombinant clone, PR-E-95c, were examined in more detail by gel electrophoresis and tryptic peptide mapping. These methods have allowed us to distinguish between the gag proteins of the two parental viruses and to determine from which virus the proteins of the recombinant virus were derived. PR-E-95c virions were found to contain p27₀, an electrophoretically distinguishable variant of p27 which is found in isolates of RAV-0. This recombinant virus also contains p12/15, which is electrophoretically indistinguishable from the p12/15 of both of the parental viruses. However, tryptic peptide analysis of p15 indicates that PR-E-95c has inherited PR-RSV-C-specific p15 sequences. These observations suggest that at least one cross-over has occurred between p15 and p27 in PR-E-95c. A striking difference between the proteins of PR-E-95c virus and those of the parental viruses is that the recombinant lacks polypeptides migrating in the position of p19 and contains two novel polypeptides termed p19α (MW 20,000) and p19β (MW 15,000). Both of these polypeptides are phosphorylated and share antigenic determinants and some tryptic peptides with parental p19. As determined by peptide analysis and radioimmunoassay, these p19-related proteins contain information from both parental viruses, suggesting that PR-E-95c has another cross-over within p19. The altered p19 proteins bind to viral RNA specifically and are associated with genomic RNA in the virion. Neither the stability nor the specific infectivity of the recombinant viruses appears to be significantly affected by the altered proteins.     

Detection and enumeration of transformation-defective strains of avian sarcoma virus with molecular hybridization.

Serial propagation of avian sarcoma viruses generates deletions in the viral gene responsible for cellular transformation (src). We have devised an assay for these deletion mutants which utilizes molecular hybridization and exploits the availability of DNA (cDNA_sarc) complementary to the nucleotide sequences affected by the deletion in src. Our procedure is also applicable to deletions in other viral genes and offers several advantages over conventional bioassays for the deletion mutants; moreover, it can be used to detect deletions in virus-specific intracellular nucleic acids. In order to illustrate the utility of the assay, we demonstrate that all 20 copies of the proviral DNA for avian sarcoma viruses in XC cells contain src, and we show that single avian cells can contain functioning proviruses for both avian sarcoma virus and a congenic deletion mutant. It should now be possible to use molecular hybridization to study the mechanism by which deletions in src are generated.     

Recombinant avian oncoviruses. I. Alterations in the precursor to the internal structural proteins.

The synthesis of the gag precursor protein (Pr76) was studied in a number of recombinant avian oncoviruses, which were selected for recombination between the env and pol genes or the env and src genes. Such studies show that the electrophoretic mobility of the gag precursor protein of recombinant viruses (ΔPr76) was greater than that of the parental gene product (Pr76) in 16 of 24 cases. Viruses derived from recombination between endogenous (RAV-0) and exogenous viruses (RSV), as well as between two exogenous viruses, showed the ΔPr76 phenotype. In an mRNA-dependent rabbit reticulocyte translation system, 35 S RNA isolated from PR-RSV-C directed the synthesis of Pr76, while RNA isolated from a recombinant between PR-RSV-C and RAV-0 directed the synthesis of ΔPr76. These observations show that the synthesis of ΔPr76 is due to an alteration in the genome related to recombination. An analysis of the RNase T₁-resistant oligonucleotides demonstrated a crossover near the 5′ end of the genome (which may be within the gag gene) in two recombinant virus clones which synthesize ΔPr76 in infected cells; but no crossover was detected near the 5′ end of the genome in a third recombinant virus clone which synthesizes Pr76 in infected cells. Our data suggest that the synthesis of ΔPr76 is a consequence of recombination near the 5′ end of the genome.     

Specific RNA sequences of Rous sarcoma virus (RSV) recovered from tumors induced by transformation-defective RSV deletion mutants.

We have investigated the RNAs of two avian sarcoma viruses recovered (rASV) from tumors induced in chickens by a deletion mutant of Schmidt-Ruppin Rous sarcoma virus (SR-RSV) that had lost part, but not all, of its sarcoma gene (src). The RNAs of the rASVs had the same size as SR-RSV RNA and were larger than the predominant RNA species of the partial src deletion mutant, if measured by electrophoresis in polyacrylamide gels. Fingerprinting of RNase T,-resistant oligonucleotides indicated that the rASVs shared one src gene oligonucleotide with SR-D which was also present in the partial src deletion mutant of SR-RSV. The two rASVs shared one other, probable src oligonucleotide, that was not found in SR-RSV, and SR-RSV contained a src oligonucleotide not found in the rASVs. However, the distinctive src oligonucleotide of the rASVs was structurally closely related to that of SR-RSV. We conclude that the src genes of the rASVs and that of SR-RSV are closely related. Possible mechanisms by which a partial src deletion may recover a complete src gene are discussed in view of our results.     

Replication of avian sarcoma viruses in chicken macrophages

Avian sarcoma viruses of subgroups B and C were able to replicate in chicken embryonic macrophages (derived from yolk sac) and in adult macrophages (obtained from peripheral blood), while avian sarcoma viruses of subgroups A and D were not. Infectious center assays indicated that the proportion of macrophages infected by viruses representative of subgroups B and C which registered as infectious centers was significantly lower than that of fibroblasts infected by the same viruses. Moreover, the comparative growth of avian sarcoma virus B77 (subgroup C) in fibroblasts and in yolk sac cells showed that macrophages have a reduced ability to replicate this virus. In addition, viruses of subgroups B and C and mainly helper-independent viruses of these two subgroups induced morphological alterations (formation of giant cells) and biochemical changes (increased rate of sugar transport), indicating that the infection of chicken macrophages by avian sarcoma viruses of subgroups B and C led to the malignant transformation of these cells.     

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.     

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.     

Phosphoproteins of Rous sarcoma viruses.

³²P-labeled proteins from Rous sarcoma viruses were extracted with phenol and analyzed by SDS-polyacrylamide gel electrophoresis. p19 was found to be the major phosphorylated protein. p12, a ribonucleoprotein, was also phosphorylated. Phosphorylation took place at both serine and threonine. The phosphorylation patterns of viral proteins were studied with respect to subgroup specificity, transforming ability, and maturation ability of the viruses. It was found that some of the viruses belonging to subgroup A, including RAV-3, RSV(RAV-1), and PR-B:RAV-3, contain a novel p23 phosphoprotein in addition to phosphorylated p19 and p12. It could not be determined whether p23 phosphoprotein was cellular or viral in origin. The phosphorylation patterns did not vary with regard to other viral properties. Furthermore, the in vivo phosphorylated proteins were found to be different from the viral proteins phosphorylated in vitro by virion-associated protein kinases. It was suggested that the phosphorylation of viral proteins in vivo is a specific process.     

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

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