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.     

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.     

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.     

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.     

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.     

Subgroup-specific antigenic determinants of avian RNA tumor virls structural proteins: analysis of virus recombinants.

Two nonglycosylated structural proteins of avian RNA tumor viruses, p15 and p19, were examined for the presence of subgroup-specific antigenic determinants by using competition radioimmunoassays. A comparison of viruses from subgroups A, B, and C revealed that subgroup B and C virus proteins were equally efficient in inhibiting the homologous radioimmunoassays which used antiserum against B77 and iodinated p15 and p19 from B77. On the other hand, subgroup A virus proteins were less efficient than subgroup C viruses in causing inhibition in these assays. That these differences in inhibition were due to true immunological differences was confirmed by using heterologous competition radioimmunoassays. Since it was possible to distinguish the p19 or p15 of subgroup A viruses from the corresponding proteins of either subgroup B or subgroup C virus, recombinant viruses from crosses between leukosis viruses of subgroup A and sarcoma viruses of subgroups B or C were examined. The recombinant viruses have the envelope glycoprotein gene (env) of the subgroup A virus and the sarcoma gene (src) of the subgroup B or C virus. The results show that 14 clones out of 17 examined had p19 from the sarcoma virus. RNA fingerprinting has shown that the src gene is located close to the 3′ end of the genome and is closely followed towards the 5′ end by the env gene. The observed linkage between src and p19 can be explained by postulating that the gene for p19 is located close to the 5′ end of the genome and that recombination takes place between circular forms of the virus genome.     

Structural and nonstructural proteins of strain Colburn cytomegalovirus

The growth of most Rous sarcoma viruses (RSV) is severely restricted on MSB-1 cells (a line of chicken T lymphoblasts) in comparison to growth on chicken embryo fibroblast (CEF). Nonconditional transformation defective mutants of RSV from which the complete src region has been deleted (td RSV) are not subject to growth restriction. We examined the formation and integration of RSV and td RSV in MSB-1 cells following high multiplicity infection. Nearly equivalent quantities of the linear form of unintegrated RSV and td RSV DNA were formed in these cells during the first 10 hr after infection. Linear RSV DNA from MSB-1 cells could not be distinguished from linear RSV from CEF by restriction endonuclease analysis and by previously described transfection assays (P. E. Neiman, C. McMillin-Helsel, and G. M. Cooper, 1978, Virology 89,360–371). Beyond 10 hr after infection, and with progressive cell growth in the MSB-1 cultures, the level of RSV linear DNA rapidly decreased. Presumptive circular RSV DNA was detected only transiently, and at very low levels, about 15 hr after infection. Association of RSV DNA with high-molecular-weight chromosomal DNA, i.e., integration, was not detected in this study. In contrast, nearly constant levels of td RSV unintegrated linear DNA and, after 20 hr, circular DNA persisted in MSB-1 cells for at least 7 days after infection. Integration of td RSV proviral DNA was inefficient, occurring in only about 5% of MSB-1 cells (even at very high multiplicities of infection) in the first round of infection, and in 25–40% of cells by 3 days after infection. Almost all MSB-1 cells containing td RSV DNA produced virus. Analysis of eight nonconditional transformation defective mutants of RSV which retain the src region to different extents showed that all of these mutants replicated to the same normal titer on MSB-1 cells as on CEF without further deletion of the src region. Two temperature sensitive src mutants that thermal inactivation of the scr gene on MSB-1 cells at both 35° and 41°, indicating that thermal inactivation of the src gene product could not abrogate the replication block. These studies clearly demonstrate that the presence of the src region in RSV impedes the formation and/or integration of provirus in some types of host cells.     

Specific restriction of avian sarcoma viruses by a line of transformed lymphoid cells.

MSB-1 cells are a line of transformed chicken lymphoid cells derived from tumors induced by Marek's disease viruses and free of exogenous avian leukosis viruses (ALV). They can be infected by ALV of subgroups A and C including transformation-defective (td) deletion mutants of avian sarcoma viruses (ASV). In terms of virus titers in supernatant culture medium, proportion of virus-producing cells, and levels of viral RNA detected by hybridization with a cDNA probe, infection by td ASV of MSB-1 cells was indistinguishable from infection of chicken embryo fibroblasts. In contrast, wild type ASV was restricted in its growth on MSB-1 cells. Different clones of ASV varied in their restriction by all these parameters of viral growth by factors of 10^?1 to 10^?4 Studies of a severely restricted viral clone showed equal quantities of hybridizable viral DNA in Hirt supernatant fractions of both fibroblasts and MSB-1 cells at 10 hr after high multiplicity infection, and transfection assays indicated infectious viral DNA in both cell types. Viral DNA largely disappeared from Hirt supernatant fractions of MSB-1 cells by 48 hr after infection, and sarcoma virus-specific DNA was not detected in Hirt pellet fractions from MSB-1 cells at levels found in comparably infected fibroblasts. Infectious ASV DNA, while easily detected in fibroblasts, could not be detected on MSB-1 cells at 48 hr or later times after infection. Because replication of td ASV does not appear restricted in MSB-1 cells, the failure of ASV DNA to integrate normally in these cells seems to be related to the presence of src sequences in the viral genome.     

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.     

The genomic RNA of avian reticuloendotheliosis virus REV

Purified virus obtained from a subline of chicken bone marrow cells transformed by avian reticuloendotheliosis virus (REV) was found to contain the RNA of REV in excess over the RNA of its associated helper virus REV-A. Electrophoretic and sedimentation analyses resolved these RNAs into a 28 S and a 34 S component, respectively. Comparison of these RNA species with the RNA obtained from plaque-purified preparations of REV-A confirmed that the 28 S RNA represents the genome of transforming REV. The small size of 28 S REV RNA suggests that the defectiveness of REV is due to a deletion of replicative sequences. Hybridization experiments indicated that about 25–30% of REV RNA sequences are unrelated to REV-A. These may include the putative transforming sequences of REV. REV shared 12–15 of 42 identifiable large RNase T₁-resistant oligonucleotides with REV-A. The 28 S REV RNA did not contain the transformation-specific oligonucleotides which are largely conserved among avian acute leukemia viruses MC29, MH2, and CMII or the src-specific oligonucleotides of avian sarcoma viruses. It is concluded that the sequences which are unique for REV contain a new class of avian tumor virus transforming genes.     

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.     

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.     

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.     

A src-related 120,000-dalton protein expressed in an avian sarcoma virus-transformed rat cell line

A Schmidt-Ruppin strain of Rous sarcoma virus-transformed rat cell line, SRYI, produced a 120,000-dalton phosphoprotein (P120) immunoprecipitated by a tumor-bearing rabbit serum in addition to the src gene product, pp60^src and the gag gene product. Antisera against viral structure components did not precipitate P120, and preabsorption of the tumor-bearing rabbit serum with disrupted virions did not affect the precipitation of P120 and pp60^src. One-dimensional peptide mapping by partial proteolysis revealed that P120 is a protein consisting of peptides common with pp60^src SRYl cells contained 28 and 21 S virus-specific RNA species and P120 was translated in vitro from 28 S polyadenylic acid-containing RNA. The expression of P120 in all subclones of SRY1 suggested that the genome coding P120 is integrated in the SRY1 cell DNA. The rescued virus from SRYl cells, however, failed to produce P120 in chick embryo cells.     

RNA tumor viruses of pheasants: Characterization of avian leukosis subgroups F and G

Endogenous leukosis-like viruses of ring-necked pheasants (Phasianus colchicus) and golden pheasants (Chrysolophus pictus) have been isolated and characterized. The majority of the normal pheasant embryo cultures contain helper activity for the defective Bryan high titer strain of Rous sarcoma virus. The Rous sarcoma pseudotypes produced with endogenous helper activity from ring-necked pheasants belong to subgroup F. The pseudotypes from golden pheasant cells constitute subgroup G. Subgroup F and G pseudotypes can infect all known genetic types of chicken fibroblasts as well as pheasant and Japanese quail cells, but do not plate on goose cells. Duck cells are resistant to subgroup G but not to F.The subgroup F and G helper viruses isolated from Rous sarcoma viral pseudotypes show interference with their homologous subgroup. RAV-61, a standard of subgroup F, interferes with pseudotypes produced with endogenous helper activity from ring-necked pheasant cells but not with subgroup G pseudotypes.Subgroups F and G do not cross-react with subgroup A to E in neutralization tests. Some normal ring-necked pheasant sera have anti-F activity.Subgroup F and probably also G leukosis-like viruses can undergo genetic recombination with nondefective avian sarcoma viruses.     

Transformation-enhancing factor(s) released from chicken Rous sarcoma cells: effect on some transformation parameters

The medium of chick embryo fibroblasts (CEF) transformed by Schmidt-Ruppin strain of Rous sarcoma virus (SR-RSV) contains a factor(s) which complements the expression of some transformation parameters depending on the src gene. Notably, it reverses the block by puromycin of morphological transformation of cells infected with three ts-T mutants after shift-down from restrictive (41.5°) to permissive (37°) temperature. This reversal is not due to the release of inhibition of protein synthesis produced by puromycin, and is accompanied by the expression of two other src-dependent transformation parameters: disorganization of the cytoskeleton and loss of cell surface-associated fibronectin. The factor(s) able to overcome the puromycin block of morphological transformation was operationally called transformation-enhancing factor (TEF) like a previously reported factor favoring transformation by RSV (Krycève et al., Int. J. Cancer17, 370–379, 1976). It is lacking in media of untransformed cells, uninfected or infected with a nontransforming virus (RAV-1), and its production by RSV-infected cells seems to depend on the acquisition of the transformed phenotype, therefore on the expression of the src gene. Its effect was also shown to persist beyond the period of contact with the cells. It appears to be a glycoprotein which can be resolved by gel filtration into two peaks of 250K and 190K, apparently distinct from other known factors spontaneously released by transformed cells. A similar activity was also found in the medium of mammalian (rodent) cells transformed by SR-RSV and by other RNA and DNA oncogenic viruses, but not in the medium of untransformed controls.     

Endogenous proviruses of random-bred chickens and ring-necked pheasants: analysis with restriction endonucleases

We have examined, by digestion with restriction endonucleases and nucleic acid hybridization, sequences homologous to avian sarcoma virus (ASV) DNA in DNA from 18 random-bred chickens of the brown leghorn and brown nick flocks and 8 ring-necked pheasants. Both species have sequences related to the replicative genes (gag, pol, andenv) and to the transforming gene (src) of ASV. The disposition of these sequences in random-bred chickens is reminiscent of the situation in inbred white leghorn flocks; the sequences related togag, pol, andenv appear to reside in structures which closely resemble proviruses of the endogenous chicken virus RAV-O, and thesrc-related sequences appear to be a cellular gene (or genes). The number of endogenous proviruses present in the random-bred flocks is highly variable, and there are proviruses present at positions in the genomes of the random-bred birds different from those described for white leghorns. The endogenous ASV-related sequences in ring-necked pheasants fall into the same two categories; sequences related to the replicative genes of ASV probably reside in proviruses, and thesrc-related sequences in a cellular gene (or genes). However, the endogenous pheasant viruses are clearly distinct from those of chickens both by analysis with restriction endonucleases and by hybridization. These observations support the hypothesis that cellularsrc (c-src) has had a separate evolutionary history from the endogenous proviruses, which apparently arise by germ line infections. The endogenous viruses of chickens and pheasants, while clearly related, appear to have undergone significant independent evolution, which suggests that the frequency with which these viruses achieve a successful germ line infection across species boundaries is low compared with the rate of successful germ line infections within a species.     

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.     

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.