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.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

Effects of the src gene product on microfilament and microtubule organization in avian and mammalian cells infected with the same temperature-sensitive mutant of Rous sarcoma virus.

We have studied cytoskeletal organization in both mammalian (rat kidney cells) and avian cells (chick embryo fibroblasts) that have been transformed with temperature-sensitive src gene mutants of Rous sarcoma virus. The functioning of the src gene in rat cells affected the organization of actin-containing microfilament bundles, but not microtubules at the permissive temperature for transformation. These cells formed colonies in soft agar at permissive temperature, but not at nonpermissive temperature. In contrast, the src gene product affected both microtubule and microfilament organization in chick embryo fibroblasts at permissive temperature.     

A nonconditional replication-defective mutant of the Schmidt-Ruppin strain of Rous sarcoma virus.

A nonconditional mutant of the Schmidt-Ruppin strain of Rous sarcoma virus (subgroup A) that fails to synthesize infectious progeny virus is described. Nonproducing cells transformed by this mutant LA7365 have functional viral src and env genes. The gag gene of LA7365 is defective. There is no complementation with a gag ts mutant, and pulse-chase experiments show that the proteolytic processing of the gag precursor pr76 is strongly inhibited in mutant-infected cells. Whether there is an additional lesion in the pol gene of LA 7365 could not be determined from the available data.     

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.     

The virion RNA species of the Kirsten murine sarcoma-leukemia virus complex released from a clonally related series of mouse cells

Summary We have characterized the virion RNA species of Kirsten sarcoma (KiSV) and Kirsten leukemia (KiLV) viruses released from a clonally related series of mouse cells (14). We have identified the KiLV and KiSV genome RNAs. In addition to the viral RNA species we find large amounts of a virus-like RNA (VL30 RNA), which is heterogeneous and shows variability in its expression. The amount of VL30 RNA in virions does not correlate with the state of transformation of the cells releasing the virus or the ability of the virus to transform other cells. Characterization of RNA rescued from non-producer cells has revealed a sarcoma virus (KiSV_CB3) with an oligonucleotide finger-print different from that of a standard KiSV RNA, suggesting that it has lost some viral sequences. The oligonucleotide fingerprints of KiLV and VL30 RNAs are distinct from each other and from those reported for other murine leukemia virus RNAs.With 7 Figures     

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.     

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.     

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.     

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.     

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.     

The Conserved, 5′ Termini of RNAs 1 and 2 of <Emphasis Type="Italic">Tomato aspermy virus</Emphasis> are Dispensable for Infection but Affect Virulence

The 5 terminus of each of the three genomic RNAs (RNAs 1, 2 and 3) of Tomato aspermy virus (TAV) begins with the sequence 5-GUUU, which is also shared by a number of other viruses. Mutagenic analyses showed that the 5-GUUU sequence of RNAs 1 and 2 of TAV was dispensable for viral infection and did not prevent symptom induction. On the other hand, substitution of U at position 5 for G in RNA 1, but not RNA 2, induced veinal necrosis symptoms in Nicotiana glutinosa. The mutants constructed included insertion of UUU into the 5-GUUU sequence of TAV RNAs 1 and/or 2. All RNA 2 mutants induced more severe symptoms than viral RNAs containing either mutated RNA 1 or most combinations of mutated RNAs 1 and 2. Some combinations of mutated RNAs 1 and 2 also induced veinal necrosis in N. glutinosa. Virulence was unrelated to the levels of viral RNA accumulation. Sequence analysis of progeny viral RNAs showed that only the mutant viral RNAs with a G to U substitution in RNA 1 and the deletion of the 5-GUUU in both RNAs 1 and 2 were able to maintain the same sequence as the inoculum. The other mutants either reverted to the wildtype sequence or underwent further deletion or insertion. None of the constructed mutants were able to compete for accumulation with the wildtype virus after co-inoculation to the plant species tested.