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.     

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.     

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.     

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.     

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.     

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.     

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.     

Intracellular murine hepatitis virus-specific RNAs contain common sequences

A major polyadenylated viral RNA of approximately 0.8 × 10⁶ daltons was isolated from murine hepatitis virus (A59)-infected cells by preparative polyacrylamide gel electrophoresis in formamide. This RNA was shown to encode the viral nucleocapsid protein by direct in vitro translation in a cell-free, reticulocyte-derived system. Single stranded ³²P-labeled complementary DNA was prepared from this RNA and was demonstrated to be virus specific. Using this complementary DNA in a Northern blotting procedure, we were able to identify six major virus-specific intracellular RNA species with estimated molecular weights of 0.8, 1.1, 1.4, 1.6, 3, and 4 × 10⁶ daltons. All of these RNA species were polyadenylated. Our results support the idea that coronavirus-infected cells contain multiple intracellular polyadenylated RNAs which share common sequences.     

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.     

Organization of the endogenous proviruses of chickens: implications for origin and expression

Eleven of the endogenous proviruses of white leghorn chickens have been mapped with restriction endonucleases and specific nucleic acid hybridization reagents. The restriction maps of these endogenous proviruses have been compared with restriction maps of avian sarcoma virus (ASV) and Rous-associated virus O (RAV-O), an endogenous virus which is spontaneously released by cells from certain lines of chickens. Endogenous proviruses have the same basic structure as proviruses acquired by exogenous infection; the gene order is the same in the provirus as in viral RNA, and the ends of the provirus form a characteristic direct repeat which contains sequences derived from both ends of viral RNA. The endogenous proviruses can thus be described “cell DNA-3′5′-gag-pol-env-3′5′-cell DNA,” where 3′ and 5′ denote sequences homologous to the 3′ and 5′ ends of viral RNA. The endogenous proviruses of chickens are more closely related to RAV-O than to ASV, based on restriction maps and on hybridization with reagents specific for the 3′ ends of RAV-O and ASV. However, all but two of the endogenous proviruses lack at least one of the twoSstI sites in RAV-O DNA (see the preceding paper) and can thus be distinguished from RAV-O by digestion withSstI. One of the two exceptions,ev-2, is found in the DNA of line 7₂ and line 100 chickens and is genetically linked to the production of RAV-O. The only other provirus (which we call B) having both theSstI sites in RAV-O DNA was seen only once in a line 100 sample. Of the nine remaining elements, six had large deletions. Three proviruses (ev-4,ev-6, and an element we call A) are missing the left 3′5′ repeat and have sustained deletions extending varying distances intogag orpol. One of these,ev-6, is associated with the gs^?chf⁺ phenotype; the phenotype can be explained from the structure of the provirus.ev-3 has both terminal repeats intact but has sustained a deletion near thegag-pol boundary. This provirus is associated with the gs⁺chf⁺ phenotype and the structure of the provirus could account for the peculiar RNA and protein associated with this phenotype. We have also found two elements which apparently consist of sequences present only in the 3′5′ terminal repeat unit, with no other associated virus specific sequences. Such structures might arise by homologous recombination between the terminal repeats of a normal provirus.     

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.     

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.     

Herpesvirus-associated nuclear antigen(s) in cells biochemically transformed by fragments of herpesvirus DNA and in somatic cell hybrids

Human and mouse cells biochemically transformed by ultraviolet light (UV)-irradiated HSV-1 express HSV-1 thymidine kinase (TK) activity and also express type-specific herpesvirus-associated nuclear antigen(s) (HANA). To identify the HSV-1 DNA sequences coding for HANA and their location on the viral genome, studies were carried out on: (i) somatic cell hybrid clones obtained by fusing mouse [LM(TK^?)] cells with UV-irradiated HSV-1-transformed human [HeLa(BU25)/KOS 8-1] cells; and (ii) LM(TK^?) cells biochemically transformed with restriction endonuclease fragments of DNA which code for HSV-1 TK. Molecular hybridization experiments were also carried out and demonstrated that HSV-1 DNA sequences coding for TK were integrated in the biochemically transformed cells. The human-mouse somatic cell hybrid clones (LH81) which were HSV-1 TK+ were also HANA⁺, while clones counterselected in bromodeoxyuridine which had lost HSV-1 TK activity and DNA sequences likewise lost HANA. Previous studies had shown that the HSV-1 TK gene of LH81 hybrid clones was associated with a marker chromosome, designated M7, which consists of a human chromosome 17 translocated to the short arm of chromosome 3, or a modified M7 chromosome containing a translocation from a mouse chromosome. The present results indicate that at least one HANA gene was integrated in the same chromosome as the HSV-1 TK gene. LM(TK^?) cells biochemically transformed by HSV-1 DNA restriction nuclease fragments of diminishing size, which map in the HpaI-I region (26.2 to 31.7) of the HSV-1 genome, were HANA⁺ as well as TK⁺. The HANA⁺ cells included LM(TK^?)/TF pAGO PP and LM(TK^?)/TF pAGO PS clones. The latter are clones of LM(TK^?) cells biochemically transformed, respectively, by a PvuII fragment (1.35 × 10⁶ daltons) and a PvuII-SmaI fragment (0.9 × 10⁶ daltons; 30.2 to 31.1 map units) of HSV-1 DNA derived from Escherichia coli plasmid, pAGO. Since the PvuII-SmaI DNA fragment has only enough genetic information to code for a polypeptide of about 53,000 daltons and the HSV-1 TK polypeptide is about 40,000 daltons, the findings indicate that the genes for HSV-1 TK and one herpesvirus-associated nuclear antigen are either contiguous or overlapping, or HSV-1 TK and one HANA gene are identical.     

Mechanism of infection by Epstein-Barr virus. II. Comparison of viral DNA from HR-1 and superinfected Raji cells by restriction enzymes.

Virus DNA (RS virus DNA) was directly isolated from Raji cells superinfected with Epstein-Barr virus derived from P3HR-1 cells and compared with original superinfecting virus DNA from P3HR-1 cells (HR-1 virus DNA) in agarose-gel electrophoresis after digestion with various restriction enzymes. EcoR-1 digestion of RS virus DNA produced 15 fragments identical to those from HR-1 virus DNA. However, two fragments, EcoR1 No. 6 (10 × 10⁶ daltons) and EcoR1 No. 11 (4.6 × 10⁶ daltons), observed in HR-1 virus DNA were not detected in RS virus DNA from superinfected Raji cells. In addition, the EcoR1 No. 4 (13.5 × 10⁶ daltons) fragment of RS virus DNA showed a molar ratio of 2 whereas HR-1 virus DNA produced the same fragment with a molar ratio of 1. Electrophoresis patterns of virus DNA digested with Hind III, Bam H-I, Hpa I, and Sal I were also examined. In general, both types of virus DNA produced similar patterns after gel electrophoresis, with minor differences in molar ratios after being treated with the restriction enzymes suggesting that RS virus DNA obtained by superinfection of Raji cells is basically identical to HR-1 virus DNA but may contain a population of DNA a little more heterogenous than HR-1 virus DNA.     

Restriction endonuclease mapping of the DNA of Rous-associated virus O reveals extensive homology in structure and sequence with avian sarcoma virus DNA

We have prepared a physical map of the DNA of Rous-associated virus O (RAV-O), an endogenous virus released by certain chicken lines, in order to examine the relationship between the genome of this virus and closely related proviruses endogenous to chickens. Nineteen recognition sties for 11 restriction endonucleases have been mapped on the unintegrated linear and circular forms of RAV-O DNA isolated from acutely infected quail cells.PvuI is the only enzyme tested which does not cleave RAV-O DNA. Most of the sites (18 of 19) occur at similar or identical positions in the DNA of avian sarcoma virus (ASV). Significant differences between the maps of corrseponding regions of RAV-O and ASV DNA are observed only with those endonucleases which recognize sites encoded near the 3′ terminus of ASV RNA (PvuI andEcoRI). Both ends of RAV-O linear DNA contain sequences copied from both the 3′ and the 5′ ends of viral RNA. Two species of closed circular DNA were found: one the same size as the terminally redundant linear DNA (5.0 × 10⁶M_r and the other lacking ca. 0.35 kb from an end of linear DNA. Thus the unintegrated forms of RAV-O DNA appear structurally similar to those of ASV DNA[Shank, P. R.,et al. (1978b).Cell15, 1383–1395;Hsu, W.,et al. (1978).J. Virol.28, 810–818]; presumably one copy of a ca. 0.35-kb terminal repeat unit is lost during formation of the smaller circle from linear DNA. The following paper illustrates the utility of the physical map for differentiating between endogenous proviruses which might or might not have sequence identity with the RAV-O genome.     

Revertants of an ASV-transformed rat cell line have lost the complete provius or sustained mutations in src

We have isolated and characterized 12 revertants of a clonal line (B31) of avian sarcoma virus (ASV)-transformed rat-1 cells. The B31 cells contain a single normal ASV provirus, display the classical features of virally transformed cells, and revert to normal phenotype at low frequency. Revertants isolated after selective killing of transformed cells resemble uninfected rat-1 cells morphologically, fail to grow in suspension, and are at least 100-fold less tumorigenic than B31 cells. Two mechanisms of reversion have been identified in these cells. (i) Three of the revertant lines have lost the entire provirus, including both copies of the sequences repeated at the ends of the provirus; the manner in which the provirus is lost is not known. (ii) The other nine revertants retain a provirus of normal size and unaltered flanking cellular DNA: contain the same species of viral RNA at the same concentrations as in the parental line, B31; are susceptible to retransformation by wild-type ASV; and yield transformation-defective (td) virus after fusion with chicken cells. In one case, the rescued virus transforms chicken cells, but produces fusiform rather than normal foci and does not retransform rat cells morphologically. Hence these revertants arise as a consequence of nonconditional mutations (base substitutions or small deletions) in the viral transforming gene,src. In several cases, the revertant cells retransform spontaneously, or transforming virus appears in stocks of rescued td virus after passage through chicken cells, indicating back mutations to wild-type. Several of the rescued td viruses can also recombine to restore a wild-type phenotype. Analysis of the structure and enzymatic activity of products ofsrc confirms that the revertant cells bear various mutations insrc.     

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.