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.     

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.     

Biological and biochemical studies on the inactivation of avian oncoviruses by ultraviolet irradiation.

The effects of ultraviolet (uv) irradiation on transforming and replicating capacities of avian oncoviruses and on the synthesis of virus specific products after infection with irradiated virus were studied. Different strains of nondefective avian sarcoma viruses were inactivated at the same rate following single-hit kinetics. The 37% survival dose D₃₇ (1/e) was 736 erg mm^?2 on average. A comparison of the inactivation kinetics in a focus assay (transforming capacity) and an infectious center assay (replicating and transforming capacity) showed no partial inactivation of the virus genome; focus and infectious center formation were inactivated at the same rate. Similar results were obtained when the replicating capacity of the avian sarcoma virus was measured in a plaque assay; focus and plaque formation were inactivated at the same rate. No repair of the uv damage by either complementation or recombination with exogenous or endogenous avian leukosis virus could be demonstrated. The rates of inactivation of avian sarcoma virus assayed in focus and infectious center tests on chick embryo fibroblasts expressing or not expressing chicken helper factor, on chick embryo cells preinfected with RAV-1, and on Peking duck cells were identical. Nondefective avian sarcoma virus and deletion mutants of avian sarcoma virus defective for replication or transformation were inactivated at the same rate. Biochemical analysis of the DNA extracted from a Japanese quail tumor cell line (QT-6) 26 hr after infection with irradiated avian sarcoma virus strain B77 showed a decrease of total virus specific DNA and of full-length covalently closed circular (form I) viral DNA synthesis with increase of the uv dose. Virus-specific RNA synthesis, measured by hybridization of labeled RNA extracted from chicken embryo fibroblasts infected with irradiated virus to viral DNA, and particle production, assayed by uridine incorporation, were also inhibited with increasing uv dose. The inactivation rates for virus-specific DNA and RNA synthesis and for particle production were very similar, but lower than the rate for the loss of infectivity.     

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.     

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.     

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

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

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

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

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.     

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.     

Isolation of complementary DNA unique to the genome of avian myeloblastosis virus (AMV)

Avian myeloblastosis virus (AMV) can transform avian cells of hemopoietic origin, such as bone marrow, embryonic yolk sac, and circulating macrophages. The AMV is defective in its replication and can only replicate in the presence of helper viruses. This defectiveness in replication is probably due to a deletion or substitution of nucleotide sequences in its genome. The AMV genome contains no sequences homologous to the src gene, which is responsible for the transforming function of the sarcoma viruses. We attempted to identify the AMV sequences that may contain sequences which are responsible for its transforming function. We isolated a complementary DNA (cDNA_AMV) that hybridized preferentially to the RNA of the transforming AMV but not to the RNA of the helper virus. Using this cDNA_AMV as a probe, we determined the size of the AMV genome to be 33–34 S with a molecular weight of 2.6 × 10⁶. A similar molecular weight estimation of the AMV genome size was obtained by methylmercury-agarose gel electrophoresis of AMV RNA. In AMV-producer myeloblasts we can detect about 6000–7000 copies per cell of AMV-specific RNA, whereas fewer than 2 copies per cell of AMV RNA are found in helper virus-infected cells. In AMV nonproducer myeloblasts, about 2000 copies of AMV-specific RNA are detected. Furthermore, we find that RNA of AMV NP myeloblasts can only hybridize to 55% of cDNA complementary to helper virus genome. In uninfected hemopoietic cells, e.g., bone marrow cells, about 20 copies per cell of AMV-specific RNA are present, whereas in uninfected chick embryo fibroblasts less than 1 copy per cell is found.     

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.     

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.     

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.     

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.     

The isolation of SV40 tsA/deletion, double mutants and the induction of host DNA synthesis

Simian virus 40 mutants were constructed that contain both a tsA mutation leading to temperature sensitivity of the 92K T-antigen, and deletions of 20–200 base pairs leading to a loss in the expression of the 20K t-antigen. As expected, these mutants were temperature sensitive for viral growth and viral DNA replication in lytically infected cells. At nonpermissive temperatures, the ts/deletion mutants stimulated the incorporation of nucleosides into host DNA as did the tsA mutant alone. This induction of incorporation by the tsA mutants resulted from semiconservative DNA replication, not repair synthesis. At 200 μg/ml caffeine the induction of host DNA by A209 was inhibited by 30 to 50%, whereas induction by the ts/deletion mutants was abolished.     

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.