Guanine nucleotide binding activity of the src gene product of rat-derived murine sarcoma viruses.
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Biomedical subjects
E M Scolnick
Publications and source records attributed to E M Scolnick.
Transfection of molecularly cloned Friend murine leukemia virus DNA yields a highly leukemogenic helper-independent type C virus.
Unintegrated viral DNA was isolated via the Hirt procedure from mouse fibroblasts newly infected with Friend murine leukemia virus (F-MuLV) clone 201, a biologically cloned helper virus isolated from stocks of F-MuLV complex. A physical map of the unintegrated in vivo linear viral DNA was generated for several restriction endonucleases. The supercoiled viral DNA was digested with EcoRI, which cleaved the viral DNA at a unique site. The linearized viral DNA was then inserted into lambda gtWES.lambda B at the EcoRI site and cloned in an approved EK2 host. Eight independent lambda-mouse recombinants were identified as containing F-MuLV DNA inserts by hybridization with F-MuLV 32P-labeled complementary DNA. One of the F-MuLV DNA inserts was 9.1 kilobases (kb) and had the same restriction enzyme sites as the unintegrated linear F-MuLV DNA. Six inserts were 8.5 kb; each lacked a single copy of the terminally redundant sequences of the unintegrated linear viral DNA. One insert was 8.2 kb and contained a 0.9-kb deletion. After digestion with EcoRI, one recombinant DNA preparation containing an 8.5-kb insert was infectious for NIH 3T3 cells. Undigested recombinant DNA was not infectious. The infectivity of the EcoRI-digested DNA followed multihit kinetics, indicating that more than one molecule was required to register as an infectious unit. The virus isolated from this transfection (F-MuLV-57) was NB-ecotropic, helper-independent, and formed XC plaques. Inoculation of this virus into newborn NIH Swiss mice induced leukemia and splenomegaly in greater than 90% of animals within 3 to 4 weeks. The gross and microscopic abnormalities induced by F-MuLV clone 57 were identical to those seen with the original parent stocks of F-MuLV clone 201. These results indicate that this helper-independent F-MuLV can induce a rapid nonthymic leukemia in the absence of the spleen focus-forming virus.
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Molecular cloning of the Harvey sarcoma virus circular DNA intermediates. II. Further structural analyses.
Three species of unintegrated supercoiled Harvey sarcoma virus DNA (6.6, 6.0, and 5.4 kilobase pairs) have been molecularly cloned from Harvey sarcoma virus-infected cells. On the basis of restriction enzyme analyses, the 6.6- and 6.0-kilobase pair viral DNAs contain two and one copies, respectively, of a 650-base pair DNA segment which contains sequences present at the 3' and 5' termini of the viral genome. R-loop structures formed between Moloney leukemia virus RNA and the cloned Harvey sarcoma virus DNA indicated that about 500 base pairs of the 650-base pair repeating segment was complementary to the 3' end of the viral RNA. During amplification in the Escherichia coli host, some recombinants containing the 6.6- or the 6.0-kilobase pair Harvey sarcoma virus DNA insert acquired or lost the complete 650-base pair DNA segment. These changes occurred in both recA+ and recA- E. coli.
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Functional organization of the Harvey murine sarcoma virus genome.
The comparative infectivity of Harvey murine sarcoma virus (Ha-MuSV) DNA for NIH 3T3 cells was determined for supercoiled Ha-MuSV DNA molecularly cloned in lambda phage and pBR322 at its unique EcoRI site (which is located near the middle of the 6-kilobase pair [kbp] unintegrated linear viral DNA) and for two cloned subgenomic fragments: one was 3.8 kbp and lacked about 1 kbp from each side of the EcoRI site, and the second did not contain the 3 kbp of the unintegrated linear viral DNA located on the 3' side of the EcoRI site. Each subgenomic DNA induced foci of transformed cells, but with a lower relative efficiency then genomic DNA. Transfection with intact vector Ha-MuSV DNA yielded results similar to those obtained after separation of Ha-MuSV DNA from vector DNA. Cells lines were then derived from individual foci transformed with each type of viral DNA. Focus-forming virus was recovered from transformed cells after superinfection with a helper-independent virus, but the efficiency varied by several orders of magnitude. For several transformed lines, the efficiency of recovery of focus-forming virus was correlated with the structure of the Ha-MuSV DNA in the cells before superinfection. When 32P-labeled Ha-MuSV DNA probes specific for sequences on either the 3' or 5' side of the EcoRI site were used to analyze the viral RNA in the transformed cell lines, all lines were found to hybridize with the 5' probe, but some lines did not hybridize with the 3' probe. The transformed lines contained high levels of the Ha-MuSV-coded p21 or its associated GDP-binding activity. We conclude that the transforming region and the sequences that code for the viral p21 protein are both located within the 2 kilobases closest to the 5' end of the Ha-MuSV genome.
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Recovery of biologically active spleen focus-forming virus from molecularly cloned spleen focus-forming virus-pBR322 circular DNA by cotransfection with infectious type C retroviral DNA.
The genome of the Lilly-Steeves strain of spleen focus-forming virus (SFFV) was molecularly cloned in the plasmid vector pBR322. Infectious SFFV could be recovered by releasing the SFFV DNA from the vector, transfecting the released DNA onto NIH 3T3 cells, and rescuing the SFFV either by superinfection with helper virus or by cotransfection with molecularly cloned infectious helper viral DNA. By using transfections with SFFV DNA still attached to the plasmid vector, infectious SFFV activity could also be recovered with either method of rescue. Studies performed with these latter types of transfections indicated that only a portion of the SFFV genome was required for biological activity. Since gp52, a marker protein for SFFV, could be detected in all cultures from which adequate titers of biologically active SFFV were recovered, the results are consistent with the hypothesis that gp52 is necessary for SFFV-induced erythroblastosis and polycythemia.
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Dual evolutionary origin for the rat genetic sequences of Harvey murine sarcoma virus.
Detailed restriction endonuclease maps were developed for Harvey murine sarcoma virus (Ha-MuSV) DNA (clone H-1), molecularly closed at its unique EcoRI site in pBR322, for three nonoverlapping subgenomic HindIII clones which together span the entire H-1 clone and for a molecularly cloned DNA copy of a portion of rat 30S RNA (which represents the majority of the rat genetic sequences in Ha-MuSV). Molecular hybridization of the 30S clone to small restriction fragments of clone H-1 revealed a 0.9-to-1.0-kilobase pair region in the 5' half of the Ha-MuSV genome not homologous to the 30S clone, although the 30S clone did contain related sequences in Ha-MuSV on both sides of this nonhomologous region. By using cloned sequences from a segment of the Ha-MuSV nonhomology region as a probe for hybridization to Southern blots of DNA from rat, mouse, bat, and chicken cells, one to three bands were detected in DNA of each species. By contrast, the 30S clone DNA was highly related to many sequences in rat DNA, partially related to fewer mouse DNA sequences, and homologous only to one to three bands in bat and chicken DNA. Earlier work had shown that the 5' half of the Ha-MuSV genome coded for transformation and for the viral p21 protein (Chang et al., J. Virol. 35: 76--92, 1980; Wei et al., Proc. Natl. Acad. Sci. U.S.A., in press). We used two subgenomic HindIII clones whose shared HindIII site mapped within the 5' region of clone H-1 nonhomologous to the 30S clone to test whether the nonhomologous segment might encode the transforming and p21 functions. Although neither of the subgenomic HindIII fragments by themselves induced transformation, ligation of these two nontransforming DNAs to each other did restore p21-mediated transformation. A conclusion consistent with these results is that a region in the 5' half of the Ha-MuSV genome evolutionarily distinct from and not present in rat 30S RNA is essential for transformation and for p21 encoding.
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The spleen focus-forming virus (SFFV)-specific neoantigen shares cross-reactive determinants with mink cell focus-inducing (MCF) virus gp70.
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Animals↗
Guanine nucleotide-binding activity as an assay for src protein of rat-derived murine sarcoma viruses.
We have recently identified a 21,000-dalton protein, p21, coded for by Kirsten or Harvey murine sarcoma virus. On the basis of the results obtained with the p21 of a mutant of Kirsten sarcoma virus, temperature sensitive for the maintenance of transformation, we concluded that the p21 was required for the maintenance of transformation induced by either virus. We report herein that when extracts from cells transformed by Kirsten or Harvey sarcoma virus are incubated with [(3)H]GDP or [alpha-(32)P]GTP, picomole quantities of guanine nucleotide can be immunoprecipitated with antisera that contain antibodies to the p21. Previously we have shown that the immunoprecipitability of [(35)S]methionine-labeled p21 of the temperature-sensitive mutant of Kirsten sarcoma virus is thermolabile. The binding of guanine nucleotide is shown herein also to be thermolabile in extracts of cells transformed by the same mutant. However, the immunoprecipitability of the [(35)S]methionine-labeled p21 in such extracts of the temperature-sensitive mutant can be preserved if the extracts containing labeled p21 are incubated with added GDP or GTP prior to heating. The results suggest an interaction between p21 and certain guanine nucleotides, and the possible roles of guanine nucleotides and p21 in the maintenance of transformation are discussed.
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Different rat-derived transforming retroviruses code for an immunologically related intracellular phosphoprotein.
Kirsten sarcoma virus (Ki-MSV) and Harvey sarcoma virus (Ha-MSV) are mouse-rat recombinant viruses that were originally isolated by experimental inoculation of rats with helper-independent mouse type C viruses. We have recently identified in cells transformed by Ki-MSV or Ha-MSV, a phosphoprotein, p21, coded for by Ki-MSV and Ha-MSV [Shih, T.Y., Weeks, M.O., Young, H.A. & Scolnick, E.M. (1979) Virology 95, in press]. The p21, which is not a virion structural protein, was identified with antisera prepared by transplantation in rats of syngeneic Ha-MSV- or Ki-MSV-transformed nonproducer cells. In this study, we have applied the same methodology to examine a purely rat sarcoma virus (RaSV), which was isolated in cell culture by using helper-independent rat type C viruses [Rasheed, S., Gardner, M.B. & Huebner, R.J. (1978) Proc. Natl. Acad. Sci. USA 75, 2972-2976]. We report here that this new, purely rat sarcoma virus apparently codes for a p29, which shares immunological determinants and common V-8 protease-generated peptides with the p21 of Ha-MSV. The data suggest that the RaSV has acquired genetic information with similar coding capacity to some rat genetic information with similar coding combinant viruses, Ki-MSV and Ha-MSV. Based on data obtained on the p21 of a mutant of Ki-MSV temperature-sensitive for the maintenance of transformation, we suggest that the gene in RaSV that codes for the p29 is also required for the maintenance of RaSV-induced fibroblast transformation.
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Defective retrovirus-like 30S RNA species of rat and mouse cells are infectious if packaged by type C helper virus.
RNA species with properties of defective retrovirus-like 30S RNA genomes have previously been detected in both rats and mice and in some rat and mouse retroviruses. Using cell lines which express high levels of this retrovirus-like RNA, we formed pseudotypes of the 30S RNAs with helper-independent type C viruses. A pseudotype virus complex containing a mouse 30S subunit was transmitted to rat cells, and a pseudotype virus complex containing a rat 30S subunit was transmitted to bat cells. In other transmission experiments, a rat 30S subunit was isolated in nonproducer bat cells without detectable expression of the helper-independent type C virus used to pseudotype it. The results provide further support for the retrovirus-like nature of the rat 30S subunit and provide evidence which supports the protovirus hypothesis proposed by Temin.
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Characterization of a protein found in cells infected with the spleen focus-forming virus that shares immunological cross-reactivity with the gp70 found in mink cell focus-inducing virus particles.
Previously we detected an antigen in cells infected with the spleen focus-forming virus (SFFV) with a radioimmunoassay specific for the gp 70's of murine leukemia mink cell focus-inducing (MCF) viruses. This antigen has now been characterized in competition radioimmunoassays with limiting dilutions of antibody and in pulse-labeling studies under conditions of antibody excess. Both methods of analysis indicate that the SFFV-encoded antigen is a glycoprotein with a molecular weight of approximately 52,000. The gp52 shared immunological reactivity and methionine-containing tryptic peptides with the gp70 of a Friend MCF virus and was expressed on the surface of SFFV-infected cells as well as in the cytoplasm. The gp52 could be detected (i) in fibroblastic cell lines from several species when these cells were infected with SFFV; (ii) in several established erythroleukemic cell lines; and (iii) in the spleens of mice recently infected with SFFV. Although it shared immunochemical properties with the gp70 of Friend MCF virus, the gp52 could be distinguished from the MCF gp70 (i) by its apparent lack of group and interspecies immunological determinants compared with MCF virus-derived gp70's; (ii) by its failure to be released from cells infected with SFFV or SFFV plus helper virus; (iii) by its molecular weight; and (iv) by tryptic peptide analysis. The results indicate that SFFV codes for an MCF gp70-related gp52 which is apparently no longer a virion structural protein like the MCF gp70 from which it was originally derived.
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Nature and distribution of feline sarcoma virus nucleotide sequences.
The genomes of three independent isolates of feline sarcoma virus (FeSV) were compared by molecular hybridization techniques. Using complementary DNAs prepared from two strains, SM- and ST-FeSV, common complementary DNA'S were selected by sequential hybridization to FeSV and feline leukemia virus RNAs. These DNAs were shown to be highly related among the three independent sarcoma virus isolates. FeSV-specific complementary DNAs were prepared by selection for hybridization by the homologous FeSV RNA and against hybridization by fline leukemia virus RNA. Sarcoma virus-specific sequences of SM-FeSV were shown to differ from those of either ST- or GA-FeSV strains, whereas ST-FeSV-specific DNA shared extensive sequence homology with GA-FeSV. By molecular hybridization, each set of FeSV-specific sequences was demonstrated to be present in normal cat cellular DNA in approximately one copy per haploid genome and was conserved throughout Felidae. In contrast, FeSV-common sequences were present in multiple DNA copies and were found only in Mediterranean cats. The present results are consistent with the concept that each FeSV strain has arisen by a mechanism involving recombination between feline leukemia virus and cat cellular DNA sequences, the latter represented within the cat genome in a manner analogous to that of a cellular gene.
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Spleen focus-forming Friend virus: identification of genomic RNA and its relationship to helper virus RNA.
The genome of the defective, murine spleen focus-forming Friend virus (SFFV) was identified as a 50S RNA complex consisting of 32S RNA monomers. Electrophoretic mobility and the molecular weights of unique RNase T1-resistant oligonucleotides (T1-oligonucleotides) indicated that the 32S RNA had a complexity of about 7.4 kilobases. Hybridization with DNA complementary to Friend murine leukemia virus (Fr-MLV) has distinguished two sets of nucleotide sequences in 32S SFFV RNA, 74% which were Fr-MLV related and 26% which were SFFV specific. By the same method, SFFV RNA was 48% related to Moloney MLV. We have resolved 23 large T1-oligonucleotides of SFFV RNA and 43 of Fr-MLV RNA. On the basis of the relationship between SFFV and Fr-MLV RNAs, the 23 SFFV oligonucleotides fell into four classes: (i) seven which had homologous equivalents in Fr-MLV RNA; (ii) six more which could be isolated from SFFV RNA-Fr-MLV cDNA hybrids treated with RNases A and T1; (iii) eight more which were isolated from hybrids treated with RNases A and T1; and (iv) two which did not have Fr-MLV-related counterparts. Surprisingly, the two class iv oligonucleotides had homologous counterparts in the RNA of six amphotropic MLV's including mink cell focus-forming and HIX-MLVs analyzed previously. The map locations of the 23 SFFV T1-oligonucleotides relative to the 3' polyadenylic acid coordinate of SFFV RNA were deduced from the size of the smallest polyadenylic acid-tagged RNA fragment from which a given oligonucleotide was isolated. The resulting oligonucleotide map could be divided roughly into three segments: two terminal segments which are mosaics of oligonucleotides of classes i, ii, and iii and an internal segment between 2 and 2.5 kilobases from the 3' end containing the two oligonucleotides shared with amphotropic MLVs. Since SFFV RNA consists predominantly of sequence elements related to ecotropic and amphotropic helper-independent MLVs, it would appear that the transforming gene of SFFV is not a major specific sequence unrelated to genes of helper viruses, as is the case with Rous sarcoma and probably withe other defective sarcoma and acute leukemia viruses.
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p21 of Kirsten murine sarcoma virus is thermolabile in a viral mutant temperature sensitive for the maintenance of transformation.
We have recently described an intracellular protein, p21, in nonproducer cells transformed by either the Kirsten (Ki-MSV) or Harvey (Ha-MSV) strain of murine sarcoma virus (Shih et al., Virology, in press). The p21 is phosphorylated and has been shown to be coded for by either Ki-MSV or Ha-MSV. In this report, we compare the thermal stability of the newly synthesized [35S]methionine-labeled p21 in cells transformed by the wild-type Ki-MSV or by a mutant of Ki-MSV (ts 371) which is temperature sensitive in a viral function required for the maintenance of several properties of the transformed phenotype. The immunoprecipitability of the p21 coded for by the ts 371 Ki-MSV was markedly more thermolabile than the p21 of the wild-type Ki-MSV when the cell extracts are heated in vitro. The present finding suggests that the p21 is required for the maintenance of transformation induced by Ki-MSV.
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Heteroduplex analysis of the sequence relationships between the genomes of Kirsten and Harvey sarcoma viruses, their respective parental murine leukemia viruses, and the rat endogenous 30S RNA.
The sequence relations between Kirsten murine sarcoma virus (Ki-SV), Harvey murine sarcoma virus (Ha-SV), and a rat endogenous 30S RNA were studied by electron microscope heteroduplex analysis. The sequence relationships between the sarcoma viruses and their respective parental murine leukemia viruses (Kirsten and Moloney murine leukemia viruses), as well as between the two murine leukemia viruses, were also studied. The only observed nonhomology feature of the Kirsten murine leukemia virus/Moloney murine leukemia virus heteroduplexes was a substitution loop with two arms of equal length extending from 1.80 +/- 0.18 kilobases (kb) to 2.65 +/- 0.27 kb from the 3' end of the RNA. It is believed that this feature lies in the env gene region of the viral genomes. The Ha-SV and Moloney murine leukemia virus genomes (respective lengths, 6.0 and 9.0 kb) were homologous in a 1.0 +/- 0.05-kb region at the 3' end and possibly over a 200-nucleotide region at the 5' ends; otherwise, they were nonhomologous. Ha-SV and Ki-SV (length, 7.5 kb) were homologous in the first 4.36 +/- 0.37-kb region from the 3' end and in a 0.70 +/- 0.15-kb region at the 5' end. In between, there was a nonhomology region, possibly containing a short (0.23-kb) region of partial or total homology. The heteroduplex analysis between rat endogenous 30S RNA and Ki-SV shows that there are mixed regions of sequence homology and nonhomology at both the 5' and 3' ends. However, there is a large (4-kb) region of homology between Ki-SV and the rat 30S RNA in the center of the genomes, with only a small nonhomology hairpin feature. These studies help to define the regions of homology between the Ha-SV and Ki-SV genomes with each other and with the rat endogenous 30S RNA. These regions may be related to the sarcoma genicity of the viruses. In particular, the 0.7-kb region of homology of Ha-SV with Ki-SV at the 5' ends may be related to the formation of a 21,000-dalton phosphoprotein in cells transformed by either virus.
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Molecular cloning of the Harvey sarcoma virus closed circular DNA intermediates: initial structural and biological characterization.
Supercoiled Harvey sarcoma virus (Ha-SV) DNA was extracted from newly infected cells by the Hirt procedure, enriched by preparative agarose gel electrophoresis, and digested with EcoRI, which cleaved the viral DNA at a unique site. The linearized Ha-SV DNA was then inserted into lambda gtWESlambda B at the EcoRI site and cloned in an approved EK2 host. Ha-SV DNA inserts from six independently derived recombinant clones have been analyzed by restriction endonuclease digestion, molecular hybridization, electron microscopy, and infectivity. Four of the Ha-SV DNA inserts were identical, contained about 6.0 kilobase pairs (kbp), and comigrated in agarose gels with the infectious, unintegrated, linear Ha-SV DNA. One insert was approximately 0.65 kbp smaller (5.35 kbp) and one was approximately 0.65 kpb larger (6.65 kpb) than the 6.0 kpb inserts. R-looping with Ha-SV RNA revealed that the small (5.35 kbp) insert contained one copy of the Ha-SV RNA. Preliminary restriction endonuclease digestion of the recombinant DNAs suggested that the middle-size inserts contained a 0.65-kbp tandem duplication of sequences present only one in the small-size insert; this duplication corresponded to the 0.65-kpb terminal duplication of the unintegrated linear Ha-SV DNA. The large-size insert apparently contained a tandem triplication of these terminally located sequences. DNA of all three sized inserts induced foci in NIH 3T3 cells, and focus-forming activity could be rescued from the transformed cells by superinfection with helper virus. Infectivity followed single-hit kinetics, suggesting that the foci were induced by a single molecule.
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Helper-independent mink cell focus-inducing strains of Friend murine type-C virus: potential relationship to the origin of replication-defective spleen focus-forming virus.
Recent studies have indicated that both the replication-defective spleen focus-forming virus (SFFV) in the Friend virus complex and the helper-independent mink cell focus-inducing (MCF) viruses derived from AKR-murine leukemia virus (MuLV) are env gene recombinants between ecotropic virus and xenotropic virus. In an attempt to isolate additional env gene recombinants between Friend murine leukemia virus (F-MuLV) and xenotropic virus, we have inoculated cloned ecotropic F-MuLV into newborn NIH Swiss mice and analyzed MuLV released from preleukemic and leukemic spleens of infected mice. Two helper-independent MCF strains of F-MuLV have been isolated. Like the previously described AKR-MCF viruses, the Friend MCF viruses are env gene recombinants between an ecotropic virus (F-MuLV) and a mouse xenotropic virus, as shown by host range, interference pattern, and tryptic peptide analysis of the gp70s of these MuLV. Furthermore, RNA from the Friend MCF viruses hybridizes completely to cDNAsffv, a nucleic acid probe which detects that portion of SFFV which was not derived from P-MuLV. The ability to isolate replicating MCF viruses derived from F-MuLV FURTHER strengthens the parallels between the Friend erythroleukemia system and the AKR thymic leukemia system. Finally, the potential relationship of helper-independent env gene recombinants between F-MuLV and xenotropic virus to be highly leukemogenic SFFV is discussed.
AKR murine leukemia virus↗
Rapid leukemia induced by cloned friend strain of replicating murine type-C virus. Association with induction of xenotropic-related RNA sequences contained in spleen focus-forming virus.
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