Nucleotide sequence and formation of the transforming gene of a mouse sarcoma virus

Beveren, Charles Van; Galleshaw, J.; Jonas, Vivian; Berns, Anton; Doolittle, Russell F.; Donoghue, Daniel J.; Verma, Inder M.

doi:10.1038/289258a0

Cited by 193 publications

(115 citation statements)

References 39 publications

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“…However, together the helper and vector genomes still retain all essential viral functions and could generate competent virus by an illegitimate recombination. Detailed studies of Moloney murine sarcoma virus (47,48) and Moloney mink cell focus-forming virus (2) suggest that recombination can involve both cellular and viral information and that extensive nucleic acid sequence homology is not an absolute prerequisite for formation of recombinant virus. Although nonhomologous recombination is not well understood, it has been shown that unrelated DNAs transfected into L cells recombine close to regions of partial sequence homology (3).…”

Section: Resultsmentioning

confidence: 99%

Replication-defective chimeric helper proviruses and factors affecting generation of competent virus: expression of Moloney murine leukemia virus structural genes via the metallothionein promoter.

Bosselman¹,

Hsu²,

Bruszewski³

et al. 1987

Mol. Cell. Biol.

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Two chimeric helper proviruses were derived from the provirus of the ecotropic Moloney murine leukemia virus by replacing the 5' long terminal repeat and adjacent proviral sequences with the mouse metallothionein I promoter. One of these chimeric proviruses was designed to express the gag-pol genes of the virus, whereas the other was designed to express only the env gene. When transfected into NIH 3T3 cells, these helper proviruses failed to generate competent virus but did express Zn2 -inducible trans-acting viral functions needed to assemble infectious vectors. One helper cell line (clone 32) supported vector assembly at levels comparable to those supported by the Psi-2 and PA317 cell lines transfected with the same vector. Defective proviruses which carry the neomycin phosphotransferase gene and which lack overlapping sequence homology with the 5' end of the chimeric helper proviruses could be transfected into the helper cell line without generation of replication-competent virus. Mass cultures of transfected helper cells produced titers of about 104 G418r CFU/ml, whereas individual clones produced titers between 0 and 2.6 x 104 CFU/ml. In contrast, defective proviruses which share homologous overlapping viral sequences with the 5' end of the chimeric helper proviruses readily generated infectious virus when transfected into the helper cell line. The deletion of multiple cis-acting functions from the helper provirus and elimination of sequence homology overlapping at the 5' ends of helper and vector proviruses both contribute to the increased genetic stability of this system.

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Section: Resultsmentioning

confidence: 99%

Replication-defective chimeric helper proviruses and factors affecting generation of competent virus: expression of Moloney murine leukemia virus structural genes via the metallothionein promoter.

Bosselman¹,

Hsu²,

Bruszewski³

et al. 1987

Mol. Cell. Biol.

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“…Our data do not indicate whether strict conservation of this residue is required for stability or whether replacement with other hydrophobic residues might preserve protein stability and activity. For (39), Xenopus c-src (54), Drosophila src and ash (20), v-src (11,46,58), v-fqr (36), v-yes (26), v-ros (37), v-fps (52), v-fes (19), v-fms (18), c-fms (9), v-mil (v-mht) (25,56), v-raf(32), IskT (tck) (33,63), human insulin receptor (61), v-mos (62), human epidermal growth factor (EGF) receptor (12,61), c-erbB-2 (67), v-erbB (66), neu (2), and v-abl (44). The chicken c-src, v-src, v-fgr, v-yes, v-ros, v-fps, v-fes, v-abl, human EGF receptor, and v-erbB sequences were aligned as in Hunter and Cooper (21).…”

Section: Discussionmentioning

confidence: 99%

Features of the pp60v-src carboxyl terminus that are required for transformation.

Yaciuk¹,

Shalloway²

1986

Mol. Cell. Biol.

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Analysis of the biological and biochemical activities of pp6orecombinant-src proteins encoded by 12 carboxylterminal mutants showed that a wide family of alternate src carboxyl termini permit complete transforming and kinase activities. src proteins having carboxyl termini which are up to 10 amino acids longer than that of pp60 c-src (17 amino acids longer than that of pp6Ov-sc) still permit transformation. Transformation-positive mutations preserve leucine-516, a residue which is highly conserved in protein-tyrosine kinase sequences; removal causes in vivo protein instability. Successive deletion mutants show that this residue is at the boundary of a region required for kinase activity. pp6OSrC which is truncated just outside this point still transforms cells and binds both pp5O and pp9O cellular proteins.The v-src retroviral oncogene arose by transduction and modification of the c-src cellular gene (for a review, see reference 3). Unlike the viral product pp60v-", the cellular product pp6oc-rc does not induce transformation even when overexpressed in fibroblasts (23,40,50) although it cafi induce focus formation at high expression levels (24). This may be due to the reduced protein-tyrosine kinase activity of pp6Oc-s relative to pp6oV-"' (7,10,22). The catalytic domain for this activity resides in the carboxyl half of the molecule which is strongly homologous to domains in the other sequenced protein-tyrosine kinases (for a review, see reference 21). Two cellular phosphoproteins, pp90 (a major cytoplasmic heat shock protein [38]) and pp50, specifically interact with pp60v-" (4) much more extensively than with pp6Oc-s" (22).Structurally, the viral and cellular proteins differ by the substitution of 12 different carboxyl-terminal amino acids in pp6Ov-"" for the 19 carboxyl-terminal amino acids of pp6Oc-'( and, depending on the strain of v-src, 8 to 15 isolated substitutions (11,46,59) scattered throughout the remainder of the proteins. In pp60c'r', this carboxyl-terminal region contains the major site of in vivo tyrosine phosphorylation (6) which may play a role in negatively regulating pp60c-Xrc protein-tyrosine kinase activity (8). Most of the DNA sequence encoding the v-src carboxyl terminus is found in the chicken genome about 900 base pairs (bp) downstream from the c-src termination codon (57, 59).The functional significance of the existence of multiple mutations between v-src and c-src has not yet been resolved.Single point mutations in pp60c-%'r enable it to transform chicken embryo cells (J. B. Levy, H. Iba, and H. Hanafusa, Proc. Natl. Acad. Sci. USA, in press), and chimeric genes encoding the amino region of pp60c-A' and carboxyl region of pp60'r-"' (clv-src chimeras) or encoding the amino region of pp60v-s and the carboxyl region of pp60c-r (vlc-src chime- (49).We studied the carboxyl-terminal region using a series of deletion, substitution, and addition mutants of SR-D pp60"vA(. We report that a wide variety of SR-D pp6O-vS(' carboxyl-terminal mutants can transform fibroblasts efficiently and comple...

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“…Not all viral transforming proteins that display associated kinase activity phosphorylate tyrosine. The guanine nucleotide-binding protein (p21) encoded by Harvey MuSV v-mos gene autophosphorylates at threonine (30), A computerized search for similarities among the predicted amino acid sequences of avian sarcoma virus p6src, the MoMuSV v-mos gene product first observed by Van Beveren et al (2), and the catalytic subunit ofcAMP-dependent protein kinase has revealed potential ATP-binding sites in all three proteins (31). Such a binding'site within the mos gene is consistent with our results which would suggest that the mos portion of P85gag-ms is a protein kinase.…”

Section: Discussionmentioning

confidence: 99%

P85 ^gag-mos encoded by ts110 Moloney murine sarcoma virus has an associated protein kinase activity

Kloetzer

Maxwell

Arlinghaus

1983

Proc. Natl. Acad. Sci. U.S.A.

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A protein identified as P859'9-°was shown to be phosphorylated when immunoprecipitates from tsllO Moloney murine sarcoma virus transformed nonproducer cells (clone 6m2) were incubated with [y-32P]ATP. The in vitro-labeled 85,000-dalton phosphoprotein comigrated on NaDodSO4/polyacrylamide gels with authentic phosphorylated P859'9-°. Immunoprecipitates obtained with antisera prepared against Rauscher murine leukemia virus core protein p30 were active in the immune complex kinase assay but anti-murine leukemia virus plO precipitates were not. Previous studies have shown that anti-p30 but not antiplO antisera recognize P859'9-°. The 6m2 clone has been shown to express P85gag-"w8 at 330C but not at 39TC. Anti-p30 immune complexes from 6m2 cells maintained at 390C failed to phosphorylate the 85,000-dalton protein. Furthermore, the in vitro phosphorylated 85,000-dalton protein gave the same pattern of V8 protease-generated cleavage products as in vivo 32P-labeled P85gag-'.8, We conclude from these results that P85gag`m06 is phosphorylated in anti-p30 immune complex kinase reactions. Phosphoamino acid analyses indicated that the in vitro phosphorylated P85gag-tno contained phosphoserine and phosphothreonine. Our findings indicate that incubation of anti-p30 immunoprecipitates at 39°C drastically reduced, in a specific way, the kinase activity associated with P85gag-1l8. This result and other data suggest that the kinase is virus-encoded. Because P85gag-"1w8, but not Pr65gag, is phosphorylated in anti-p30 immunoprecipitates from MuLVMuSV tsllO producer cells, the kinase enzyme is associated with P85gag-' and not gag gene products. A second major polypeptide of the size of P58gag was also phosphorylated in anti-p30 immunoprecipitates from cells maintained at 33°C but not at 39°C. Since 6m2 cells at 39°C contain P58wag, this is also consistent with the kinase activity being associated with P85gag-11W.

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Nucleotide sequence and formation of the transforming gene of a mouse sarcoma virus

Cited by 193 publications

References 39 publications

Replication-defective chimeric helper proviruses and factors affecting generation of competent virus: expression of Moloney murine leukemia virus structural genes via the metallothionein promoter.

Replication-defective chimeric helper proviruses and factors affecting generation of competent virus: expression of Moloney murine leukemia virus structural genes via the metallothionein promoter.

Features of the pp60v-src carboxyl terminus that are required for transformation.

P85 ^gag-mos encoded by ts110 Moloney murine sarcoma virus has an associated protein kinase activity

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Nucleotide sequence and formation of the transforming gene of a mouse sarcoma virus

Cited by 193 publications

References 39 publications

Replication-defective chimeric helper proviruses and factors affecting generation of competent virus: expression of Moloney murine leukemia virus structural genes via the metallothionein promoter.

Replication-defective chimeric helper proviruses and factors affecting generation of competent virus: expression of Moloney murine leukemia virus structural genes via the metallothionein promoter.

Features of the pp60v-src carboxyl terminus that are required for transformation.

P85 gag-mos encoded by ts110 Moloney murine sarcoma virus has an associated protein kinase activity

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P85 ^gag-mos encoded by ts110 Moloney murine sarcoma virus has an associated protein kinase activity