Generation of avian myeloblastosis virus structural proteins by proteolytic cleavage of a precursor polypeptide

Vogt, Volker M.; Eisenman, Robert N.; Diggelmann, Heidi

doi:10.1016/0022-2836(75)90174-6

Cited by 295 publications

(153 citation statements)

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“…Other virus-specific proteins in these immunoprecipitates are the precursors to virus structural proteins (15), pr76 and prl80. Because the lysates were precipitated with sera against structural proteins before immunoprecipitation with the sera from tumor-bearing animals, the precursors pr66 and pr62 (15) :P2p043-labeled cell extracts and detection of kinase activity. Radioactively labeled lysates (buffer B) were adjusted to contain (within 10%) similar specific radioactivities by adding unlabeled extracts.…”

Section: Methodsmentioning

confidence: 99%

src gene product from different strains of avain sarcoma virus: Kinetics and possible mechanism of heat inactivation of protein kinase activity from cells infected by transformation-defective, temperature-sensitive mutant and wild-type virus

Rübsamen

Friis

Bauer

1979

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

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Sera from certain rabbits bearing SchmidtRuppin strain Rous sarcoma virus (RSV)induced tumors precipitated p6Osrc from chicken cells transformed by the homologous virus as well as by other strains [Prague strain RSV, Bryan high-titer strain RSV, and Bratislava 77 strain of avian sarcoma virus (ASV)J, the molecular weights (Mrs) In order to test whether the protein kinase reaction is directly catalyzed by p6Osrc, we compared the in vitro temperature sensitivities of the kinase activities from cells infected by transformation-temperature-sensitive mutant and parental wild-type virus. The first-order rate constant for the inactivation of the kinase from extracts of cells infected by the mutant virus was 2-fold greater than that from cells infected by wild-type virus. This result implicates the protein kinase as an enzymatic activity of the src gene product, the p60src. Concomitant with the loss of the kinase activity by heat inactivation, p60src loses 60-70% of its phosphate content. The kinetics of dephosphorylation exactly parallel those for the inactivation of the kinase activity, suggesting that the p6osrc kinase is itself dependent on phosphorylation for its activity. In recent papers, Erikson and coworkers have shown that sera from rabbits bearing tumors induced by the Schmidt-Ruppin (SR) strain of Rous sarcoma virus (RSV) precipitated a sarcoma virus-specific protein of molecular weight (Mr) 60,000 from extracts of SR-transformed chicken embryo cells (1), as well as from various mammalian cells transformed by the same virus (2). The in vitro cell-free translation of the 3' one-third of the RSV genome, the region known to. contain the viral transforming information [the src gene (3, 4)1, yielded a protein of the same molecular weight and similar peptide composition (5, 6). Hence, this protein is thought to be responsible for RSVinduced cell transformation and has been named p6Osrc. A protein kinase activity is associated with the p6Osrc, because immunoprecipitates of p60src incorporate 32P from [y-32P]ATP into the Mr 53,000 subunit of IgG (7). This activity was greatly reduced (though not completely lost) in immunoprecipitates prepared from cells that were infected by a transformationdefective temperature-sensitive (ts) mutant of avian sarcoma virus (ASV) when the cells were grown at the nonpermissive temperature for transformation. These data were interpreted to indicate that the kinase activity found in immunoprecipitates of p60src is an enzymatic activity of the protein and not of a contaminant (7).In the present study we show that p60srC can be precipitated by certain rabbit sera from extracts of cells infected by several different strains of ASV and that all these immunoprecipitates exhibit a kinase activity. p60src proteins from cells infected with wild-type and transformation-defective (T class) ts mutants of RSV were compared with respect to protein kinase inactivation kinetics at 42°C. The results obtained show that, after in vitro incubations, the protein kinase activity from cells infecte...

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Section: Methodsmentioning

confidence: 99%

src gene product from different strains of avain sarcoma virus: Kinetics and possible mechanism of heat inactivation of protein kinase activity from cells infected by transformation-defective, temperature-sensitive mutant and wild-type virus

Rübsamen

Friis

Bauer

1979

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

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“…3(a) are shown the results obtained using antiserum to detergentdisrupted A M V virions. Two major virus-specific polypeptides were immunoprecipitated under these conditions: Pr76gao, the major product, and a cleavage intermediate Pr66 gag (Vogt et al, 1975). Assuming that the amount of incorporation is proportional to the amount of m R N A present, we calculated a half-life for gag m R N A (Fig.…”

Section: Decay Of Cellular and B77 Asv Mrna Activities After The Addimentioning

confidence: 99%

Stabilities of Avian Sarcoma Virus RNAs: Comparison of Subgenomic and Genomic Species with Cellular mRNAs

Dimock

Horikami

et al. 1983

Journal of General Virology

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SUMMARYThe stabilities of B77 avian sarcoma virus intracellular RNAs were compared to the stability of the total cellular poly(A)-containing RNA by labelling infected chicken embryo fibroblasts with [3H]uridine for 15 h, adding actinomycin D (1 ~tg per ml) to block further transcription of viral RNA, and selecting virus-specific RNA from the total cellular poly(A)-containing RNA at 3 hourly intervals. The three virus-specific RNA species (9-3, 3.3 and 5.4 kilobases) decayed with half-lives of 7-5, 10, and 15 h, respectively, whereas the bulk of the cellular mRNA decayed with a half-life of 13 h. To correlate these decay rates with the disappearance of mRNA activities, the actinomycin D-treated cells were pulse-labelled with [3H]leucine at 3 hourly intervals after the addition of the drug and virus-specific protein synthesis was assayed by immunoprecipitation. The mRNA activity for the precursor to the non-glycosylated viral structural proteins (Pr76g ag) decayed with a half-life of approximately 6 h, whereas the mRNA activity coding for the precursor to the envelope proteins (gPr92 env) decayed with a half-life of 14 h. Thus, the rate of decay of the individual mRNA species corresponded reasonably well with the decay rate for the synthesis of two of the corresponding gene products. The results indicated that the 5-4 kb env mRNA is more stable under these conditions than the 9.3 kb gag mRNA but was not significantly more stable than the bulk of the cellular mRNA. Virus particle production following the addition of actinomycin D was determined by the reverse transcriptase assay and by the incorporation of viral genomic 70S RNA into extracellular virions. Both assays yielded similar results and indicated that particle production was inhibited at a rate (t~ = 4 h) somewhat faster than the decay of Pr769ag synthesis or the disappearance of 9.3 kb RNA. It was established by two independent methods (pulse and chase, and approach to isotope equilibrium), however, that the intracellular halflife of the RNA that is packaged into virions is 6 to 7 h. Tt~us, these results suggest that a single metabolic pool of 9.3 kb RNA exists in avian sarcoma virus-infected cells and is used both as mRNA and as genome RNA.

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“…Radioactivity was located with Du Pont Cronex 4 medical x-ray film, or with Kodak X-Omat R film for fluorography. To locate the polypeptides in preparative gels, we dried a small strip cut from the edge of the gel after it had been destained and exposed it to film, while the remainder of the gel was stored in water at 0 For tryptic fingerprinting, the pertinent regions were cut from preparative gels and the polypeptides were eluted, precipitated with trichloroacetic acid after the addition of 100 Mug of bovine serum albumin, and oxidized with performic acid (27). After two lyophilizations, the polypeptides were digested with trypsin and the peptides were separated by ascending chromatography followed by electrophoresis as described (25).…”

mentioning

confidence: 99%

Identification of a polypeptide encoded by the avian sarcoma virus src gene.

Purchio¹,

Erikson²,

Brugge³

et al. 1978

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

224

102

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Two techniques were used to search for the polypeptide encoded by the avian sarcoma virus (ASV) src gene. First, antiserum from rabbits bearing ASV-induced fibrosarcomas was used to immunoprecipitate a transformation-specific antigen from ASV-transformed chick embryo fibroblasts. This antigen has an apparent molecular weight (Mr) of 60,000. Second, the 3' one-third of the ASV genome, selected by oligo(dTcellulose chromatography and sucrose gradient sedimentation, was translated in a mRNA-dependent reticulocyte cell-free lysate. This RNA species programmed the synthesis of a polypeptide that comigrated with the transformation-specific antigen of Mr 60,000 immunoprecipitated from transformed cells. The methionine-containing tryptic peptides from the polypeptides of Mr 60,000 obtained from translation in vitro and from immunoprecipitation were found to be identical upon twodimensional fractionation.Some RNA-containing tumor viruses are able to cause rapid and efficient transformation of cells in culture as well as the production of sarcomas in animals. The avian sarcoma viruses (ASV) are particularly interesting because they produce tumors in both avian and mammalian species (1, 2). The high efficiency of cell transformation by these viruses, the availability of viral mutants, and the considerable structural information about the viral genome make ASV useful for the study of the molecular events that occur during viral oncogenesis.Although analyses of viral mutants indicate that a polypeptide encoded by the src gene of ASV is likely to be responsible for the maintenance of cell transformation, such a polypeptide had not been identified biochemically or immunologically when we began our studies. Several ASV conditional, temperature-sensitive (ts) mutants, and nonconditional, deletion mutants, termed transformation-defective (td), have been isolated that have an altered capacity to transform cells (3-5). Studies with ts mutant-infected cells indicate that a virus-encoded product affects gross cytoskeletal structure, growth in soft agar, and rate of sugar uptake, characteristics that are phenotypic markers of transformation in culture. Avian or mammalian cells infected with a ts mutant can be reversibly switched to either the transformed or "normal" state within a few-hours after the appropriate temperature shift. Inhibitors of protein synthesis do not affect the conversion from the transformed to normal state upon shift to the nonpermissive temperature, but do interfere with the appearance of the transformed phenotype when cultures are shifted from the nonpermissive to permissive temperature. These virus mutants can replicate normally at both temperatures, suggesting that a virus-encoded, heat-labile protein, which is not an essential structural component of the virus, is responsible for cell transformation (6).All ts transformation mutations occur in a region of the ASVThe costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "...

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Generation of avian myeloblastosis virus structural proteins by proteolytic cleavage of a precursor polypeptide

Cited by 295 publications

References 44 publications

src gene product from different strains of avain sarcoma virus: Kinetics and possible mechanism of heat inactivation of protein kinase activity from cells infected by transformation-defective, temperature-sensitive mutant and wild-type virus

src gene product from different strains of avain sarcoma virus: Kinetics and possible mechanism of heat inactivation of protein kinase activity from cells infected by transformation-defective, temperature-sensitive mutant and wild-type virus

Stabilities of Avian Sarcoma Virus RNAs: Comparison of Subgenomic and Genomic Species with Cellular mRNAs

Identification of a polypeptide encoded by the avian sarcoma virus src gene.

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