Isolation and characterization of proteins from Rous sarcoma virus

Hung, Paul P.; Robinson, Harriet L.; Robinson, William S.

doi:10.1016/0042-6822(71)90243-1

Cited by 88 publications

(58 citation statements)

References 24 publications

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“…This procedure was repeated once a day for several days, with the labeled medium being used again each time, until at least three-quarters of the label had been incorporated. The spent labeled and unlabeled media were then pooled, and the labeled virus was purified by banding to equilibrium in sucrose gradients (2). For preparation of individual virion polypeptides, the purified virus was dialyzed against ammonium carbonate (pH 8.5), lyophilized, redissolved in buffer containing dodecyl sulfate, and electrophoresed as described below.…”

Section: Methodsmentioning

confidence: 99%

“…In the avian oncornaviruses, two of the proteins account for 60% of the total aminoacid label in virions. These two proteins (2), and perhaps two or three lesser ones, are the so-called group-specific antigens that carry determinants common to all avian oncornaviruses (3). Two or three additional proteins with carbohydrate moieties (1, 2, 4) are found on the surface of the virus particles (5) and appear to carry the type specificity (4).…”

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confidence: 99%

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Identification of a Large Polypeptide Precursor of Avian Oncornavirus Proteins

Vogt¹,

Eisenman²

1973

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

104

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Antibody to partially disrupted avian myeloblastosis virus was used to selectively precipitate newly synthesized intracellular viral polypeptides from extracts of infected chicken cells. When analyzed by sodium dodecyl sulfategel electrophoresis, immune precipitates from extracts of cells pulse-labeled for 10 min with [36SImethionine contain none of the major virion polypeptides. Instead they show prominent viral specific polypeptides of molecular weight 76,000 and 12,000, as well as minor quantities of other labeled polypeptides. From pulse-chase kinetics and two-dimensional tryptic fingerprints it appears that the large polypeptide is a precursor of at least the two major virion proteins of molecular weights 24,000 and 11,000, while the smaller is a precursor of the 11,000-dalton virion protein.The oncornaviruses, or RNA tumor viruses, consist of singlestranded RNA encapsidated by four to eight major proteins (1, 2, 2a) and a lipid-containing coat. In the avian oncornaviruses, two of the proteins account for 60% of the total aminoacid label in virions. These two proteins (2), and perhaps two or three lesser ones, are the so-called group-specific antigens that carry determinants common to all avian oncornaviruses (3). Two or three additional proteins with carbohydrate moieties (1, 2, 4) are found on the surface of the virus particles (5) and appear to carry the type specificity (4).Little is known about the synthesis of oncornavirus proteins. Cells infected with these viruses continue to grow, devoting only a small percentage of cellular protein synthesis to viral proteins. Thus, an investigation of viral-specific translation requires a specific probe. We have prepared a rabbit antiserum to disrupted avian myeloblastosis virus (AMV) and have used this antiserum to selectively precipitate intracellular viral polypeptides from extracts of infected cells labeled with amino acids. From the analysis of these immune precipitates, we conclude that at least the two most numerous AMV proteins are synthesized together as a single large precursor polypeptide. MATERIALS AND METHODSGrowth and Labeling of Cells and Virus. Primary fibroblasts from 11-day leukosis-free chick embryos (Villejuif, Paris) were infected in suspension by incubation of 50 uA of leukemic chicken serum (approximate titer 1012 AMV particles per ml) with 107 cells in 1 ml of Dulbecco's modified Eagle's medium containing 10% calf serum. After 45 min at 370, the cells were diluted with the same medium and serum and seeded onto 10-cm plastic dishes. On the third to fifth day after seeding, cells were washed twice with 5 ml of warm phosphatebuffered saline (pH 7.4) and then incubated for 2 hr with modified Eagle's medium containing 5% dialyzed calf serum and 0.02 mM unlabeled or 3H-labeled methionine (50 ,Ci/ml).Subsequent to this long-term labeling, the cell monolayer was washed as before and then incubated for 10 min at 370 with 1.5 ml of Earle's saline (6) containing [35S]methionine (about 100 Ci/mmol) at 30 uCi/m], or 500 MCi/ml for preparation o...

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Section: Methodsmentioning

confidence: 99%

mentioning

confidence: 99%

Identification of a Large Polypeptide Precursor of Avian Oncornavirus Proteins

Vogt¹,

Eisenman²

1973

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

104

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“…RSV(RAV-1) was recovered from large volumes of culture medium and purified (27); unlabeled viral RNA was extracted and purified (28). Sendai virus RNA was isolated and purified as described by Robinson (29).…”

Section: Methodsmentioning

confidence: 99%

“…It consisted of medium 199 free of purines, pyrimidines, ATP, and AMP (GIBCO, Berkeley, Calif.) and was supplemented with 1% dialyzed chicken serum, 4% dialyzed calf serum, 1% tryptose phosphate broth, 0.12%0o HEPES buffer (pH 7.5), and 60 ,uCi/ml each of [5-3H]uridine, [2,8-3H]adenosine, and [5-3H]cytidine (specific activities 20-30 Ci/mmol). Medium from the first 12 hr was discarded and virus was purified from the remainder by three successive purifications on discontinuous sucrose gradients (27). [3H]RNA was extracted from the purified virus by the sodium dodecyl sulfate-phenol method (28) DNA-RNA Hybridization.…”

Section: Methodsmentioning

confidence: 99%

DNA in Uninfected and Virus-Infected Cells Complementary to Avian Tumor Virus RNA

Rosenthal

Robinson

et al. 1971

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

Self Cite

100

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The 70S RNA component of several avian tumor viruses was hybridized with DNA extracted from avian tumor virus-infected and uninfected chicken and Japanese quail cells. Tritium-labeled 70S RNAs from Rous sarcoma virus (RSV), Rous associated vinus-I (RAV-1), RAV-60, and Schmidt-Ruppin-RSV (SR-RSV) hybridize from 3 to 10 times more with DNA from uninfected chicken cells than with DNA from Escherichia coli, calfthymus, or baby hamster kidney cells. (24) and was grown on K813-C/B chicken-embryo fibroblasts.

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“…Isoelectric~focusing may be used for analytical or preparative separation from heterogenous mixtures of individual ampholytes, particularly proteins, and the technique has recently been extended to the study of the protein components of viruses (Shortridge & Biddle, 197o;Hung, Robinson & Robinson, 1971). The technique offers almost complete recovery of total protein after separation.…”

Section: Electrofocusing Of Hepatitis B Antigenmentioning

confidence: 99%