Isolation and characterization of proteins from Rous sarcoma virus

Abstract

When the Bryan high-titer strain of Rous sarcoma virus RSV (RAV-1) labeled with a mixture of 14C amino acids was dissociated with a neutral detergent (Brij 35), mercaptoethanol, and urea and analyzed by isoelectric focusing in urea, seven radioactive peaks with pIs between 3.5 and 9.9 were found. The peaks were further analyzed by polyacrylamide gel electrophoresis in SDS to determine the number and molecular weight of the individual protein components in each peak. A total of eight amino acid- 14C-labeled proteins were identified in RSV (RAV-1) with molecular weights between 14,000 and 96,000 daltons. When 3H-glucosamine labeled RSV (RAV-1) was dissociated with SDS and the viral components separated by SDS-gel electrophoresis, four radioactive components were observed. Analysis of glucosamine- 3H-labeled virus together with amino acid- 14C-labeled virus by dissociation with Brij 35, urea, and mercaptoethanol and separation of components by isoelectric focusing followed by electrophoresis in SDS-containing gels revealed that three of the glucosamine labeled components were glycoproteins corresponding to the three largest amino acid-labeled proteins. The fourth glucosamine-labeled component did did not contain radioactive amino acids suggesting that it was protein-free carbohydrate. Separation of virion components on Bio-Gel columns revealed that the glycoproteins were released from virus with Brij 35, urea, and mercaptoethanol in a large complex which was further dissociated into smaller units by SDS. Two of the eight protein components had high complement fixation titers and formed crossing precipitin lines in agar gel diffusion with hamster antiserum to the avian tumor virus group-specific antigen.