The NH 2-terminal sequence of the avian oncovirus gag precursor polyprotein (Pr76 gag)

Abstract

Pr76 gag, the polyprotein precursor to avian oncovirus internal structural proteins ( gag proteins) was isolated by immunoprecipitation from a mRNA-dependent in vitro translation system programmed with genomic (35 S) RNA purified from the Prague strain of Rous sarcoma virus (PR-RSV-C). Attempts to sequence Pr76 were initially unsuccessful due to the presence of a blocked NH 2-terminus. However, when virion 35 S RNA was translated under conditions that prevent NH 2-terminal acetylation, the sequence: Met-Glu-Ala-Val-Ile-Lys-Val-Ile-X-X-Ala-X-Lys was obtained by automated Edman degradation. Since the NH 2-terminal methionine is derived from Met-tRNA f Met we conclude that this sequence repesents a primary translation product. Sequence analysis of the supernatant remaining after immunoprecipitation suggests that no viral-related proteins are synthesized which are not precipitated by anti- gag serum. Previous studies employing pactamycin mapping of viral proteins synthesized in infected chick cells indicated that the virion protein p19 was located close to the NH 2-terminus of Pr76 gag (Vogt, et al., J. Mol. Biol. 96, 471–493, 1975). We therefore prepared tryptic peptides of p19 purified from virions, separated them by cation exchange chromatography, and determined the amino acid composition of the 19a and 19d peptides. The amino acid composition of the 19a, but not the 19d, peptide correlated with the first six amino acids determined by sequencing. In addition, 19a was the onyl p19-derived peptide not susceptible to digestion by leucine amino peptidase, indicating the presence of a blocked NH 2-terminus. We conclude that the NH 2-terminal sequence of Pr76 gag represents the NH 2-terminus of p19. Comparison of the amino acid sequence with the previously published nucleotide sequence of the 5′ end of 35S RNA from PR-RSV-C (Haseltine et al., Proc. Nat. Acad. Sci. USA 74, 989–993, 1977; Shine et al., Proc. Nat. Acad. Sci. USA 74, 1473–1477, 1977) shows that synthesis of Pr76 gag is not initiated within the first 119 nucleotides of the viral genome.