Analysis of Rous sarcoma virus Gag protein by mass spectrometry indicates trimming by host exopeptidase.
We have used electrospray ionization-mass spectrometry to investigate Gag protein structure and processing in Rous sarcoma virus, the prototype of the avian sarcoma and leukemia viruses. Molecular masses determined for the mature virion proteins MA, CA, NC, and PR agree closely with those predicted by currently accepted models for their structures. However, the data for p10 imply that only about 10% of the product has the predicted mass while the remainder is missing the C-terminal methionine residue. Molecular masses also were obtained for products generated by PR cleavage in vitro of a Gag precursor polyprotein expressed in Escherichia coli. The data confirm the predicted Gag cleavage sites for PR. Thus, carboxypeptidase activity appears to be responsible for generating the des-Met form of p10. The same activity may account for the small amount of the mature des-Met CA, as previously reported. Analysis of cleavage products generated in vitro also serves to define the PR processing site separating the p2a and p2b peptides, Asn-164-Cys-165. In conjunction with published characterizations of these two peptides processed from the segment of Gag between MA and p10, these data suggest trimming of p2b by an aminopeptidase. Finally, the molecular masses determined for the MA-related species p19f, p23, and p35 now accurately define the structures of these proteins.
Pepinsky
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RB
Department of Protein Chemistry, Biogen, Inc., Cambridge, Massachusetts 02142, USA.
Papayannopoulos
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IA
Campbell
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Vogt
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VM
D000595:Amino Acid Sequence; D000818:Animals; D001358:Avian Sarcoma Viruses / Q000378:metabolism*; D002851:Chromatography, High Pressure Liquid; D003001:Cloning, Molecular; D004926:Escherichia coli; D020689:Exopeptidases; D015683:Gene Products, gag / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D015750:Genes, gag; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010446:Peptide Fragments / Q000737:chemistry / Q000302:isolation & purification; D010447:Peptide Hydrolases / Q000378:metabolism*; D011499:Protein Processing, Post-Translational; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D014771:Virion / Q000378:metabolism
D015683:Gene Products, gag; D010446:Peptide Fragments; D011994:Recombinant Proteins; D020689:Exopeptidases; D010447:Peptide Hydrolases