ANALYSIS OF ROUS-SARCOMA VIRUS GAG PROTEINS 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 protot ype of the avian sarcoma and leukemia viruses. Molecular masses determ ined for the mature virion proteins MA, CA, NC, and PR agree closely w ith those predicted by currently accepted models for their structures. However, the data for p10 imply that only about 10% of the product ha s the predicted mass while the remainder is missing the C-terminal met hionine residue. Molecular masses also were obtained for products gene rated by PR cleavage in vitro of a Gag precursor polyprotein expressed in Escherichia coli. The data confirm the predicted Gag cleavage site s for PR. Thus, carboxypeptidase activity appears to be responsible fo r generating the des-Met form of p10. The same activity may account fo r the small amount of the mature des-Met CA, as previously reported. A nalysis of cleavage products generated in vitro also serves to define the PR processing site separating the p2a and p2b peptides, Asn-164-Cy s-165. In conjunction with published characterizations of these two pe ptides processed from the segment of Gag between RIA and pill, these d ata suggest trimming of p2b by an aminopeptidase. Finally, the molecul ar masses determined for the MA-related species p19f, p23, and p35 now accurately define the structures of these proteins.