ANALYSIS OF CLEAVAGE SITE MUTATIONS BETWEEN THE NC AND PR GAG DOMAINSOF ROUS-SARCOMA VIRUS

In retroviruses, the viral protease (PR) is released as a mature prote in by cleavage of Gag, Gag-Pro, or Gag-Pro-Pol precursor polypeptides. In avian sarcoma and leukemia viruses (ASLV), PR farms the C-terminal domain of Gag, Based on the properties of a mutation (cs22) in the cl eavage site between the upstream NC domain and the PR domain, the prot eolytic liberation of PR previously was inferred to he essential for p rocessing of Gag and Pol proteins. To study this process in more detai l, we have analyzed the effects that several mutations at the NC-PR cl eavage site have on proteolytic processing in virus-like particles exp ressed in COS and quail cells, Mutant Gag proteins carrying the same m utations also were synthesized in vitro and tested for processing with purified PR, In both types of studies, N-terminal sequencing of the l iberated PR domain was carried out to exactly identify the site of cle avage, Finally, synthetic peptides corresponding to the mutant protein s were assessed for the ability to act as substrates for PR The result s were all consistent and led to the following conclusions, (i) In viv o, if normal processing between NC and PR is prevented by mutations, l imited cleavage occurs at a previously unrecognized alternative site t hree amino acids downstream, i.e., in PR, This N-terminally truncated PR is inactive as an enzyme, as inferred from the global processing de fect in cs22 and a similar mutant, iii) In Gag proteins translated in vitro, purified PR cleaves this alternative site as rapidly as it does the wild-type site, (iii) Contrary to previously accepted rules descr ibing retroviral cleavage sites, an isoleucine residue placed at the P 1 position of the NC-PR cleavage site does not hinder normal processin g, (iv) A proline residue placed at the P2 position in this cleavage s ite blocks normal processing.