POINT MUTATION IN AVIAN-SARCOMA LEUKEMIA-VIRUS PROTEASE WHICH INCREASES ITS ACTIVITY BUT IMPAIRS INFECTIOUS VIRUS PRODUCTION

The retrovirus protease (PR), an aspartic PR, is composed of two ident ical subunits, each containing a conserved tripeptide sequence present at the active site of the enzyme. Asp-Ser-Gly is found in avian sarco ma leukaemia viruses (ASLV) and Asp-Thr-Gly in mammalian oncoretroviru ses. We have mutated the conserved sequence at the active site of ASLV PR by converting the Ser Gly residues to Thr and Ala, respectively. R eplacement of Gly with Ala yielded an ASLV PR devoid of proteolytic ac tivity. The Ser to Thr conversion did not alter the substrate specific ity of the enzyme. Both wild-type and mutated PRs correctly cleaved vi ral precursors expressed in bacterial cells, as well as synthetic pept ides homologous to ASLV and human immunodeficiency virus type 1 cleava ge sites. Bacterially produced ASLV PR with Thr instead of Ser had inc reased enzymatic activity, as shown by hydrolysis of synthetic peptide s. However, this mutation reduced the production of reverse transcript ase-containing particles and infectious virus following transfection o f permissive cells with virus DNA.