PROGRAMMING THE ROUS-SARCOMA VIRUS PROTEASE TO CLEAVE NEW SUBSTRATE SEQUENCES

The Rous sarcoma virus protease displays a high degree of specificity and catalyzes the cleavage of only a limited number of amino acid sequ ences. This specificity is governed by interactions between side chain s of eight substrate amino acids and eight corresponding subsite pocke ts within the homodimeric enzyme, We have examined these complex inter actions in order to learn how to introduce changes into the retroviral protease (PR) that direct it to cleave new substrates. Mutant enzymes with altered substrate specificity and wild-type or greater catalytic rates have been constructed previously by substituting single key ami no acids in each of the eight enzyme subsites with those residues foun d in structurally related positions of human immunodeficiency virus (H IV)-1 PR. These individual amino acid substitutions have now been comb ined into one enzyme, resulting in a highly active mutant Rous sarcoma virus (RSV) protease that displays many characteristics associated wi th the HIV-1 enzyme, The hybrid protease is capable of catalyzing the cleavage of a set of HIV-1 viral polyprotein substrates that are not r ecognized by the wild-type RSV enzyme. Additionally, the modified PR i s inhibited completely by the HIV-1 PR-specific inhibitor KNI-272 at c oncentrations where wild-type RSV PR is unaffected, These results indi cate that the major determinants that dictate RSV and HIV-1 PR substra te specificity have been identified, Since the viral protease is a hom odimer, the rational design of enzymes with altered specificity also r equires a thorough understanding of the importance of enzyme symmetry in substrate selection, We demonstrate here that the enzyme homodimer acts symmetrically in substrate selection with each enzyme subunit bei ng capable of recognizing both halves of a peptide substrate equally.