Ž . Biochimica et Biophysica Acta 1339 1997 113–125 Escape mutants of HIV-1 proteinase: enzymic efficiency and susceptibility to inhibition Sara I. Wilson a , Lowri H. Phylip a , John S. Mills b , Sergei V. Gulnik c , John W. Erickson c , Ben M. Dunn d , John Kay a, ) a School of Molecular and Medical Biosciences, UniÕersity of Wales College of Cardiff, P.O. Box 911, Cardiff, CF1 3US, UK b Roche Research Centre, Welwyn Garden City, Herts. AL7 3AY, UK c Structural Biochemistry Program, NCI-Frederick Cancer Research and DeÕelopment Center, Frederick, MD 21702, USA d Department of Biochemistry and Molecular Biology, J. Hillis Miller Health Center, UniÕersity of Florida, GainesÕille, FL 32610, USA Received 30 September 1996; revised 5 December 1996; accepted 5 December 1996 Abstract Genes encoding a number of mutants of HIV-1 proteinase were sub-cloned and expressed in E. coli. The proteinases Ž . containing mutations of single residues e.g., G48V, V82F, I84V and L90M were purified and their catalytic efficiencies Ž . relative to that of wild-type proteinase were examined using a polyprotein recombinant HIV-1 gag substrate and several series of synthetic peptides based on the -Hydrophobic ) Hydrophobic-, -Aromatic ) Pro- and pseudo-symmetrical types of cleavage junction. The L90M proteinase showed only small changes, whereas the activity of the other mutant enzymes was compromised more severely, particularly towards substrates of the -Aromatic ) Pro- and pseudo-symmetrical types. The susceptibility of the mutants and the wild-type proteinase to inhibition by eleven different compounds was compared. The L90M proteinase again showed only marginal changes in its susceptibility to all except one of the inhibitors examined. The Ž . K values determined for one inhibitor Ro31-8959 showed that its potency towards the V82F, L90M, I84V and G48V i mutant proteinases respectively was 2-, 3-, 17- and 27-fold less than against the wild-type proteinase. Several of the other inhibitors examined form a systematic series with Ro31-8959. The inhibition constants derived with these and a number of other inhibitors, including ABT-538 and L-735,524, are used in conjunction with the data on enzymic efficiency to assess whether each mutation in the proteinase confers an advantage for viral replication in the presence of any given inhibitor. Ž . Keywords: Mutant HIV proteinase; HIV proteinase; Substrate specificity; Inhibitor binding; E. coli Abbreviations: HIV, human immunodeficiency virus; IPTG, isopropyl b-D-thiogalactopyranoside; Nph, p-nitrophenylalanine; Ž Nle, norleucine; the nomenclature e.g. MA, matrix, CA, capsid, . PR, proteinase used for retroviral gag and gag-pol proteins w x follows the conventions described in 39 ) Corresponding author. Fax: q44 1222 874116; E-mail: kayj@cardiff.ac.uk 1. Introduction The aspartic proteinase encoded by HIV-1 has been recognised as a suitable target for antiviral therapy and extensive efforts have been made to produce and evaluate selective inhibitors of this en- wx zyme 2 . In December 1995, the first of these in- Ž wx. Ž hibitors Ro31-8959; 3 was licensed under the 0167-4838r97r$17.00 Copyright q 1997 Elsevier Science B.V. All rights reserved. Ž . PII S0167-4838 96 00224-5