CHARACTERIZATION OF HIV-1 PROTEASE MUTANTS FROM THE VIRAL VARIANTS WITH REDUCED SENSITIVITY TO A PROTEASE INHIBITOR

Abstract

Human immunodeficiency virus type-1 (HIV) protease has been an important target for seeking effective therapeutic agents against the acquired immunodeficiency syndrome (AIDS). Up until now, numerous drugs have been selected or designed to inhibit the HIV protease activity. A-77003, a potent inhibitor of HIV protease, showed promising antiviral activity in tissue culture and it is being evaluated in clinical trials as a potential drug for AIDS. Because of the concern over the possibility of drug resistance, in vitro tissue culture selection led to the discovery that resistant HIV variants containing one or more mutations in their protease genes develop over a short period of time to A-77003. However, the mechanisms of how these protease mutations affect the sensitivity of HIV to the drug is not well understood. As part of the team effort to design more potent protease inhibitors targeting these HIV variant strains, recombinant techniques were used to produce four protease mutants with the following single amino acid changes: Arg 8 to Gin, Val 32 to Ile, V82 to either or Ile and Ala. The recombinant mutant enzymes were characterized for their enzymatic function and catalytic efficiency, as well as for inhibition by A-77003. In addition, crystallization of the V82A HIV protease mutant in complex with A-77003 was also reported here for X-ray structure analysis. These four mutations in the viral protease all resulted in weaker inhibition of the enzyme by A-77003 with Ki increases of up to 25 fold. Although the Kₘ values of the four protease mutants did not change more than 2 fold compared to the wild type protease, the K꜀ₐₜ values of the mutants were 2.5 to 13 fold lower than the wild-type enzyme. The mutant enzymes are not as efficient catalytically as the wild-type protease, but they are still functional for viral propagation. This study suggests that amino acid residues 8, 32 and 82 of HIV- 1 protease are important for enzyme-inhibitor interaction. This is confirmed by X-ray structural analysis on a complex of HIV- 1 protease with A-77003. Further structure analysis along with the enzymaticcharacterization of the protease mutants reported here will be useful for the design of novel drugs for AIDS therapy that target a wide spectrum of HIV strains or are less likely to select resistant HIV variants.