Characterization of HIV Reverse Transcriptases with Tyr181→Cys and Leu100→lle Mutations

Abstract

Two mutants of human immunodeficiency virus (HIV) reverse transcriptase (RT), Tyr181 to Cys and Leu100 to He, have been prepared and characterized by use of various inhibitors. As compared to wild type RT the mutant RT's had lower Kcat/Kmvalues. The Kmvalues were lower with heteropolymeric than with homopolymeric template-primers. Inhibition by phosphonoformate was of mixed type with both wild-type and mutant RT's and the mutants were less sensitive to phosphonoformate than the wild type RT. The non-nucleoside RT inhibitors 9-CI-TIBO and L-697,661 gave a non-competitive inhibition with respect to substrate of the wild type RT. The mutant RT's were inhibited at higher concentrations, showing a mixed type of inhibition with respect to substrate. ddGTP caused a competitive inhibition of wild type and mutant RT's with respect to substrate. RT preparations with different mutations are useful in rapidly characterizing the interaction between various inhibitors and HIV RT and thus facilitate the development of new inhibitors.