Site-directed mutagenesis of human immunodeficiency virus type 1 reverse transcriptase at amino acid position 138

TSAO derivatives represent a class of nonnucleoside reverse transcriptase i nhibitors (NNRTls) that consistently select for the Glu138Lys resistance mu tation in HIV-1 reverse transcriptase (RT). Seven RT mutants (i.e., Ala, As p, Gin, Gly, Lys, Phe, and Tyr) were constructed by site-directed mutagenes is. The mutant Glu138Asp, Glu138Lys, Glu138Gln, Glu138Ala, and Glu138Gly RT s retained marked catalytic activity. In contrast, the Glu138Phe and Glu138 Tyr RT mutants showed poor RNA-dependent DNA polymerase activity (30 and 4% of wild-type, respectively). TSAO derivatives lost their inhibitory activi ty against all mutant enzymes, except against the closely related Glu138Asp RT mutant that remained as sensitive to TSAOs as did wild-type RT. Other N NRTls, including delavirdine, emivirine, and UC-781, and the NRTI ddGTP ret ained pronounced inhibitory activity against all mutant enzymes. When the a mino acid mutations at position 138 of RT were introduced in recombinant vi rus clones, the sensitivity/resistance spectrum obtained toward the TSAOs a nd other NNRTls was similar to those observed for the isolated recombinant mutant enzymes. The Glu138Lys RT mutant virus had the most marked resistanc e to TSAOs. followed by the Glu138Gln, Glu138Phe, Glu138Gly, Glu138Tyr, and Glu138Ala virus mutants. The Glu138Asp RT mutant virus kept full sensitivi ty to the TSAO derivatives. Mixtures of Glu138Lys RT mutant virus with the other virus clones mutated at the 138 position resulted in all cases, excep t for the Glu138Asp and Glu138Gly RT mutant viruses, in an outgrowth of the Glu138Lys RT mutant virus. Since the Glu138Lys RT proved most resistant to TSAO derivatives, was among the most catalytically efficient enzymes, and resulted in highly replication-competent virus, our data explain why the Gl u138Lys RT mutant virus strains but not virus strains containing other amin o acids at position 138 invariably emerge in cell cultures under TSAO drug pressure. (C) 2001 Academic Press.