Mutational analysis of Tyr-318 within the non-nucleoside reverse transcriptase inhibitor binding pocket of human immunodeficiency virus type I reverse transcriptase

The highly conserved Tyr-318 is part of the non-nucleoside reverse transcri ptase inhibitor (NNRTI)-specific lipophilic pocket of human immunodeficienc y virus type I reverse transcriptase (RT) and makes contact within 4 Angstr om with the NNRTIs in all reported RT/NNRTI complexes. Using site-directed mutagenesis, six mutant RTs were constructed bearing the mutations Y318H, Y 318K, Y318L, Y318C, Y318W, and Y318F. We found that only the Y318W and Y318 F mutant RTs retained substantial RT activity, whereas the catalytic activi ties of the Y318K, Y318C, Y318H, and Y318L RT mutants were less than 5% of the wild-type activity, The Y318F mutant RT retained substantial sensitivit y to the majority of NNRTIs tested, whereas the Y318W mutant RT showed vary ing degrees of resistance to NNRTIs, Subunit-specific site-directed mutagen esis revealed that there was no difference in the catalytic activity or res istance/sensitivity spectrum toward NNRTIs regardless of whether the Tyr-31 8 mutation was introduced in both subunits or only in the p66 subunit of RT . Recombinant viruses harboring the Y318F or Y318W mutation in the RT showe d a similar resistance/sensitivity pattern to NNRTIs as their corresponding 318 mutant recombinant RTs, Our findings stress a functional or structural role for Tyr-318 in wild-type RT and argue for the design of novel NNRTIs that interact more closely with this amino acid in the NNRTI-specific pocke t of human immunodeficiency virus type I RT.