Mutational Analysis of Tyr-318 within the Non-nucleoside Reverse Transcriptase Inhibitor Binding Pocket of Human Immunodeficiency Virus Type I Reverse Transcriptase

Abstract

The highly conserved Tyr-318 is part of the non-nucleoside reverse transcriptase inhibitor (NNRTI)-specific lipophilic pocket of human immunodeficiency virus type I reverse transcriptase (RT) and makes contact within 4 Å with the NNRTIs in all reported RT/NNRTI complexes. Using site-directed mutagenesis, six mutant RTs were constructed bearing the mutations Y318H, Y318K, Y318L, Y318C, Y318W, and Y318F. We found that only the Y318W and Y318F mutant RTs retained substantial RT activity, whereas the catalytic activities of the Y318K, Y318C, Y318H, and Y318L RT mutants were less than 5% of the wild-type activity. The Y318F mutant RT retained substantial sensitivity to the majority of NNRTIs tested, whereas the Y318W mutant RT showed varying degrees of resistance to NNRTIs. Subunit-specific site-directed mutagenesis revealed that there was no difference in the catalytic activity or resistance/sensitivity spectrum toward NNRTIs regardless of whether the Tyr-318 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 showed 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 pocket of human immunodeficiency virus type I RT.

The N137 and P140 amino acids in the p51 and the P95 amino acid in the p66 subunit of human immunodeficiency virus type 1 (HIV‐1) reverse transcriptase are instrumental to maintain catalytic activity and to design new classes of anti‐HIV‐1 drugs

“…Site-directed mutagenesis was performed using the QuickChange Site-Directed Mutagenesis Kit (Stratagene, Westburg, Leusden, The Netherlands), as described before [6]. The two synthetic oligonucleotide primers (Invitrogen Life Technologies, Merelbeke, Belgium) used contained the desired mutation at amino acid position 137, 140 or 95 of HIV-1 RT.…”

“…Briefly, the supernatant of the lysed bacterial cell culture was incubated with Ni-NTA resin (Qiagen). After sedimentation of the Ni-NTA resin with the bound (His) 6 -tagged proteins, a column was formed and washed twice with sodium phosphate buffer containing 10 mM imidazole. Then, the RT was eluted from the column with sodium phosphate buffer containing 125 mM imidazole.…”

“…The imidazole-containing buffer was exchanged by a Tris-HCl buffer and the eluate was concentrated to 2 ml using Ultrafree-15 centrifugal filtration devices (Millipore, Brussels, Belgium). The (His) 6 -tagged RT was further purified to about 98% purity over a Hitrap Heparin column (Amersham Biosciences, Roosendaal, The Netherlands). All fractions containing heterodimer RT were pooled and stored in a 50% glycerol buffer at À20°C.…”

“…Previous studies have revealed that mutations of conserved amino acids such as W229 [5], Y318 [6] or W401 [7] of HIV-1 RT result in a severe decrease of the catalytic activity of the enzyme. Interestingly, the W229 and Y318 mutations did not result in a markedly altered sensitivity of the RT to most NNRTIs.…”

“…Interestingly, the W229 and Y318 mutations did not result in a markedly altered sensitivity of the RT to most NNRTIs. Therefore, it would be unlikely that treatment of HIV-1 infected cells with NNRTIs targeting these conserved amino acids would result in selection of resistance mutations at these amino acid positions [5,6].…”