Pelemans H, Esnouf R M, Jonckheere H, De Clercq E, Balzarini J
Rega Institute for Medical Research, Katholieke Universiteit Leuven, B-3000 Leuven, Belgium.
J Biol Chem. 1998 Dec 18;273(51):34234-9. doi: 10.1074/jbc.273.51.34234.
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 A 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.
高度保守的酪氨酸-318是I型人类免疫缺陷病毒逆转录酶（RT）非核苷类逆转录酶抑制剂（NNRTI）特异性亲脂口袋的一部分，在所有已报道的RT/NNRTI复合物中，它与NNRTI在4埃范围内相互作用。通过定点诱变，构建了六个携带Y318H、Y318K、Y318L、Y318C、Y318W和Y318F突变的突变型RT。我们发现，只有Y318W和Y318F突变型RT保留了大量的RT活性，而Y318K、Y318C、Y318H和Y318L RT突变体的催化活性不到野生型活性的5%。Y318F突变型RT对大多数测试的NNRTI仍保持高度敏感性，而Y318W突变型RT对NNRTI表现出不同程度的抗性。亚基特异性定点诱变表明，无论酪氨酸-318突变是在两个亚基中引入还是仅在RT的p66亚基中引入，其对NNRTI的催化活性或抗性/敏感性谱均无差异。在RT中携带Y318F或Y318W突变的重组病毒对NNRTI的抗性/敏感性模式与其相应的318突变重组RT相似。我们的研究结果强调了酪氨酸-318在野生型RT中的功能或结构作用，并支持设计与I型人类免疫缺陷病毒RT的NNRTI特异性口袋中的该氨基酸更紧密相互作用的新型NNRTI。
