Simultaneous mutations at Tyr‐181 and Tyr‐188 in HIV‐1 reverse transcriptase prevents inhibition of RNA‐dependent DNA polymerase activity by the bisheteroarylpiperazine (BHAP) U‐90152s

[摘要]

The replacement of either Tyr-181 or Tyr-188 of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) by the corresponding HIV-2 RT amino acids Ile-181 or Leu-188 is known to result in active mutant enzymes (Y181I; Y188L) with virtual loss of sensitivity towards three structural classes of nonnucleoside RT inhibitors; L-697,661, nevirapine, and TIBO R82913. The bisheteroarylpiperazine (BHAP) U-90152S, a highly specific inhibitor (IC₅₀, 0.29 ± 0.01 μM) of HIV-1 RT, inhibited the recombinant Y181I and Y188L HIV-1 RT mutants with IC₅₀ values of 3.6 ± 0.15 μM and 0.71 ± 0.02 μM, respectively. Construction and in vitro analysis of double mutants Y181I/Y188L and Y181C/Y188L of HIV-1 RT showed > 150-fold resistance to U-90152S. An HIV-2 mutant containing amino acids 176–190 from HIV-1 RT acquired full sensitivity to U-90152S (IC₅₀, 0.29 ± 0.01 μM). It is concluded tha simultaneous mutations at Tyr-181 and Tyr-188 of HIV-1 RT promotes resistance to U-90152S.

[学科分类] 生物化学/生物物理

[关键词] HIV-1 reverse transcriptase;HIV-1 RT mutant;Resistance to U-90152S;HIV-2 RT;Double mutant;HIV-1;human immunodeficiency virus type 1;HIV-2;human immunodeficiency virus type 2;RT;reverse transcriptase;AIDS;acquired immunodeficiency syndrome;IMAC;immobilized metal affinity chromatography;SDS;sodium dodecyl sulfate;PAGE;polyacrylamide gel electrophoresis;BSA;bovine serum albumin;TCA;trichloroacetic acid;TIBO R82913;(+)-(5S)-4;5;6;7-tetrahydro-9-chloro-5-methyl-6-(3-methyl-2-butenylimidazo[4;5;1-j;k][1;4;]benzodiazepin-2(1H)- thione;BHAP;bisheteroarylpiperazine;NNRTIs;nonnucleoside reverse transcriptase inhibitors;PCR;polymerase chain reaction