Mutational analysis of Tyr-501 of HIV-1 reverse transcriptase -: Effects on ribonuclease H activity and inhibition of this activity by N-acylhydrazones
Mutational analysis of Tyr-501 of HIV-1 reverse transcriptase -: Effects on ribonuclease H activity and inhibition of this activity by N-acylhydrazones
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DOI：
10.1074/jbc.m110254200
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发表时间：
2002-01-11
影响因子：
4.8
通讯作者：
Parniak, MA
中科院分区：
文献类型：
--
作者：
Arion, D;Sluis-Cremer, N;Parniak, MA
N-(4-叔丁基苯甲酰基)-2-羟基萘乙醛腙 (BBNH) 是人类免疫缺陷病毒 (HIV)-1 逆转录酶 (RT) 核糖核酸酶 H (RNase H) 活性的有效抑制剂。分子模型预测 BBNH 通过两种主要相互作用与 HIV-1 RT RNase H 活性位点结合，即与酶活性位点中的金属离子辅因子（Mg2+ 或 Mn2+）配位，以及 BBNH 的萘环与氨基酸 Tyr-501 之间的芳香环堆积相互作用。后一个残基等效物在几乎所有 RNase H 中都是保守的，表明该区域需要芳香族或 pi 堆积相互作用。为了评估 Tyr-501 在结合 BBNH 抑制 RT RNase H 活性中的重要性，我们使用位点特异性诱变来生成在该位置具有多种取代的 RT。大多数替换实际上导致 RNase H 活性完全丧失。然而，三个突变体 Y501F、Y501W 和 Y501R 具有与野生型酶相当的 RNase H 活性。虽然 BBNH 抑制 Y501F RT RNase H 活性的效力与野生型 RT 相当，但 Y501W 突变体对 BBNH 的抑制表现出 6 倍的抵抗力，而 Y501R 突变体则完全抵抗 BBNH 的抑制作用。与野生型病毒相比，具有Y501W或Y510R突变的HIV分子克隆的复制“适应性”显着受损。重要的是，BBNH 是所有测试的 Y501X 突变体 DNA 聚合酶活性的有效抑制剂。我们的结果强调了 Tyr-501 在 RT RNase H 活性和 N-酰腙抑制剂结合中的重要性，并表明针对 HIV-1 蛋白中关键残基的药物可能是新抗病毒开发的有用方法。
N-(4-tert-Butylbenzoyl)-2-hydroxynaphthaidehyde hydrazone (BBNH) is a potent inhibitor of the ribonuclease H (RNase H) activity of human immunodeficiency virus (HIV)-1 reverse transcriptase (RT). Molecular modeling predicted that BBNH binds to the HIV-1 RT RNase H active site via two major interactions, coordination to the metal ion cofactor (Mg2+ or Mn2+) in the enzyme active site and aromatic ring-stacking interaction between the naphthyl ring of BBNH and amino acid Tyr-501. The latter residue equivalent is conserved in virtually all RNases H, suggesting the need for an aromatic or pi-stacking interaction in this region. To assess the importance of Tyr-501 in the binding of BBNH for the inhibition of RT RNase H activity, we used site-specific mutagenesis to generate RT with a variety of substitutions at this position. Most substitutions resulted virtually in a complete loss of RNase H activity. However, three mutants, Y501F, Y501W, and Y501R, possessed RNase H activities comparable with wild-type enzyme. Whereas BBNH inhibited Y501F RT RNase H activity with potency equivalent to wild-type RT, the Y501W mutant showed a 6-fold resistance to inhibition by BBNH, and the Y501R mutant was completely resistant to inhibition by BBNH. The replication "fitness" of HIV molecular clones with the Y501W or Y510R mutation was significantly compromised compared with wild-type virus. Importantly, BBNH was an effective inhibitor of the DNA polymerase activity of all Y501X mutants tested. Our results highlight the importance of Tyr-501 in RT RNase H activity and in N-acylhydrazone inhibitor binding and suggest that drugs that target critical residues in HIV-1 proteins may be a useful approach in new antiviral development.