Hooker D J, Tachedjian G, Solomon A E, Gurusinghe A D, Land S, Birch C, Anderson J L, Roy B M, Arnold E, Deacon N J
AIDS Molecular Biology Laboratory, Macfarlane Burnet Center for Medical Research, Fairfield, Australia.
J Virol. 1996 Nov;70(11):8010-8. doi: 10.1128/JVI.70.11.8010-8018.1996.
Sequencing of the reverse transcriptase (RT) region of 26 human immunodeficiency virus type 1 (HIV-1) isolates from eight patients treated with 3'-azido-3'-deoxythymidine (AZT) revealed a mutation at codon 210 from TTG (leucine) to TGG (tryptophan) exclusively in association with resistance to AZT. The mutation Trp-210 was observed in 15 of the 20 isolates phenotypically resistant to AZT, being more commonly observed than resistance-associated mutations at codons 67, 70, and 219. Trp-210 was never observed before the emergence of resistance-associated mutations Leu-41 and Tyr-215, and in a sequential series of five isolates from one patient the order of emergence of mutations was found to be Tyr-215, Leu-41, and then Trp-210. Trp-210 was also found in association with the Leu-41, Asn-67, Arg-70, and Tyr-215 resistance genotype. To define the role of Trp-210 in AZT resistance, molecular HIV-1 clones were constructed with various combinations of RT mutations at codons 41, 67, 70, 210, and 215 and tested for susceptibility to AZT. In clones with polymerase genes derived either from HXB2-D or clinical isolates, Trp-210 alone did not increase AZT resistance, whereas in conjunction with Leu-41 and Tyr-215, Trp-210 contributed to high-level resistance (50% inhibitory concentration of >1 microM). In HXB2-D, Trp-210 with Tyr-215 generated a virus with resistance comparable to one with Leu-41, Tyr-215, and Trp-210. Inserting Trp-210 into the genetic context of mutations at codons 41, 67, 70, and 215 further enhanced resistance from a 50% inhibitory concentration of 1.44 microM to 8.41 microM. Molecular modeling of the tertiary structure of HIV-1 RT revealed that the distance between the side chains of Trp-210 (in helix alphaF) and Tyr-215 (in strand beta11a) approximated 4 A (1 A = 0.1 nm), sufficiently close to result in significant energetic interaction between these two aromatic side chains. In conclusion, Trp-210 contributes significantly to phenotypic AZT resistance of HIV-1 by augmenting resistance at least three- to sixfold in the context of two resistant genotypes, and its effect may require an interaction with an aromatic amino acid at position 215.
对8例接受3'-叠氮-3'-脱氧胸苷（AZT）治疗的患者的26株1型人类免疫缺陷病毒（HIV-1）分离株的逆转录酶（RT）区域进行测序，结果显示，仅在对AZT耐药的情况下，密码子210处发生了从TTG（亮氨酸）到TGG（色氨酸）的突变。在20株对AZT表型耐药的分离株中，有15株观察到Trp-210突变，其比密码子67、70和219处与耐药相关的突变更常见。在与耐药相关的Leu-41和Tyr-215突变出现之前，从未观察到Trp-210突变，并且在来自一名患者的连续5株分离株系列中，发现突变出现的顺序为Tyr-215、Leu-41，然后是Trp-210。还发现Trp-210与Leu-41、Asn-67、Arg-70和Tyr-215耐药基因型相关。为了确定Trp-210在AZT耐药中的作用，构建了具有密码子41、67、70、210和215处RT突变的各种组合的HIV-1分子克隆，并测试了它们对AZT的敏感性。在具有源自HXB2-D或临床分离株的聚合酶基因的克隆中，单独的Trp-210不会增加AZT耐药性，而与Leu-41和Tyr-215一起时，Trp-210会导致高水平耐药（50%抑制浓度>1 microM）。在HXB2-D中，Trp-210与Tyr-215产生的病毒耐药性与具有Leu-41、Tyr-215和Trp-210的病毒相当。将Trp-210插入密码子41、67、70和215处突变的遗传背景中，可将耐药性从50%抑制浓度1.44 microM进一步提高到8.41 microM。HIV-1 RT三级结构的分子建模显示，Trp-210（在αF螺旋中）和Tyr-215（在β11a链中）侧链之间的距离约为4 Å（1 Å = 0.1 nm），足够接近，导致这两个芳香族侧链之间产生显著的能量相互作用。总之，Trp-210通过在两种耐药基因型的背景下将耐药性至少提高三到六倍，对HIV-1的表型AZT耐药性有显著贡献，其作用可能需要与215位的芳香族氨基酸相互作用。
