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Characterization of resistance to a potent d-peptide HIV entry inhibitor
Retrovirology ( IF 3.3 ) Pub Date : 2019-10-22 , DOI: 10.1186/s12977-019-0489-7
Amanda R Smith 1 , Matthew T Weinstock 1 , Amanda E Siglin 2 , Frank G Whitby 1 , J Nicholas Francis 1 , Christopher P Hill 1 , Debra M Eckert 1 , Michael J Root 2 , Michael S Kay 1
Affiliation  

BackgroundPIE12-trimer is a highly potent d-peptide HIV-1 entry inhibitor that broadly targets group M isolates. It specifically binds the three identical conserved hydrophobic pockets at the base of the gp41 N-trimer with sub-femtomolar affinity. This extremely high affinity for the transiently exposed gp41 trimer provides a reserve of binding energy (resistance capacitor) to prevent the viral resistance pathway of stepwise accumulation of modest affinity-disrupting mutations. Such modest mutations would not affect PIE12-trimer potency and therefore not confer a selective advantage. Viral passaging in the presence of escalating PIE12-trimer concentrations ultimately selected for PIE12-trimer resistant populations, but required an extremely extended timeframe (> 1 year) in comparison to other entry inhibitors. Eventually, HIV developed resistance to PIE12-trimer by mutating Q577 in the gp41 pocket.ResultsUsing deep sequence analysis, we identified three mutations at Q577 (R, N and K) in our two PIE12-trimer resistant pools. Each point mutant is capable of conferring the majority of PIE12-trimer resistance seen in the polyclonal pools. Surface plasmon resonance studies demonstrated substantial affinity loss between PIE12-trimer and the Q577R-mutated gp41 pocket. A high-resolution X-ray crystal structure of PIE12 bound to the Q577R pocket revealed the loss of two hydrogen bonds, the repositioning of neighboring residues, and a small decrease in buried surface area. The Q577 mutations in an NL4-3 backbone decreased viral growth rates. Fitness was ultimately rescued in resistant viral pools by a suite of compensatory mutations in gp120 and gp41, of which we identified seven candidates from our sequencing data.ConclusionsThese data show that PIE12-trimer exhibits a high barrier to resistance, as extended passaging was required to develop resistant virus with normal growth rates. The primary resistance mutation, Q577R/N/K, found in the conserved gp41 pocket, substantially decreases inhibitor affinity but also damages viral fitness, and candidate compensatory mutations in gp160 have been identified.

中文翻译:

对强效 d 肽 HIV 进入抑制剂的耐药性表征

背景PIE12-三聚体是一种高效的 d 肽 HIV-1 进入抑制剂,广泛靶向 M 组分离株。它以亚飞摩尔亲和力特异性结合 gp41 N-三聚体底部的三个相同的保守疏水口袋。这种对瞬时暴露的 gp41 三聚体的极高亲和力提供了结合能储备(电阻电容器),以防止适度亲和力破坏突变逐步积累的病毒抗性途径。这种适度的突变不会影响 PIE12 三聚体的效力,因此不会赋予选择性优势。在 PIE12-三聚体浓度不断升高的情况下进行病毒传代,最终选择用于 PIE12-三聚体抗性群体,但与其他进入抑制剂相比需要极长的时间范围(> 1 年)。最终,HIV 通过突变 gp41 口袋中的 Q577 产生了对 PIE12-三聚体的抗性。结果使用深度序列分析,我们在我们的两个 PIE12-三聚体抗性库中鉴定了 Q577(R、N 和 K)处的三个突变。每个点突变体都能够赋予在多克隆池中看到的大多数 PIE12 三聚体抗性。表面等离子体共振研究表明 PIE12 三聚体和 Q577R 突变的 gp41 口袋之间存在大量亲和力损失。与 Q577R 口袋结合的 PIE12 的高分辨率 X 射线晶体结构揭示了两个氢键的丢失、相邻残基的重新定位以及掩埋表面积的小幅减少。NL4-3 骨干中的 Q577 突变降低了病毒生长速率。通过 gp120 和 gp41 中的一系列补偿性突变,健身最终在耐药病毒库中得以挽救,其中我们从我们的测序数据中确定了 7 个候选者。结论这些数据表明 PIE12-三聚体表现出高抗性屏障,因为需要延长传代才能开发出具有正常生长速率的抗性病毒。在保守的 gp41 口袋中发现的主要抗性突变 Q577R/N/K 大大降低了抑制剂亲和力,但也损害了病毒适应性,并且已经确定了 gp160 中的候选代偿性突变。
更新日期:2019-10-22
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