In Vivo Emergence of a Novel Protease Inhibitor Resistance Signature in HIV-1 Matrix.
In Vivo Emergence of a Novel Protease Inhibitor Resistance Signature in HIV-1 Matrix.
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DOI：
10.1128/mbio.02036-20
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发表时间：
2020-11-03
期刊：
影响因子：
6.4
通讯作者：
Gupta RK
中科院分区：
文献类型：
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作者：
Datir R;Kemp S;El Bouzidi K;Mlchocova P;Goldstein R;Breuer J;Towers GJ;Jolly C;Quiñones-Mateu ME;Dakum PS;Ndembi N;Gupta RK
蛋白酶抑制剂（pi）是全世界大多数hiv感染患者的二线和最后一线治疗。在PI治疗失败的患者中，只有大约20%的人在蛋白酶中发生了重大的耐药突变。我们试图探索突变在6名尼日利亚患者的病毒gag-pro基因型和表型变化中的作用，这些患者在没有已知耐药性相关蛋白酶突变的情况下，未能接受基于PI的方案，以确定PI耐药的新决定因素。蛋白酶抑制剂（pi）是全世界大多数hiv感染患者的二线和最后一线治疗。在PI治疗失败的患者中，只有大约20%的人在蛋白酶中发生了重大的耐药突变。我们试图探索突变在6名尼日利亚患者的病毒gag-pro基因型和表型变化中的作用，这些患者在没有已知耐药性相关蛋白酶突变的情况下，未能接受基于PI的方案，以确定PI耐药的新决定因素。利用Illumina MiSeq系统进行目标富集和下一代测序（NGS），然后进行单倍型重建。从基线（pre-PI）和病毒学失败（VF）样本中扩增出全长gag-蛋白酶基因区域，进行测序，并用于构建gag-前假型病毒。系统发育分析采用最大似然方法。采用单周期重复试验测定对洛匹那韦（LPV）和达那韦（DRV）的敏感性。Western blotting分析Gag裂解。在六名参与者中的一名（CRF02_AG亚型）中，我们发现在二线治疗失败期间病毒克隆的LPV易感性降低了4倍。基线病毒p17基质中的四种突变（S126del、H127del、T122A和G123E）的组合使LPV易感性降低了4倍，但达那韦没有。这四种氨基酸的变化也能够赋予LPV对B亚型gag -蛋白酶主干的抗性。Western blotting结果显示，在药物存在的情况下，敏感株和耐药株的Gag裂解存在显著差异。在没有药物的情况下，耐药病毒的传染性比敏感克隆低约2倍。NGS结合单倍型重建显示，抗性较差的克隆出现在基线时的少数种群中，此后与敏感的滤过病毒一起持续存在。我们使用多管齐下的基因型和表型方法来记录HIV-1基质中新的蛋白酶抑制剂抗性特征的出现和时间动态，揭示gag相关抗性和适应度之间的相互作用。
Protease inhibitors (PIs) are the second- and last-line therapy for the majority of HIV-infected patients worldwide. Only around 20% of individuals who fail PI regimens develop major resistance mutations in protease. We sought to explore the role of mutations in gag-pro genotypic and phenotypic changes in viruses from six Nigerian patients who failed PI-based regimens without known drug resistance-associated protease mutations in order to identify novel determinants of PI resistance. Protease inhibitors (PIs) are the second- and last-line therapy for the majority of HIV-infected patients worldwide. Only around 20% of individuals who fail PI regimens develop major resistance mutations in protease. We sought to explore the role of mutations in gag-pro genotypic and phenotypic changes in viruses from six Nigerian patients who failed PI-based regimens without known drug resistance-associated protease mutations in order to identify novel determinants of PI resistance. Target enrichment and next-generation sequencing (NGS) with the Illumina MiSeq system were followed by haplotype reconstruction. Full-length Gag-protease gene regions were amplified from baseline (pre-PI) and virologic failure (VF) samples, sequenced, and used to construct gag-pro-pseudotyped viruses. Phylogenetic analysis was performed using maximum-likelihood methods. Susceptibility to lopinavir (LPV) and darunavir (DRV) was measured using a single-cycle replication assay. Western blotting was used to analyze Gag cleavage. In one of six participants (subtype CRF02_AG), we found 4-fold-lower LPV susceptibility in viral clones during failure of second-line treatment. A combination of four mutations (S126del, H127del, T122A, and G123E) in the p17 matrix of baseline virus generated a similar 4-fold decrease in susceptibility to LPV but not darunavir. These four amino acid changes were also able to confer LPV resistance to a subtype B Gag-protease backbone. Western blotting demonstrated significant Gag cleavage differences between sensitive and resistant isolates in the presence of drug. Resistant viruses had around 2-fold-lower infectivity than sensitive clones in the absence of drug. NGS combined with haplotype reconstruction revealed that resistant, less fit clones emerged from a minority population at baseline and thereafter persisted alongside sensitive fitter viruses. We used a multipronged genotypic and phenotypic approach to document emergence and temporal dynamics of a novel protease inhibitor resistance signature in HIV-1 matrix, revealing the interplay between Gag-associated resistance and fitness.