Mechanism of Darunavir (DRV)'s High Genetic Barrier to HIV-1 Resistance: A Key V32I Substitution in Protease Rarely Occurs, but Once It Occurs, It Predisposes HIV-1 To Develop DRV Resistance.
Mechanism of Darunavir (DRV)'s High Genetic Barrier to HIV-1 Resistance: A Key V32I Substitution in Protease Rarely Occurs, but Once It Occurs, It Predisposes HIV-1 To Develop DRV Resistance.
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DOI：
10.1128/mbio.02425-17
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发表时间：
2018-03-06
期刊：
影响因子：
6.4
通讯作者：
Mitsuya H
中科院分区：
文献类型：
--
作者：
Aoki M;Das D;Hayashi H;Aoki-Ogata H;Takamatsu Y;Ghosh AK;Mitsuya H
达芦那韦（DRV）具有抗HIV-1蛋白酶、酶抑制和蛋白酶二聚化抑制的双峰活性，并具有极高的耐药性遗传屏障。我们先前产生了一种高度耐DRV的HIV-1变体（HIV DRVRP 51）。我们还报告说，在HIVDRVRP 51蛋白酶中发现的四个氨基酸取代（V32 I、L33 F、I54 M和I84 V）是其对DRV高水平耐药性的主要原因。在这里，我们试图阐明每一个的四个氨基酸取代的DRV抗性的发展中的作用。我们发现V32 I是一个关键的替换，它很少发生，但一旦发生，它就容易使HIV-1产生高水平的DRV耐药性。当在存在DRV的情况下选择携带I54 M和I84 V的两个感染性重组HIV-1克隆（分别为rHIVI 54 M和rHIVI 84 V）时，出现V32 I，并且病毒迅速发展出高水平的DRV抗性。rHIVV 32 I也产生了高水平的DRV抗性。然而，野生型HIVNL 4 -3（rHIVWT）未能获得V32 I，也未产生DRV抗性。与rHIVWT相比，rHIVV 32 I对DRV高度敏感，适应性显著降低，这解释了为什么在用DRV选择rHIVWT时V32 I没有出现。当唯一的取代是在残基32处时，结构分析显示DRV与I-32之间的货车范德华相互作用比DRV与V-32之间的强得多。这些结果表明，V32 I是HIV-1 DRV耐药性发展的多个途径中的关键氨基酸取代，并至少部分阐明了DRV对耐药性发展的高遗传屏障的机制。结果还表明，应注意在含有V32 I替代的HIV-1感染者中开始或继续使用含DRV的方案。达芦那韦（DRV）是唯一被推荐作为一线治疗药物的蛋白酶抑制剂（PI），是治疗HIV-1感染者最广泛使用的PI。DRV对HIV-1耐药性的发展具有很高的遗传屏障。然而，DRV高遗传屏障的机制仍不清楚。在这里，我们表明，预先存在的某些单一的氨基酸取代，如V32 I，I54 M，A71 V，和I84 V的HIV-1蛋白酶促进高水平的DRV耐药的发展。有趣的是，所有体外选择的高度耐DRV的HIV-1变异体都获得了V32 I，但从未在野生型HIV（HIVWT）中出现，与HIVWT相比，V32 I本身使HIV-1对DRV更敏感，并降低了病毒适应性，这强烈表明V32 I的出现在HIV-1对DRV的耐药性发展中起着关键作用。我们的研究结果将有利于治疗HIV-1感染的患者接受含DRV的方案。
Darunavir (DRV) has bimodal activity against HIV-1 protease, enzymatic inhibition and protease dimerization inhibition, and has an extremely high genetic barrier against development of drug resistance. We previously generated a highly DRV-resistant HIV-1 variant (HIVDRVRP51). We also reported that four amino acid substitutions (V32I, L33F, I54M, and I84V) identified in the protease of HIVDRVRP51 are largely responsible for its high-level resistance to DRV. Here, we attempted to elucidate the role of each of the four amino acid substitutions in the development of DRV resistance. We found that V32I is a key substitution, which rarely occurs, but once it occurs, it predisposes HIV-1 to develop high-level DRV resistance. When two infectious recombinant HIV-1 clones carrying I54M and I84V (rHIVI54M and rHIVI84V, respectively) were selected in the presence of DRV, V32I emerged, and the virus rapidly developed high-level DRV resistance. rHIVV32I also developed high-level DRV resistance. However, wild-type HIVNL4-3 (rHIVWT) failed to acquire V32I and did not develop DRV resistance. Compared to rHIVWT, rHIVV32I was highly susceptible to DRV and had significantly reduced fitness, explaining why V32I did not emerge upon selection of rHIVWT with DRV. When the only substitution is at residue 32, structural analysis revealed much stronger van der Waals interactions between DRV and I-32 than between DRV and V-32. These results suggest that V32I is a critical amino acid substitution in multiple pathways toward HIV-1’s DRV resistance development and elucidate, at least in part, a mechanism of DRV’s high genetic barrier to development of drug resistance. The results also show that attention should be paid to the initiation or continuation of DRV-containing regimens in people with HIV-1 containing the V32I substitution. Darunavir (DRV) is the only protease inhibitor (PI) recommended as a first-line therapeutic and represents the most widely used PI for treating HIV-1-infected individuals. DRV possesses a high genetic barrier to development of HIV-1’s drug resistance. However, the mechanism(s) of the DRV’s high genetic barrier remains unclear. Here, we show that the preexistence of certain single amino acid substitutions such as V32I, I54M, A71V, and I84V in HIV-1 protease facilitates the development of high-level DRV resistance. Interestingly, all in vitro-selected highly DRV-resistant HIV-1 variants acquired V32I but never emerged in wild-type HIV (HIVWT), and V32I itself rendered HIV-1 more sensitive to DRV and reduced viral fitness compared to HIVWT, strongly suggesting that the emergence of V32I plays a critical role in the development of HIV-1’s resistance to DRV. Our results would be of benefit in the treatment of HIV-1-infected patients receiving DRV-containing regimens.