Secondary mutations M36I and A71V in the human immunodeficiency virus type 1 protease can provide an advantage for the emergence of the primary mutation D30N
Secondary mutations M36I and A71V in the human immunodeficiency virus type 1 protease can provide an advantage for the emergence of the primary mutation D30N
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DOI：
10.1021/bi035701y
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发表时间：
2003-12-30
期刊：
影响因子：
2.9
通讯作者：
Dunn, BA
中科院分区：
文献类型：
--
作者：
Clemente, JC;Hemrajani, R;Dunn, BA
人类免疫缺陷病毒1（HIV-1）酶靶点的耐药性突变阻碍了提供适当治疗的能力。特别感兴趣的是HIV-1蛋白酶活性位点外的突变对抑制剂结合和病毒活力的影响。我们工程化的蛋白酶突变体含有活性位点突变D30 N单独和非活性位点多态性M36 I和/或A71 V。我们测定了抑制剂奈非那韦、利托那韦、茚地那韦、KNI 272和AG 1776的Ki值以及突变体的催化效率。单突变和双突变组合表现出催化效率的降低，而三重突变体显示催化效率大于野生型。单独含有M36 I或A71 V的变体对所测试的抑制剂的结合亲和力未显示出显著变化。对于所有测试的抑制剂，含有突变D30 N的变体显示Ki增加2-6倍，其中奈非那韦显示最大增加。含有突变D30 N、M36 I和A71 V的组合的双突变体显示所有测试的抑制剂的Ki的-0.5倍至+6倍变化，其中利托那韦和奈非那韦受影响最大。只有三重突变体显示出对抑制剂奈非那韦、利托那韦或AG-1776的Ki的显著增加（> 10倍），分别显示出22倍、19倍或15倍的增加。我们的研究表明，M36 I和A71 V突变与活性位点突变D30 N相结合，提供了更高水平的抑制剂交叉耐药性。M361和A71 V，当作为天然多态性存在时，可以帮助病毒发展活性位点突变以逃避抑制剂结合，同时保持催化效率。
Development of resistance mutations in enzymatic targets of human immunodeficiency virus 1 (HIV-1) hampers the ability to provide adequate therapy. Of special interest is the effect mutations outside the active site of HIV-1 protease have on inhibitor binding and virus viability. We engineered protease mutants containing the active site mutation D30N alone and with the nonactive site polymorphisms M36I and/or A71V. We determined the K-i values for the inhibitors nelfinavir, ritonavir, indinavir, KNI272, and AG1776 as well as the catalytic efficiency of the mutants. Single and double mutation combinations exhibited a decrease in catalytic efficiency, while the triple mutant displayed catalytic efficiency greater than that of the wild type. Variants containing M36I or A71V alone did not display a significant change in binding affinities to the inhibitors tested. The variant containing mutation D30N displayed a 2-6-fold increase in K-i for all inhibitors tested, with nelfinavir showing the greatest increase. The double mutants containing a combination of mutations D30N, M36I, and A71V displayed -0.5-fold to +6-fold changes in the K-i of all inhibitors tested, with ritonavir and nelfinavir most affected. Only the triple mutant showed a significant increase (> 10-fold) in K-i for inhibitor nelfinavir, ritonavir, or AG-1776 displaying 22-, 19-, or 15-fold increases, respectively. Our study shows that the M36I and A71V mutations provide a greater level of inhibitor cross-resistance combined with active site mutation D30N. M361 and A71V, when present as natural polymorphisms, could aid the virus in developing active site mutations to escape inhibitor binding while maintaining catalytic efficiency.