The pyrophosphate analogue foscarnet traps the pre-translocational state of HIV-1 reverse transcriptase in a Brownian ratchet model of polymerase translocation
The pyrophosphate analogue foscarnet traps the pre-translocational state of HIV-1 reverse transcriptase in a Brownian ratchet model of polymerase translocation
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DOI：
10.1074/jbc.m607710200
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发表时间：
2007-02-02
影响因子：
4.8
通讯作者：
Gotte, Matthias
中科院分区：
文献类型：
--
作者：
Marchand, Bruno;Tchesnokov, Egor P.;Gotte, Matthias
焦磷酸盐（PPi）类似物膦甲酸（PFA或foscarnet）抑制人类免疫缺陷病毒1型（HIV-1）的逆转录酶（RT）；然而，药物作用和耐药性的机制仍然难以捉摸。我们研究了HIV-1 RT易位状态对药物结合和抑制DNA合成的影响。我们确定了活性延伸过程中抑制的“热点”。位点特异性足迹分析表明，相应复合物主要存在于易位前状态。对PFA的敏感性显著降低与序列显示偏向易位后状态。结合研究表明，PFA选择性地稳定了预易位构型的复合物。这些发现与聚合酶易位的布朗棘轮模型一致。这种酶可以在易位前和易位后状态之间快速穿梭。结合抑制剂的作用就像棘轮的爪子，阻止HIV-1 RT的向前运动，而结合的核苷酸结合到易位后复合体上，阻止反向运动。所提出的RT易位和药物作用机制与pfa耐药表型一致。我们发现某些序列和抗pfa的E89K突变体降低了预易位复合物的稳定性。在这些情况下，酶在引物3'端周围的多个位置可见，这提供了一种新的抗性机制。这些发现证实了预易位复合物是开发新的、可能毒性更小、更有效的阻断HIV-1 RT易位抑制剂的靶标。
The pyrophosphate (PPi) analogue phosphonoformic acid (PFA or foscarnet) inhibits the reverse transcriptase (RT) of the human immunodeficiency virus type 1 (HIV-1); however, the mechanisms of drug action and resistance remain elusive. Here we studied the effects of the translocational status of HIV-1 RT on drug binding and inhibition of DNA synthesis. We identified "hot spots" for inhibition during active elongation. Site-specific footprinting analyses revealed that the corresponding complexes exist predominantly in the pre-translocational state. The sensitivity to PFA is significantly reduced with sequences that show a bias toward the post-translocational state. Binding studies showed that PFA stabilizes selectively the complex in the pre-translocated configuration. These findings are consistent with a Brownian ratchet model of polymerase translocation. The enzyme can rapidly shuttle between pre-and post-translocated states. The bound inhibitor acts like a pawl of a ratchet and prevents the forward motion of HIV-1 RT, whereas the bound nucleotide binds to the post-translocated complex and prevents the reverse motion. The proposed mechanisms of RT translocation and drug action are consistent with the PFA-resistant phenotypes. We show that certain sequences and the PFA-resistant E89K mutant diminishes the stability of the pre-translocated complex. Inthesecases, the enzyme is seen at multiple positions around the 3' end of the primer, which provides a novel mechanism for resistance. These findings validate the pre-translocated complex as a target for the development of novel, perhaps less toxic and more potent inhibitors that block HIV-1 RT translocation.