Nucleoside analog resistance caused by insertions in the fingers of human immunodeficiency virus type 1 reverse transcriptase involves ATP-mediated excision
Nucleoside analog resistance caused by insertions in the fingers of human immunodeficiency virus type 1 reverse transcriptase involves ATP-mediated excision
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DOI：
10.1128/jvi.76.18.9143-9151.2002
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发表时间：
2002-09-01
影响因子：
5.4
通讯作者：
Hughes, SH
中科院分区：
文献类型：
--
作者：
Boyer, PL;Sarafianos, SG;Hughes, SH
尽管抗人类免疫缺陷病毒1型(HIV-1)疗法延长了患者的生命，但耐药性是一个重大问题。特别令人担忧的是导致对特定类别药物产生交叉耐药性的突变。在导致对几种核苷类似物产生抗药性的突变中，有一种是在HIV-1逆转录酶(RT)的Finger亚域第69和70位插入氨基酸。这些插入通常与侧翼氨基酸的变化以及215位的F或Y的变化有关。我们认为，T215F/Y突变使ATP与HIV-1RT的结合更加有效，从而在体外增加了3-叠氮-3‘-脱氧胸苷-5’-单磷酸(AZTMP)的切除，在体内增加了对齐多夫定(AZT)的耐药性。虽然AZT的抗性机制涉及到增强的切除，但对3TC的抵抗涉及到阻止类似物的掺入。我们测量了手指插入突变对几个核苷类似物的误整合和切除的影响。在手指和T215Y插入氨基酸的RT变异体降低了ddATP和3TCTP的误掺入水平。这些突变体还具有通过依赖于ATP的焦磷酸化来切除AZTMP的能力。然而，与经典的AZT耐药突变(M41L/D67N/K70R/T215Y或F/K219E或Q)不同，手指中的氨基酸插入和T215Y突变相结合，即使在反应中存在相对较高水平的脱氧核苷三磷酸，也可以有效地切除ddTMP和d4TMP。虽然其他核苷的双脱氧核苷类似物的切除速度比AZTMP、ddTMP和d4TMP慢，但插入手指和T215Y的突变体切除了所有经测试比野生型RT或携带经典AZT耐药突变的突变体RT更有效的核苷类似物。在三元复合体(RT/模板-引物/dNTP)中，结合的dNTP的存在阻止了引物末端进入发生切除的核苷酸结合位点(N位)。凝胶位移分析表明，与野生型HIV-1RT相比，手指中的氨基酸插入破坏了三元复合体的稳定性。如果三元复合体是不稳定的，引物的末端可以接近N位，从而发生切除。这可以解释核苷类似物的增强切除。
Although anti-human immunodeficiency virus type 1 (HIV-1) therapy has prolonged the lives of patients, drug resistance is a significant problem. Of particular concern are mutations that cause cross-resistance to a particular class of drugs. Among the mutations that cause resistance to several nucleoside analogs are the insertion of amino acids in the fingers subdomain of HIV-1 reverse transcriptase (RT) at positions 69 and 70. These insertions are usually associated with changes in the flanking amino acids and with a change to F or Y at position 215. We have proposed that the T215F/Y mutation makes the binding of ATP to HIV-1 RT more effective, which increases the excision of 3-azido-3'-deoxythymidine-5'-monophosphate (AZTMP) in vitro and increases zidovudine (AZT) resistance in vivo. Although the mechanism of AZT resistance involves enhanced excision, resistance to 3TC involves a block to incorporation of the analog. We measured the effects of fingers insertion mutations on the misincorporation and excision of several nucleoside analogs. RT variants with the amino acid insertions in the fingers and T215Y have a decreased level of misincorporation of ddATP and 3TCTP. These mutants also have the ability to excise AZTMP by ATP-dependent pyrophosphorylysis. However, unlike the classic AZT resistance mutations (M41L/D67N/K70R/T215Y or F/K219E or Q), the combination of the amino acid insertions in the fingers and the T215Y mutation allows efficient excision of ddTMP and d4TMP, even when relatively high levels of deoxynucleoside triphosphates are present in the reaction. Although the dideoxynucleoside analogs of other nucleosides were excised more slowly than AZTMP, ddTMP, and d4TMP, the mutants with the fingers insertion and T215Y excised all of the nucleoside analogs that were tested more efficiently than wild-type RT or a mutant RT carrying the classical AZT resistance mutations. In the ternary complex (RT/template-primer/dNTP), the presence of the bound dNTP prevents the end of the primer from gaining access to the nucleotide binding site(N site) where excision occurs. Gel shift analysis showed that the amino acid insertions in the fingers destabilized the ternary complex compared to wild-type HIV-1 RT. If the ternary complex is unstable, the end of the primer can gain access to the N site and excision can occur. This could explain the enhanced excision of the nucleoside analogs.