HIV-1 reverse transcriptase connection subdomain mutations reduce template RNA degradation and enhance AZT excision
HIV-1 reverse transcriptase connection subdomain mutations reduce template RNA degradation and enhance AZT excision
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DOI：
10.1073/pnas.0804660105
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发表时间：
2008-08-05
影响因子：
11.1
通讯作者：
Pathak, Vinay K.
中科院分区：
文献类型：
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作者：
Delviks-Frankenberry, Krista A.;Nikolenko, Galina N.;Pathak, Vinay K.
我们之前提出，HIV-1 逆转录酶连接子结构域 (cn) 的突变通过改变核苷酸切除和模板 RNA 降解之间的平衡来增加 AZT 耐药性。为了测试该模型的预测，我们分析了先前确定的 cn 突变与胸苷类似物突变（D67N、K70R、T215Y 和 K219Q）组合对体外 RNase H 活性和 AZT 单磷酸 (AZTMP) 切除的影响。我们发现 cn 突变 G335C/D、N348I、A360I/V、V365I 和 A376S 减少了初级和次级 RNase H 裂解。与 DNA 模板相比，源自患者的 cns 增加了 RNA 模板上 ATP 和 PPi 介导的 AZTMP 切除。 5 个 cns 之一导致 DNA 模板上 ATP 介导的 AZTMP 切除增加，而 3 个 cns 显示 ATP 与 PPi 介导的切除比例较高，表明一些 cn 突变也影响 DNA 底物上的切除。总体而言，结果强烈支持以下模型：cn 突变通过减少模板 RNA 降解来增加 AZT 耐药性，从而为 RT 切除 AZTMP 提供额外的时间。
We previously proposed that mutations in the connection subdomain (cn) of HIV-1 reverse transcriptase increase AZT resistance by altering the balance between nucleotide excision and template RNA degradation. To test the predictions of this model, we analyzed the effects of previously identified cn mutations in combination with thymidine analog mutations (D67N, K70R, T215Y, and K219Q) on in vitro RNase H activity and AZT monophosphate (AZTMP) excision. We found that cn mutations G335C/D, N348I, A360I/V, V365I, and A376S decreased primary and secondary RNase H cleavages. The patient-derived cns increased ATP- and PPi-mediated AZTMP excision on an RNA template compared with a DNA template. One of 5 cns caused an increase in ATP-mediated AZTMP excision on a DNA template, whereas three cns showed a higher ratio of ATP- to PPi-mediated excision, indicating that some cn mutations also affect excision on a DNA substrate. Overall, the results strongly support the model that cn mutations increase AZT resistance by reducing template RNA degradation, thereby providing additional time for RT to excise AZTMP.