HIV-1 reverse transcriptase mutants resistant to nonnucleoside reverse transcriptase inhibitors do not adversely affect DNA synthesis: pre-steady-state and steady-state kinetic studies.
HIV-1 reverse transcriptase mutants resistant to nonnucleoside reverse transcriptase inhibitors do not adversely affect DNA synthesis: pre-steady-state and steady-state kinetic studies.
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对非核苷类逆转录酶抑制剂具有抗性的 HIV-1 逆转录酶突变体不会对 DNA 合成产生不利影响：稳态前和稳态动力学研究。
DOI：
10.1097/01.qai.0000222288.90201.33
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发表时间：
2006
期刊：
影响因子：
--
通讯作者：
Demeter,LisaM
中科院分区：
文献类型：
--
作者：
Domaoal,RobertA;Bambara,RobertA;Demeter,LisaM
我们以前已经证明，非核苷类逆转录酶抑制剂（NNRTI）耐药突变体具有不同水平的复制健身相对于野生型，那些更大的健身减少不太可能在治疗过程中发展的患者。我们还发现，突变型RT的RNase H切割速率的降低与适应性的降低相关，并且NNRTI抗性RT催化聚合的持续合成能力与野生型相似。在本研究中，我们更详细地评估了3种临床发生的NNRTI耐药RT（K103 N、P236 L和V106 A）在稳态前和稳态条件下的聚合酶功能。与野生型相比，突变型RT的单核苷酸掺入的总体途径没有变化。此外，NNRTI抗性突变体的核苷酸掺入率（k pol），对dGTP的亲和力（K d），和稳态聚合率（k ss和k cat），使用RNA或DNA模板，每个类似于野生型。这些发现表明，NNRTI抗性突变与聚合酶活性位点的紧密接近不影响酶与传入核苷酸或引物模板的相互作用，足以影响聚合，并支持RNA酶H活性的降低有助于复制适应性降低的假设。
We have previously demonstrated that nonnucleoside reverse transcriptase inhibitor (NNRTI)-resistant mutants have different levels of replication fitness relative to wild type; those with greater reductions in fitness are less likely to develop during therapy in patients. We have also found that reductions in rates of RNase H cleavage by mutant RTs correlate with reductions in fitness and that NNRTI-resistant RTs catalyze polymerization with a processivity similar to wild type. In this study, we evaluated the polymerase function of 3 clinically occurring NNRTI-resistant RTs (K103N, P236L, and V106A) in greater detail, under both pre-steady-state and steady-state conditions. The overall pathway of single-nucleotide incorporation was unchanged for the mutant RTs compared with wild type. In addition, the NNRTI-resistant mutants were each similar to wild type in rate of nucleotide incorporation (k pol), affinity for dGTP (K d), and steady-state rate of polymerization (k ss and k cat), using either RNA or DNA templates. These findings suggest that the close proximity of the NNRTI-resistance mutations to the polymerase active site does not affect the interactions of the enzyme with the incoming nucleotide or the primer-template sufficiently to affect polymerization and support the hypothesis that these reductions in RNase H activity contribute to reductions in replication fitness.