Compensation by the E138K Mutation in HIV-1 Reverse Transcriptase for Deficits in Viral Replication Capacity and Enzyme Processivity Associated with the M184I/V Mutations
Compensation by the E138K Mutation in HIV-1 Reverse Transcriptase for Deficits in Viral Replication Capacity and Enzyme Processivity Associated with the M184I/V Mutations
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DOI：
10.1128/jvi.05584-11
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发表时间：
2011-11-01
影响因子：
5.4
通讯作者：
Wainberg, Mark A.
中科院分区：
文献类型：
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作者：
Xu, Hong-Tao;Asahchop, Eugene L.;Wainberg, Mark A.
最近，几项3期临床试验（ECHO和THRIVE）显示，E138 K和M184 I是利匹韦林（RPV）联合恩曲他滨（FTC）和替诺福韦（TDF）两种核苷（酸）逆转录酶抑制剂治疗失败的患者中最常见的突变。为了研究E138 K和M184 I共存的基础，我们生成了重组突变型和野生型（WT）逆转录酶（RT）酶和HIV-1（NL 4 -3）感染性克隆。在脐带血单个核细胞（CBMCs）中测定药物敏感性。进行结构建模以分析对脱氧核苷三磷酸（dNTP）结合的任何影响。表型分析结果表明，同时含有E138 K和M184 V突变的病毒比含有E138 K和M184 I的病毒对FTC、3 TC和ETR的抗性更强。具有E138 K的病毒仅显示出对ETR的中度抗性，对依法韦仑（EFV）的抗性很小，并且对FTC或3 TC没有抗性。E138 K在M184 I/V存在下恢复病毒复制能力（RC），这在无细胞RT持续合成能力测定中得到证实。在低dNTP浓度下，含有E138 K、E138 K/184 I或E138 K/184 V的RT酶表现出比WT RT更高的持续合成能力。稳态动力学分析表明E138 K突变导致dNTPs的K（m）s降低。与此相反，M184 I/V导致增加的K（m）的dNTP相比，WT RT。这些结果表明，E138 K突变弥补了缺陷的dNTP的使用和M184 I/V的复制能力的损害。结构建模表明，除了E138 K的M184 I/V促进更紧密的dNTP结合。
Recently, several phase 3 clinical trials (ECHO and THRIVE) showed that E138K and M184I were the most frequent mutations to emerge in patients who failed therapy with rilpivirine (RPV) together with two nucleos( t)ide reverse transcriptase inhibitors, emtricitabine (FTC) and tenofovir (TDF). To investigate the basis for the copresence of E138K and M184I, we generated recombinant mutated and wild-type (WT) reverse transcriptase (RT) enzymes and HIV-1(NL4-3) infectious clones. Drug susceptibilities were determined in cord blood mononuclear cells (CBMCs). Structural modeling was performed to analyze any impact on deoxynucleoside triphosphate (dNTP) binding. The results of phenotyping showed that viruses containing both the E138K and M184V mutations were more resistant to each of FTC, 3TC, and ETR than viruses containing E138K and M184I. Viruses with E138K displayed only modest resistance to ETR, little resistance to efavirenz (EFV), and no resistance to either FTC or 3TC. E138K restored viral replication capacity (RC) in the presence of M184I/V, and this was confirmed in cell-free RT processivity assays. RT enzymes containing E138K, E138K/184I, or E138K/184V exhibited higher processivity than WT RT at low dNTP concentrations. Steady-state kinetic analysis demonstrated that the E138K mutation resulted in decreased K(m)s for dNTPs. In contrast, M184I/V resulted in an increased K(m) for dNTPs compared to those for WT RT. These results indicate that the E138K mutation compensates for both the deficit in dNTP usage and impairment in replication capacity by M184I/V. Structural modeling shows that the addition of E138K to M184I/V promotes tighter dNTP binding.