Biochemical and Structural Properties of Entecavir-Resistant Hepatitis B Virus Polymerase with L180M/M204V Mutations
Biochemical and Structural Properties of Entecavir-Resistant Hepatitis B Virus Polymerase with L180M/M204V Mutations
复制标题
具有L180M/M204V突变的恩替卡韦耐药乙型肝炎病毒聚合酶的生化和结构特性
DOI：
10.1128/jvi.02401-20
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发表时间：
2021-08-01
影响因子：
5.4
通讯作者：
Toyoda, Tetsuya
中科院分区：
文献类型：
--
作者：
Nakajima, Shogo;Watashi, Koichi;Toyoda, Tetsuya
恩替卡韦（ETV）是一种广泛使用的抗乙型肝炎病毒（HBV）药物。然而，HBV逆转录酶（RT）耐药突变的出现导致治疗失败。为了了解HBV RT对ETV产生耐药性的机制，我们结合生化和结构技术分析了L180M、M204V和L180M/M204V突变体。通过Lineweaver-Burk图观察到，ETV-triphosphate （ETV-TP）在野生型（wt） RT和M204V RT中都表现出对dGTP的竞争性抑制。相比之下，RT L180M或L180M/M204V不适合竞争性、非竞争性、非竞争性或典型的混合抑制，尽管ETV-TP是dGTP的竞争性抑制剂。模拟HBV RT L180M/M204V的HIV RTY115F/F116Y/Q151M/F160M/M184V的晶体学显示，F160M突变引起的F115凸起（HBV RT中的F88）诱导dCTP偏离其正常紧密结合位置。ETV-TP在偏离的dCTP上的建模表明，ETV-TP亚甲基与3'端核苷核糖之间可能发生空间冲突。ETV-TP可能首先与HBV RT - M171相互作用，然后在dNTP结合位点最终调节（Y. Yasutake， S. Hattori， H. Hayashi， K. Matsuda等，Sci Rep 8:1624, 2018, https://doi.org/10.1038/s41598-018-19602-9）。因此，在HBV RT L180M/M204V中，ETV-TP可能卡在M171位点，这一残基在几乎所有HBV分离株中都是保守的，导致在动力学分析中观察到奇怪的抑制模式。总之，我们的研究结果为L180M和M204V突变引起HBV RT的ETV耐药机制提供了新的见解。全球有2.57亿人感染乙肝病毒，这些人患肝硬化和癌症的风险增加。ETV是最有效的抗HBV药物之一，ETV在HBV RT中的耐药突变已被广泛研究。然而，ETV抗性的机制仍然难以捉摸。我们提出了一个有吸引力的假设来解释ETV阻力和有效性，使用动力学和结构分析相结合。ETV可能有一个额外的相互作用位点M171，在HBV RT的dNTP口袋旁边；这一发现表明，识别RT内多个相互作用位点的核苷类似物（NAs）可能有效抑制该酶。对ETV进行修饰可以使其更有效，并可以合理设计高效的NA抑制剂。
Entecavir (ETV) is a widely used anti-hepatitis B virus (HBV) drug. However, the emergence of resistant mutations in HBV reverse transcriptase (RT) results in treatment failure. To understand the mechanism underlying the development of ETV resistance by HBV RT, we analyzed the L180M, M204V, and L180M/M204V mutants using a combination of biochemical and structural techniques. ETV-triphosphate (ETV-TP) exhibited competitive inhibition with dGTP in both wild-type (wt) RT and M204V RT, as observed using Lineweaver-Burk plots. In contrast, RT L180M or L180M/M204V did not fit either competitive, uncompetitive, noncompetitive, or typical mixed inhibition, although ETV-TP was a competitive inhibitor of dGTP. Crystallography of HIV RTY115F/F116Y/Q151M/F160M/M184V, mimicking HBV RT L180M/M204V, showed that the F115 bulge (F88 in HBV RT) caused by the F160M mutation induced deviated binding of dCTP from its normal tight binding position. Modeling of ETV-TP on the deviated dCTP indicated that a steric clash could occur between ETV-TP methylene and the 3'-end nucleoside ribose. ETV-TP is likely to interact primarily with HBV RT M171 prior to final accommodation at the deoxynucleoside triphosphate (dNTP) binding site (Y. Yasutake, S. Hattori, H. Hayashi, K. Matsuda, et al., Sci Rep 8:1624, 2018, https://doi.org/10.1038/s41598-018-19602-9). Therefore, in HBV RT L180M/M204V, ETV-TP may be stuck at M171, a residue that is conserved in almost all HBV isolates, leading to the strange inhibition pattern observed in the kinetic analysis. Collectively, our results provide novel insights into the mechanism of ETV resistance of HBV RT caused by L180M and M204V mutations.IMPORTANCE HBV infects 257 million people in the world, who suffer from elevated risks of liver cirrhosis and cancer. ETV is one of the most potent anti-HBV drugs, and ETV resistance mutations in HBV RT have been extensively studied. Nevertheless, the mechanisms underlying ETV resistance have remained elusive. We propose an attractive hypothesis to explain ETV resistance and effectiveness using a combination of kinetic and structural analyses. ETV is likely to have an additional interaction site, M171, beside the dNTP pocket of HBV RT; this finding indicates that nucleos(t)ide analogues (NAs) recognizing multiple interaction sites within RT may effectively inhibit the enzyme. Modification of ETV may render it more effective and enable the rational design of efficient NA inhibitors.