Resistance to a Nucleoside Analog Antiviral Drug from More Rapid Extension of Drug-Containing Primers.
Resistance to a Nucleoside Analog Antiviral Drug from More Rapid Extension of Drug-Containing Primers.
复制标题
DOI：
10.1128/mbio.03492-20
复制
发表时间：
2021-02-09
期刊：
影响因子：
6.4
通讯作者：
Coen DM
中科院分区：
文献类型：
--
作者：
Chen H;Lawler JL;Filman DJ;Hogle JM;Coen DM
虽然抗病毒药物的耐药性可能会阻碍其临床应用，但了解耐药性机制可以阐明这些药物及其靶点的作用方式。我们研究了人巨细胞病毒（HCMV）DNA聚合酶中的一个取代，该取代赋予了对主要抗HCMV药物更昔洛韦的耐药性。核苷类似物是抗病毒治疗的支柱。虽然对这些药物的耐药性阻碍了它们的使用，但了解耐药性可以阐明药物及其靶点的机制。某些核苷类似物，如更昔洛韦（GCV），一种治疗人巨细胞病毒（HCMV）的主要药物，含有3′-羟基部分的等价物，但它们的三磷酸盐可以终止基因组合成（非专性链终止）。对于更昔洛韦，链终止被延迟，直到随后的核苷酸掺入，之后病毒聚合酶空转（重复添加和去除掺入的核苷酸）阻止延伸。在这里，我们研究了如何丙氨酸甘氨酸取代残基987（A987 G），在保守的基序V中的拇指亚结构域的催化亚基（Pol）的HCMV DNA聚合酶，影响聚合酶的功能，克服延迟链终止和赋予更昔洛韦耐药。稳态酶动力学研究表明，这种取代对更昔洛韦三磷酸盐掺入DNA，可以解释耐药性没有影响。我们还发现取代对Pol的核酸外切酶活性没有影响，并且在掺入GCV和随后的核苷酸后，突变酶仍然表现出空转。然而，尽管延伸正常的DNA引物类似于野生型酶，A987 G Pol更迅速地延伸含有更昔洛韦的DNA引物，从而克服链终止。突变的Pol也更迅速地延伸RNA引物，以前未报道的活动HCMV Pol。结合引物模板的相关Pol的结构分析为这些结果提供了依据。这些研究揭示了一种新的耐药机制，可能适用于其他非专性链终止核苷类似物，并揭示了聚合酶的功能。
While resistance to antiviral drugs can hinder their clinical use, understanding resistance mechanisms can illuminate how these drugs and their targets act. We studied a substitution in the human cytomegalovirus (HCMV) DNA polymerase that confers resistance to a leading anti-HCMV drug, ganciclovir. Nucleoside analogs are mainstays of antiviral therapy. Although resistance to these drugs hinders their use, understanding resistance can illuminate mechanisms of the drugs and their targets. Certain nucleoside analogs, such as ganciclovir (GCV), a leading therapy for human cytomegalovirus (HCMV), contain the equivalent of a 3′-hydoxyl moiety, yet their triphosphates can terminate genome synthesis (nonobligate chain termination). For ganciclovir, chain termination is delayed until incorporation of the subsequent nucleotide, after which viral polymerase idling (repeated addition and removal of incorporated nucleotides) prevents extension. Here, we investigated how an alanine-to-glycine substitution at residue 987 (A987G), in conserved motif V in the thumb subdomain of the catalytic subunit (Pol) of HCMV DNA polymerase, affects polymerase function to overcome delayed chain termination and confer ganciclovir resistance. Steady-state enzyme kinetic studies revealed no effects of this substitution on incorporation of ganciclovir-triphosphate into DNA that could explain resistance. We also found no effects of the substitution on Pol’s exonuclease activity, and the mutant enzyme still exhibited idling after incorporation of GCV and the subsequent nucleotide. However, despite extending normal DNA primers similarly to wild-type enzyme, A987G Pol more rapidly extended ganciclovir-containing DNA primers, thereby overcoming chain termination. The mutant Pol also more rapidly extended RNA primers, a previously unreported activity for HCMV Pol. Structural analysis of related Pols bound to primer-templates provides a rationale for these results. These studies uncover a new drug resistance mechanism, potentially applicable to other nonobligate chain-terminating nucleoside analogs, and shed light on polymerase functions.