HBV聚合酶的新型rtM204突变降低了对阿德福韦和替诺福韦的易感性

Ke Zhang, C. Bach, Maria Neumann-Fraune, Yuchen Xia, B. Beggel, R. Kaiser, V. Schildgen, A. Krämer, O. Schildgen, U. Protzer
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引用次数: 1

摘要

背景:慢性hbv感染的治疗受到耐药性选择的限制。HBV逆转录酶YMDD基序中的rtM204I/V突变是对拉米夫定和恩替卡韦的耐药决定因素,但对阿德福韦或替诺福韦没有耐药性。后两种药物的系统表型数据有限。方法:在CMV启动子控制下,系统地将rtM204突变(rtM204A/I/K/L/Q/S/T/V)引入具有复制能力的1.1倍hbv超长构建体中。归一化瞬时转染后,采用选择性qPCR检测病毒复制适应度。采用标准化的高通量表型分析,通过测定拉米维顿、恩替卡韦、阿德福韦和替诺福韦的IC50值来评估体外药物敏感性。通过感染HepaRG细胞分析其感染性。结果:体外表型分析显示,rtM204K对阿德福韦和替诺福韦具有高水平的耐药性,但同时损害了复制能力。rtM204I/V描述的rtL180M或rtL80I不能恢复其适应度。rtM204L和rtM204Q降低了对阿德福韦/替诺福韦的敏感性,但不丧失复制能力。rtM204A/I/S/T显著降低对两种药物的敏感性。有趣的是,单突变rtM204V对这两种药物的敏感性显著降低,但与代偿突变rtL180M联合后失去耐药性。通过影响重叠的s基因,除rtM204L外,rtM204突变体对HepaRG细胞的感染性降低或减弱。结论:我们已经建立了一种具有时间和成本效益的表型测定方法,并鉴定了新的rtM204突变,使其在体外对阿德福韦和替诺福韦产生交叉抗性。尽管它们在病毒群体中的频率很低,但它们的临床意义不应被低估,因为它们可能会选择代偿性突变,从而恢复病毒的适应性。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
Novel rtM204 Mutations in HBV Polymerase Confer Reduced Susceptibility to Adefovir and Tenofovir
Background: Treatment of chronic HBV-infection is limited by selection of resistance. The rtM204I/V mutations in the YMDD motif of HBV reverse transcriptase are well documented resistance determinants against lamivudine and entecavir, but not against adefovir or tenofovir. Limited systematic phenotypic data are available for the latter two drugs. Methods: rtM204 mutations (rtM204A/I/K/L/Q/S/T/V) were systematically introduced into replication-competent 1.1-fold HBV-overlength constructs under control of a CMV promoter. Viral replication fitness was determined by selective qPCR after normalized transient transfection. In vitro drug susceptibilities were evaluated by determining IC50 values of lamividune, entecavir, adefovir, and tenofovir using standardized high-throughput phenotypic assays. Infectivity was analyzed by infection of HepaRG cells. Results: In vitro phenotyping showed that rtM204K conferred high-level resistance to adefovir and tenofovir but simultaneously impaired replication capacity. Its fitness could not be restored by rtL180M or rtL80I as described for rtM204I/V. rtM204L and rtM204Q conferred low-level reduced susceptibility to adefovir/tenofovir without loss of replication capacity. rtM204A/I/S/T reduced susceptibility to either drug substantially. Interestingly, the single mutation rtM204V showed significantly reduced susceptibility to both drugs but lost resistance in combination with the compensatory mutation rtL180M. By affecting the overlapping S-gene, rtM204 mutants except rtM204L showed reduced or diminished infectivity in HepaRG cells. Conclusions: We have established a time- and cost-effective phenotypic assay and identified novel rtM204 mutations conferring cross resistance to adefovir and tenofovir in vitro. Despite of their low frequency in the viral population, their clinical significance should not be underestimated due to the potential selection of compensatory mutations, which may restore viral fitness.
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