Evolution of resistant M414T mutants among hepatitis C virus replicon cells treated with polymerase inhibitor A-782759
Evolution of resistant M414T mutants among hepatitis C virus replicon cells treated with polymerase inhibitor A-782759
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DOI：
10.1128/aac.01004-06
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发表时间：
2007-06-01
影响因子：
4.9
通讯作者：
Molla, Akhteruzzaman
中科院分区：
文献类型：
--
作者：
Lu, Liangjun;Mo, Hongmei;Molla, Akhteruzzaman
在体外用任何单一特异性抗HCV抑制剂处理丙型肝炎病毒（HCV）复制子细胞导致快速选择抗性突变体。然而，这些耐药突变体的来源和动力学演变过程中的治疗知之甚少。在这项研究中，我们开发了等位基因特异性实时PCR检测M414 T突变体的定量检测，选择了一些苯并噻二嗪HCV聚合酶抑制剂。在1b-con 1（0.22%）和1b-N（0.18%）亚基因组复制子细胞系中检测到低水平的预先存在的M414 T突变体，以及从未经治疗的HCV感染患者血清中分离的15种HCV RNA中的6种，范围为0.11%至0.60%。用苯并噻二嗪抑制剂A-782759处理后，复制子中M414 T突变体的比例以剂量依赖性方式迅速增加。处理4天后，使用1倍、10倍或100倍其50%有效浓度的A-782759时，分别有2.5、26或60%的复制子群体含有M414 T突变体。此外，短暂的4天处理导致复制子细胞中抑制剂敏感性的显著变化。我们的研究结果表明，抗性突变体预先存在的复制子细胞中的一个较小的人口和突变体的抑制剂治疗的天内选择。这项研究的结果表明，早期应用HCV特异性抑制剂与基于干扰素的方案或其他类型的可用抑制剂的联合治疗是必要的，以避免快速病毒反弹或治疗失败。
Treatment of hepatitis C virus (HCV) replicon cells with any single specific anti-HCV inhibitor in vitro leads to a rapid selection of resistant mutants. However, the source and the kinetic evolution of these resistant mutants during treatment are poorly understood. In this study we developed allele-specific real-time PCR assays for quantitative detection of the M414T mutant that was selected by a number of benzothiadiazine HCV polymerase inhibitors. Low levels of preexisting M414T mutants were detected in both 1b-con1 (0.22%) and 1b-N (0.18%) subgenomic replicon cell lines, as well as in 6 of 15 HCV RNA isolated from the sera of treatment-naive HCV-infected patients ranging from 0.11 to 0.60%. The proportion of M414T mutants in replicons rapidly increased in a dose-dependent manner upon treatment with benzothiadiazine inhibitor A-782759. After 4 days of treatment, 2.5, 26, or 60% of the replicon population contained M414T mutants with the use of A-782759 at 1x, 10X, or 100X its 50% effective concentration, respectively. In addition, the short 4-day treatment resulted in significant changes in inhibitor susceptibility in the replicon cells. Our results indicated that the resistant mutant preexisted as a minor population in replicon cells and that the mutant was selected within days of treatment with the inhibitor. The findings from this study suggested that early application of combination therapy of an HCV-specific inhibitor with interferon-based regimens or other classes of available inhibitors will be necessary to avoid quick viral rebound or treatment failure.