Robust HCV Genotype 3a Infectious Cell Culture System Permits Identification of Escape Variants With Resistance to Sofosbuvir
Robust HCV Genotype 3a Infectious Cell Culture System Permits Identification of Escape Variants With Resistance to Sofosbuvir
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DOI：
10.1053/j.gastro.2016.07.013
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发表时间：
2016-11-01
期刊：
影响因子：
29.4
通讯作者：
Bukh, Jens
中科院分区：
文献类型：
--
作者：
Ramirez, Santseharay;Mikkelsen, Lotte S.;Bukh, Jens
背景和目的：直接作用抗病毒药物 (DAA) 可有效根除慢性丙型肝炎病毒 (HCV) 感染，尽管基因型 3a 的 HCV 对这些药物的反应较差。我们的目标是开发基因型 3a 感染培养物，并研究 NS5A 和 NS5B 抑制剂的作用以及对索非布韦（唯一被批准用于治疗慢性 HCV 感染的核苷酸类似物）的耐药性。方法：将开发的 HCV 基因型 3a 全长基因组 (DBN3a) 转染至 Huh7.5 细胞中，该基因组具有株 DBN 编码序列、修饰的 NS5B 共有序列、pS52 非翻译区以及来自培养有效的 JFH1 核心 NS5A (DBN) 重组体的编码突变。所选 DAA 的功效通过剂量反应测定来确定，其中在与不同浓度的特定 DAA 孵育后测量 HCV 感染细胞的数量。使用浓度不断增加的索磷布韦对受感染的 Huh7.5 细胞进行长期培养，以促进 HCV 抗性变体的选择。结果：我们设计了具有 17 个取代的 DBN3a 变体 (DBN3a(cc))，其在 Huh7.5 细胞中的复制和增殖动力学与原型 J6/JFH1 相当。适应性突变还产生了具有来自HCV基因型3a毒株S52和DH11的NS5B的培养有效的基于DBN的重组体。与基因型1a相比，基因型3a对达卡他韦、雷迪帕韦和艾尔巴韦的敏感性较低，但对奥比他韦、维帕他韦、贝拉布韦、达沙布韦、MK-3682和索磷布韦同样敏感。将感染 DBN3a 的 Huh7.5 细胞暴露于索磷布韦，导致鉴定出带有 NS5B 取代的逃逸变体，包括与耐药性相关的取代 S282T。与 DBN3a 相比，该变体显示 Huh7.5 细胞的感染性增强，并且在不含索磷布韦的细胞培养物中具有遗传稳定性。索磷布韦、MK-3682、达沙布韦或索磷布韦和雷迪帕韦或索磷布韦和维帕他韦的组合对 DBN3a 索磷布韦逃逸变体感染的疗效降低。结论：我们开发了一种高效培养 HCV 基因型 3a 的系统。基因型 1a 对索磷布韦耐药性具有很高的遗传屏障，而基因型 3a 可以诱导对该 DAA 的耐药性。因此，我们分离出 HCV 基因型 3a 变体，其对索磷布韦的敏感性降低，适应性增加，并且对其他 NS5B 抑制剂具有交叉耐药性。这些发现表明，索磷布韦逃逸变体可能会损害核苷酸类似物抗 HCV 的有效性。 GenBank 登录号：KX280712-KX280716。
BACKGROUND & AIMS: Direct-acting antivirals (DAAs) effectively eradicate chronic hepatitis C virus (HCV) infection, although HCV genotype 3a is less responsive to these drugs. We aimed to develop genotype 3a infectious cultures and study the effects of inhibitors of NS5A and NS5B and resistance to sofosbuvir-the only nucleotide analog approved for treatment of chronic HCV infection. METHODS: The developed HCV genotype 3a full-length genome (DBN3a), with a strain-DBN coding sequence, modified NS5B consensus sequence, pS52 untranslated regions, and coding mutations from a culture-efficient JFH1-based core-NS5A (DBN) recombinant, was transfected into Huh7.5 cells. The efficacy of selected DAAs was determined in dose-response assays, in which the number of HCV-infected cells was measured after incubation with different concentrations of the specific DAA. Long-term culture of infected Huh7.5 cells with increasing concentrations of sofosbuvir was used to promote selection of HCV-resistant variants. RESULTS: We engineered a DBN3a variant with 17 substitutions (DBN3a(cc)) that had replication and propagation kinetics in Huh7.5 cells comparable with prototype J6/JFH1. The adaptive mutations also produced culture-efficient DBN-based recombinants with NS5B from HCV genotype 3a strains S52 and DH11. Compared with genotype 1a, genotype 3a was less sensitive to daclatasvir, ledipasvir, and elbasvir, but equally sensitive to ombitasvir, velpatasvir, beclabuvir, dasabuvir, MK-3682, and sofosbuvir. Exposure of Huh7.5 cells infected with DBN3a to sofosbuvir led to identification of an escape variant with substitutions in NS5B, including the resistance-associated substitution S282T. This variant showed increased infectivity of Huh7.5 cells, compared with DBN3a, and was genetically stable in cell cultures without sofosbuvir. Sofosbuvir, MK-3682, dasabuvir, or combinations of sofosbuvir and ledipasvir or sofosbuvir and velpatasvir had decreased efficacy against infection with the DBN3a sofosbuvir escape variant. CONCLUSIONS: We developed a system for highly efficient culture of HCV genotype 3a. Genotype 1a has a high genetic barrier to resistance for sofosbuvir, whereas resistance to this DAA can be induced in genotype 3a. We therefore isolated HCV genotype 3a variants with reduced sensitivity to sofosbuvir, with increased fitness and with cross-resistance to other NS5B inhibitors. These findings indicate that sofosbuvir escape variants could compromise the effectiveness of nucleotide analogs against HCV. GenBank accession numbers: KX280712-KX280716.