Ribavirin has been used for 25 years to treat patients with chronic hepatitis C virus (HCV) infection; however, its antiviral mechanism of action remains unclear. Here, we studied virus evolution in a subset of samples from a randomized 24-week trial of ribavirin monotherapy versus placebo in chronic HCV patients, as well as the viral resistance mechanisms of the observed ribavirin-associated mutations in cell culture. Thus, we performed next-generation sequencing of the full-length coding sequences of HCV recovered from patients at weeks 0, 12, 20, 32 and 40 and analyzed novel single nucleotide polymorphisms (SNPs), diversity, and mutation-linkage. At week 20, increased genetic diversity was observed in 5 ribavirin-treated compared to 4 placebo-treated HCV patients due to new synonymous SNPs, particularly G-to-A and C-to-U ribavirin-associated transitions. Moreover, emergence of 14 nonsynonymous SNPs in HCV nonstructural 5B (NS5B) occurred in treated patients, but not in placebo controls. Most substitutions located close to the NS5B polymerase nucleotide entry site. Linkage analysis showed that putative resistance mutations were found in the majority of genomes in ribavirin-treated patients. Identified NS5B mutations from genotype 3a patients were further introduced into the genotype 3a cell-culture-adapted DBN strain for studies in Huh7.5 cells. Specific NS5B substitutions, including DBN-D148N+I363V, DBN-A150V+I363V, and DBN-T227S+S183P, conferred resistance to ribavirin in long-term cell culture treatment, possibly by reducing the HCV polymerase error rate. In conclusion, prolonged exposure of HCV to ribavirin in chronic hepatitis C patients induces NS5B resistance mutations leading to increased polymerase fidelity, which could be one mechanism for ribavirin resistance.

中文翻译：

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在利巴韦林治疗的慢性丙型肝炎患者的HCV聚合酶中鉴定出的突变会引起耐药性并影响病毒复制的保真度。

利巴韦林已经用于治疗慢性丙型肝炎病毒（HCV）患者已有25年。但是，其抗病毒作用机制仍不清楚。在这里，我们研究了在慢性HCV患者中进行的利巴韦林单药治疗与安慰剂随机24周随机试验的样本子集中的病毒进化，以及在细胞培养物中观察到的利巴韦林相关突变的病毒抗性机制。因此，我们对在第0、12、20、32和40周时从患者体内回收的HCV的全长编码序列进行了下一代测序，并分析了新颖的单核苷酸多态性（SNP），多样性和突变链接。在第20周，由于新的同义SNP，与5名接受利巴韦林治疗的患者相比，与4名接受安慰剂治疗的HCV患者相比，遗传多样性有所提高，特别是G到A和C到U的利巴韦林相关转换。此外，HCV非结构性5B（NS5B）中出现14种非同义SNP出现在接受治疗的患者中，但没有出现在安慰剂对照中。大多数取代位于NS5B聚合酶核苷酸进入位点附近。连锁分析表明，在利巴韦林治疗的患者的大多数基因组中发现了推定的抗性突变。将来自基因型3a患者的已鉴定NS5B突变进一步引入适应基因型3a细胞培养的DBN菌株，以在Huh7.5细胞中进行研究。特定的NS5B替代，包括DBN-D148N + I363V，DBN-A150V + I363V和DBN-T227S + S183P，在长期的细胞培养处理中可能通过降低HCV聚合酶错误率赋予了对病毒唑的抗性。结论，