Satoh Shinya, Mori Kyoko, Ueda Youki, Sejima Hiroe, Dansako Hiromichi, Ikeda Masanori, Kato Nobuyuki
Department of Tumor Virology, Okayama University Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences, 2-5-1 Shikata-cho, Okayama 700-8558, Japan.
PLoS One. 2015 Feb 20;10(2):e0118313. doi: 10.1371/journal.pone.0118313. eCollection 2015.
Ribavirin (RBV) is a potential partner of interferon-based therapy and recently approved therapy using direct acting antivirals for patients with chronic hepatitis C. However, the precise mechanisms underlying RBV action against hepatitis C virus (HCV) replication are not yet understood. To clarify this point, we attempted to develop RBV-resistant cells from RBV-sensitive HCV RNA-replicating cells.
METHODOLOGY/PRINCIPAL FINDINGS: By repetitive RBV (100 μM) treatment (10 weeks) of 3.5-year-cultured OL8 cells, in which genome-length HCV RNA (O strain of genotype 1b) efficiently replicates, dozens of colonies that survived RBV treatment were obtained. These colonies were mixed together and further treated with high doses of RBV (up to 200 μM). By such RBV treatment, we successfully established 12 RBV-survived genome-length HCV RNA-replicating cell lines. Among them, three representative cell lines were characterized. HCV RNA replication in these cells resisted RBV significantly more than that in the parental OL8 cells. Genetic analysis of HCV found several common and conserved amino acid substitutions in HCV proteins among the three RBV-resistant cell species. Furthermore, using cDNA microarray and quantitative RT-PCR analyses, we identified 5 host genes whose expression levels were commonly altered by more than four-fold among these RBV-resistant cells compared with the parental cells. Moreover, to determine whether viral or host factor contributes to RBV resistance, we developed newly HCV RNA-replicating cells by introducing total RNAs isolated from RBV-sensitive parental cells or RBV-resistant cells into the HCV RNA-cured-parental or -RBV-resistant cells using an electroporation method, and evaluated the degrees of RBV resistance of these developed cells. Consequently, we found that RBV-resistant phenotype was conferred mainly by host factor and partially by viral factor.
CONCLUSIONS/SIGNIFICANCE: These newly established HCV RNA-replicating cell lines should become useful tools for further understanding the anti-HCV mechanisms of RBV.
利巴韦林（RBV）是基于干扰素治疗以及近期批准的针对慢性丙型肝炎患者使用直接抗病毒药物治疗的潜在辅助药物。然而，利巴韦林抗丙型肝炎病毒（HCV）复制的确切机制尚未明确。为阐明这一点，我们试图从对利巴韦林敏感的HCV RNA复制细胞中培育出对利巴韦林耐药的细胞。
方法/主要发现：通过对培养3.5年的OL8细胞重复进行利巴韦林（100μM）处理（10周），该细胞中基因组长度的HCV RNA（1b基因型的O株）能高效复制，获得了数十个在利巴韦林处理后存活的菌落。将这些菌落混合在一起，再用高剂量的利巴韦林（高达200μM）进一步处理。通过这样的利巴韦林处理，我们成功建立了12株对利巴韦林耐药的基因组长度HCV RNA复制细胞系。其中，对三个具有代表性的细胞系进行了特性分析。这些细胞中的HCV RNA复制对利巴韦林的抗性明显高于亲代OL8细胞。对HCV的基因分析发现，在这三种对利巴韦林耐药的细胞株中，HCV蛋白存在几个常见且保守的氨基酸替换。此外，利用cDNA微阵列和定量RT-PCR分析，我们鉴定出5个宿主基因，与亲代细胞相比，这些基因在这些对利巴韦林耐药的细胞中的表达水平普遍改变超过四倍。此外，为确定病毒或宿主因素是否导致对利巴韦林的抗性，我们通过电穿孔法将从对利巴韦林敏感的亲代细胞或对利巴韦林耐药的细胞中分离出的总RNA导入HCV RNA清除的亲代细胞或对利巴韦林耐药的细胞中，培育出新的HCV RNA复制细胞，并评估这些培育出的细胞对利巴韦林的耐药程度。结果，我们发现对利巴韦林的耐药表型主要由宿主因素决定，部分由病毒因素决定。
结论/意义：这些新建立的HCV RNA复制细胞系应成为进一步了解利巴韦林抗HCV机制的有用工具。
