Coto-Llerena Mairene, Koutsoudakis George, Boix Loreto, López-Oliva Juan Manuel, Caro-Pérez Noelia, Fernández-Carrillo Carlos, González Patricia, Gastaminza Pablo, Bruix Jordi, Forns Xavier, Pérez-Del-Pulgar Sofía
Liver Unit, Hospital Clínic, IDIBAPS, CIBERehd, Barcelona, Spain.
Liver Unit, Hospital Clínic, IDIBAPS, CIBERehd, Barcelona, Spain.
Virus Res. 2017 Aug 15;240:35-46. doi: 10.1016/j.virusres.2017.07.018. Epub 2017 Jul 24.
Hepatitis C virus (HCV) is a globally prevalent pathogen and is associated with high death rates and morbidity. Since its discovery in 1989, HCV research has been impeded by the lack of a robust infectious cell culture system and thus in vitro studies on diverse genetic backgrounds are hampered because of the limited number of hepatoma cell lines which are able to support different aspects of the HCV life cycle. In the current study, we sought to expand the limited number of permissive cells capable of supporting the diverse phases of the HCV life cycle. Initially, we screened a panel of new hepatoma-derived cell lines, designated BCLC-1, -2, -3, -4, -5, -6, -9 and -10 cells, for their ability to express essential HCV receptors and subsequently to support HCV entry by using the well-characterized HCV pseudoparticle system (HCVpp). Apart from BCLC-9, all BCLC cell lines were permissive for HCVpp infection. Next, BCLC cells were subjected to short- and long-term HCV RNA replication studies using HCV subgenomic replicons. Interestingly, only BCLC-1, -5 and -9 cells, supported short-term HCV RNA replication, but the latter were excluded from further studies since they were refractory for HCV entry. BCLC-1, -5 were able to support long-term HCV replication too; yet BCLC-5 cells supported the highest long-term HCV RNA replication levels. Furthermore, cured BCLC-5 clones from HCV subgenomic replicon, showed increased permissiveness for HCV RNA replication. Strikingly, we were unable to detect endogenous BCLC-5 miR122 expression - an important HCV host factor- and as expected, the exogenous expression of miR122 in BCLC-5 cells increased their permissiveness for HCV RNA replication. However, this cell line was unable to produce HCV infectious particles despite ectopic expression of apolipoprotein E, which in other hepatoma cell lines has been shown to be sufficient to enable the HCV secretion process, suggesting a lack of other host cellular factor(s) and/or the presence of inhibitory factor(s). In conclusion, the establishment of these new permissive cell lines for HCV entry and replication, which possess a different genetic background compared to the well-established models, expands the current repertoire of hepatoma cell lines susceptible to the study of the HCV life cycle and also will aid to further elucidate the cellular determinants that modulate HCV replication, assembly and egress.
丙型肝炎病毒（HCV）是一种全球流行的病原体，与高死亡率和高发病率相关。自1989年被发现以来，HCV研究一直受到缺乏强大的感染性细胞培养系统的阻碍，因此，由于能够支持HCV生命周期不同方面的肝癌细胞系数量有限，对不同遗传背景的体外研究也受到了阻碍。在本研究中，我们试图扩大能够支持HCV生命周期不同阶段的有限数量的允许细胞。最初，我们筛选了一组新的肝癌衍生细胞系，命名为BCLC-1、-2、-3、-4、-5、-6、-9和-10细胞，检测它们表达HCV必需受体的能力，随后使用特征明确的HCV假病毒颗粒系统（HCVpp）来支持HCV进入。除了BCLC-9，所有BCLC细胞系都允许HCVpp感染。接下来，使用HCV亚基因组复制子对BCLC细胞进行短期和长期的HCV RNA复制研究。有趣的是，只有BCLC-1、-5和-9细胞支持短期HCV RNA复制，但由于它们对HCV进入具有抗性，因此BCLC-9细胞被排除在进一步研究之外。BCLC-1、-5也能够支持长期HCV复制；然而，BCLC-5细胞支持的长期HCV RNA复制水平最高。此外，从HCV亚基因组复制子中获得的治愈的BCLC-5克隆，对HCV RNA复制的允许性增加。令人惊讶的是，我们无法检测到内源性BCLC-5 miR122表达——一种重要的HCV宿主因子——正如预期的那样，BCLC-5细胞中miR122的外源表达增加了它们对HCV RNA复制的允许性。然而，尽管异位表达了载脂蛋白E，但该细胞系仍无法产生HCV感染性颗粒，在其他肝癌细胞系中，载脂蛋白E已被证明足以促进HCV分泌过程，这表明缺乏其他宿主细胞因子和/或存在抑制因子。总之，这些用于HCV进入和复制的新允许细胞系的建立，与已建立的模型相比具有不同的遗传背景，扩大了目前易用于研究HCV生命周期的肝癌细胞系的范围，也将有助于进一步阐明调节HCV复制、组装和释放的细胞决定因素。
