Department of Microbiology and Immunology, and Vaccine and Infectious Disease Organization-International Vaccine Centre, University of Saskatchewan, Saskatoon, Saskatchewan, Canada.
PLoS One. 2014 Feb 26;9(2):e89971. doi: 10.1371/journal.pone.0089971. eCollection 2014.
Hepatitis C Virus (HCV) is a serious global health problem, infecting almost 3% of the world's population. The lack of model systems for studying this virus limit research options in vaccine and therapeutic development, as well as for studying the pathogenesis of chronic HCV infection. Herein we make use of the liver-specific microRNA miR-122 to render mouse cell lines permissive to HCV replication in an attempt to develop additional model systems for the identification of new features of the virus and its life cycle. We have determined that some wild-type and knockout mouse cell lines--NCoA6 and PKR knockout embryonic fibroblasts--can be rendered permissive to transient HCV sub-genomic RNA replication upon addition of miR-122, but we did not observe replication of full-length HCV RNA in these cells. However, other wild-type and knockout cell lines cannot be rendered permissive to HCV replication by addition of miR-122, and in fact, different NCoA6 and PKR knockout cell line passages and isolates from the same mice demonstrated varying permissiveness phenotypes and eventually complete loss of permissiveness. When we tested knockdown of NCoA6 and PKR in Huh7.5 cells, we saw no substantial impact in sub-genomic HCV replication, which we would expect if these genes were inhibitory to the virus' life cycle. This leads us to conclude that along with the influence of specific gene knockouts there are additional factors within the cell lines that affect their permissiveness for HCV replication; we suggest that these may be epigenetically regulated, or modulated by cell line immortalization and transformation.
丙型肝炎病毒 (HCV) 是一个严重的全球健康问题，感染了全球近 3%的人口。缺乏用于研究该病毒的模型系统限制了疫苗和治疗开发方面的研究选择，也限制了对慢性 HCV 感染发病机制的研究。在此，我们利用肝脏特异性 microRNA miR-122 使小鼠细胞系能够允许 HCV 复制，试图开发其他模型系统以鉴定病毒及其生命周期的新特征。我们已经确定一些野生型和基因敲除小鼠细胞系——NCoA6 和 PKR 基因敲除胚胎成纤维细胞——在添加 miR-122 后可以允许瞬时 HCV 亚基因组 RNA 复制，但我们没有观察到这些细胞中全长 HCV RNA 的复制。然而，其他野生型和基因敲除细胞系不能通过添加 miR-122 使其对 HCV 复制具有许可性，事实上，来自相同小鼠的不同 NCoA6 和 PKR 基因敲除细胞系传代和分离株表现出不同的许可性表型，最终完全丧失了许可性。当我们在 Huh7.5 细胞中测试 NCoA6 和 PKR 的敲低时，我们没有看到亚基因组 HCV 复制有实质性的影响，这表明这些基因对病毒的生命周期没有抑制作用。这使我们得出结论，除了特定基因敲除的影响外，细胞系中还有其他因素影响它们对 HCV 复制的许可性；我们认为这些因素可能是表观遗传调控的，或者受到细胞系永生化和转化的调节。
