Frentzen Anne, Hueging Kathrin, Bitzegeio Julia, Pietschmann Thomas, Steinmann Eike
Division of Experimental Virology, Twincore, Centre for Experimental and Clinical Infection Research.
J Vis Exp. 2012 Jul 16(65):e4029. doi: 10.3791/4029.
Hepatitis C virus (HCV) is a hepatotropic virus with a host-range restricted to humans and chimpanzees. Although HCV RNA replication has been observed in human non-hepatic and murine cell lines, the efficiency was very low and required long-term selection procedures using HCV replicon constructs expressing dominant antibiotic-selectable markers. HCV in vitro research is therefore limited to human hepatoma cell lines permissive for virus entry and completion of the viral life cycle. Due to HCVs narrow species tropism, there is no immunocompetent small animal model available that sustains the complete HCV replication cycle. Inefficient replication of HCV in non-human cells e.g. of mouse origin is likely due to lack of genetic incompatibility of essential host dependency factors and/or expression of restriction factors. We investigated whether HCV propagation is suppressed by dominant restriction factors in either human cell lines derived from non-hepatic tissues or in mouse liver cell lines. To this end, we developed two independent conditional trans-complementation methods relying on somatic cell fusion. In both cases, completion of the viral replication cycle is only possible in the heterokaryons. Consequently, successful trans-complementation, which is determined by measuring de novo production of infectious viral progeny, indicates absence of dominant restrictions. Specifically, subgenomic HCV replicons carrying a luciferase transgene were transfected into highly permissive human hepatoma cells (Huh-7.5 cells). Subsequently, these cells were co-cultured and fused to various human and murine cells expressing HCV structural proteins core, envelope 1 and 2 (E1, E2) and accessory proteins p7 and NS2. Provided that cell fusion was initiated by treatment with polyethylene-glycol (PEG), the culture released infectious viral particles which infected naïve cells in a receptor-dependent fashion. To assess the influence of dominant restrictions on the complete viral life cycle including cell entry, RNA translation, replication and virus assembly, we took advantage of a human liver cell line (Huh-7 Lunet N cells) which lacks endogenous expression of CD81, an essential entry factor of HCV. In the absence of ectopically expressed CD81, these cells are essentially refractory to HCV infection. Importantly, when co-cultured and fused with cells that express human CD81 but lack at least another crucial cell entry factor (i.e. SR-BI, CLDN1, OCLN), only the resulting heterokaryons display the complete set of HCV entry factors requisite for infection. Therefore, to analyze if dominant restriction factors suppress completion of the HCV replication cycle, we fused Lunet N cells with various cells from human and mouse origin which fulfill the above mentioned criteria. When co-cultured cells were transfected with a highly fusogenic viral envelope protein mutant of the prototype foamy virus (PFV) and subsequently challenged with infectious HCV particles (HCVcc), de novo production of infectious virus was observed. This indicates that HCV successfully completed its replication cycle in heterokaryons thus ruling out expression of dominant restriction factors in these cell lines. These novel conditional trans-complementation methods will be useful to screen a large panel of cell lines and primary cells for expression of HCV-specific dominant restriction factors.
丙型肝炎病毒（HCV）是一种嗜肝病毒，其宿主范围仅限于人类和黑猩猩。尽管在人类非肝和鼠类细胞系中已观察到HCV RNA复制，但效率非常低，并且需要使用表达显性抗生素选择标记的HCV复制子构建体进行长期选择程序。因此，HCV的体外研究仅限于允许病毒进入并完成病毒生命周期的人肝癌细胞系。由于HCV的物种嗜性狭窄，没有可维持完整HCV复制周期的具有免疫活性的小动物模型。HCV在非人类细胞（如小鼠来源的细胞）中复制效率低下，可能是由于缺乏必需宿主依赖因子的遗传相容性和/或限制因子的表达。我们研究了HCV的传播是否受到源自非肝组织的人类细胞系或小鼠肝细胞系中显性限制因子的抑制。为此，我们开发了两种依赖体细胞融合的独立条件性反式互补方法。在这两种情况下，病毒复制周期的完成仅在异核体中才有可能。因此，通过测量传染性病毒后代的从头产生来确定的成功反式互补表明不存在显性限制。具体而言，将携带荧光素酶转基因的亚基因组HCV复制子转染到高度允许的人肝癌细胞（Huh-7.5细胞）中。随后，将这些细胞与表达HCV结构蛋白核心、包膜1和2（E1、E2）以及辅助蛋白p7和NS2的各种人类和鼠类细胞共培养并融合。如果通过聚乙二醇（PEG）处理启动细胞融合，则培养物会释放出传染性病毒颗粒，这些颗粒以受体依赖性方式感染未感染的细胞。为了评估显性限制对包括细胞进入、RNA翻译、复制和病毒组装在内的完整病毒生命周期的影响，我们利用了一种人肝细胞系（Huh-7 Lunet N细胞），该细胞系缺乏HCV的必需进入因子CD81的内源性表达。在没有异位表达的CD81的情况下，这些细胞基本上对HCV感染具有抗性。重要的是，当与表达人类CD81但至少缺乏另一种关键细胞进入因子（即SR-BI、CLDN1、OCLN）的细胞共培养并融合时，只有产生的异核体显示出感染所需的完整HCV进入因子集。因此，为了分析显性限制因子是否抑制HCV复制周期的完成，我们将Lunet N细胞与满足上述标准的各种人类和小鼠来源的细胞融合。当用原型泡沫病毒（PFV）的高度融合性病毒包膜蛋白突变体转染共培养细胞，随后用传染性HCV颗粒（HCVcc）攻击时，观察到了传染性病毒的从头产生。这表明HCV在异核体中成功完成了其复制周期，从而排除了这些细胞系中显性限制因子的表达。这些新的条件性反式互补方法将有助于筛选大量细胞系和原代细胞中HCV特异性显性限制因子的表达。
