Despite the introduction of directly acting antivirals (DAAs), for the treatment of hepatitis C virus (HCV) infection, their cost, patient compliance, and viral resistance are still important issues to be considered. Here, we describe the generation of a novel JFH1-based HCV subgenomic replicon double reporter cell line suitable for testing different antiviral drugs and therapeutic interventions. This cells line allowed a rapid and accurate quantification of cell growth/viability and HCV RNA replication, thus discriminating specific from unspecific antiviral effects caused by DAAs or cytotoxic compounds, respectively. By correlating cell number and virus replication, we could confirm the inhibitory effect on the latter of cell over confluency and characterize an array of lentiviral vectors expressing single, double, or triple cassettes containing different combinations of short hairpin (sh)RNAs, targeting both highly conserved viral genome sequences and cellular factors crucial for HCV replication. While all vectors were effective in reducing HCV replication, the ones targeting viral sequences displayed a stronger antiviral effect, without significant cytopathic effects. Such combinatorial platforms as well as the developed double reporter cell line might find application both in setting-up anti-HCV gene therapy approaches and in studies aimed at further dissecting the viral biology/pathogenesis of infection.

中文翻译：

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表达多种抗丙型肝炎病毒shRNA的组合慢病毒载体的产生及其在新型HCV复制子双重报告基因细胞系上的验证。

尽管引入了直接作用抗病毒药（DAA），但对于治疗丙型肝炎病毒（HCV）感染，其成本，患者依从性和病毒耐药性仍是要考虑的重要问题。在这里，我们描述了适用于测试不同抗病毒药物和治疗干预措施的新型基于JFH1的HCV亚基因组复制子双重报告基因细胞系的生成。该细胞系可以快速准确地定量细胞生长/存活力和HCV RNA复制，从而分别区分由DAA或细胞毒性化合物引起的非特异性抗病毒作用。通过关联细胞数和病毒复制，我们可以证实融合后对后者的抑制作用，并表征一系列表达单，双，或包含短发夹（sh）RNA的不同组合的三重盒，同时靶向高度保守的病毒基因组序列和对HCV复制至关重要的细胞因子。尽管所有载体均可有效减少HCV复制，但靶向病毒序列的载体表现出更强的抗病毒作用，而没有明显的细胞病变作用。这种组合平台以及已开发的双报告基因细胞系可能会在建立抗HCV基因治疗方法和旨在进一步剖析病毒生物学/感染发病机理的研究中找到应用。靶向病毒序列的病毒显示出更强的抗病毒作用，而没有明显的细胞病变作用。此类组合平台以及已开发的双报告基因细胞系可能会在建立抗HCV基因治疗方法和旨在进一步剖析病毒生物学/感染发病机理的研究中找到应用。靶向病毒序列的病毒显示出更强的抗病毒作用，而没有明显的细胞病变作用。这种组合平台以及已开发的双报告基因细胞系可能会在建立抗HCV基因治疗方法和旨在进一步剖析病毒生物学/感染发病机理的研究中找到应用。