Department of Infectious Diseases, Molecular Virology, University of Heidelberg, Heidelberg, Germany.
Hepatology. 2013 Mar;57(3):953-63. doi: 10.1002/hep.26260. Epub 2013 Feb 7.
Intravenous silibinin (SIL) is an approved therapeutic that has recently been applied to patients with chronic hepatitis C, successfully clearing hepatitis C virus (HCV) infection in some patients even in monotherapy. Previous studies suggested multiple antiviral mechanisms of SIL; however, the dominant mode of action has not been determined. We first analyzed the impact of SIL on replication of subgenomic replicons from different HCV genotypes in vitro and found a strong inhibition of RNA replication for genotype 1a and genotype 1b. In contrast, RNA replication and infection of genotype 2a were minimally affected by SIL. To identify the viral target of SIL we analyzed resistance to SIL in vitro and in vivo. Selection for drug resistance in cell culture identified a mutation in HCV nonstructural protein (NS) 4B conferring partial resistance to SIL. This was corroborated by sequence analyses of HCV from a liver transplant recipient experiencing viral breakthrough under SIL monotherapy. Again, we identified distinct mutations affecting highly conserved amino acid residues within NS4B, which mediated phenotypic SIL resistance also in vitro. Analyses of chimeric viral genomes suggest that SIL might target an interaction between NS4B and NS3/4A. Ultrastructural studies revealed changes in the morphology of viral membrane alterations upon SIL treatment of a susceptible genotype 1b isolate, but not of a resistant NS4B mutant or genotype 2a, indicating that SIL might interfere with the formation of HCV replication sites.
Mutations conferring partial resistance to SIL treatment in vivo and in cell culture argue for a mechanism involving NS4B. This novel mode of action renders SIL an attractive candidate for combination therapies with other directly acting antiviral drugs, particularly in difficult-to-treat patient cohorts.
静脉注射水飞蓟素（SIL）是一种已获批准的治疗药物，最近已应用于慢性丙型肝炎患者，在某些患者中甚至在单药治疗中成功清除丙型肝炎病毒（HCV）感染。先前的研究表明，SIL 具有多种抗病毒机制；然而，其主要作用模式尚未确定。我们首先分析了 SIL 对不同 HCV 基因型亚基因组复制子在体外复制的影响，发现 SIL 对基因型 1a 和 1b 具有强烈的抑制 RNA 复制作用。相比之下，SIL 对基因型 2a 的 RNA 复制和感染的影响最小。为了确定 SIL 的病毒靶标，我们分析了 SIL 在体外和体内的耐药性。在细胞培养中进行药物耐药性选择鉴定出 HCV 非结构蛋白（NS）4B 的突变赋予 SIL 部分耐药性。这一结果得到了对接受 SIL 单药治疗时发生病毒突破的肝移植受者 HCV 序列分析的证实。同样，我们鉴定出影响 NS4B 高度保守氨基酸残基的不同突变，这些突变也在体外介导了表型 SIL 耐药性。嵌合病毒基因组分析表明，SIL 可能靶向 NS4B 和 NS3/4A 之间的相互作用。超微结构研究表明，在易感基因型 1b 分离株中，SIL 处理后病毒膜形态发生变化，但在耐药性 NS4B 突变体或基因型 2a 中则没有，这表明 SIL 可能干扰 HCV 复制位点的形成。
体内和细胞培养中对 SIL 治疗产生部分耐药性的突变表明涉及 NS4B 的机制。这种新的作用模式使 SIL 成为与其他直接作用抗病毒药物联合治疗的有吸引力的候选药物，特别是在难以治疗的患者群体中。
