Occludin-Knockout Human Hepatic Huh7.5.1-8-Derived Cells Are Completely Resistant to Hepatitis C Virus Infection.
Occludin-Knockout Human Hepatic Huh7.5.1-8-Derived Cells Are Completely Resistant to Hepatitis C Virus Infection.
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DOI：
10.1248/bpb.b15-01023
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发表时间：
2016-05
影响因子：
2
通讯作者：
Yoshitaka Shirasago;Y. Shimizu;I. Tanida;Tetsuro Suzuki;R. Suzuki;K. Sugiyama;T. Wakita;K. Hanada;K. Yagi;M. Kondoh;M. Fukasawa
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文献类型：
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作者：
Yoshitaka Shirasago;Y. Shimizu;I. Tanida;Tetsuro Suzuki;R. Suzuki;K. Sugiyama;T. Wakita;K. Hanada;K. Yagi;M. Kondoh;M. Fukasawa
众所周知，阻断素(occludin，OCLN)参与了丙型肝炎病毒(HCV)进入肝细胞的过程，但尚无确凿证据表明OCLN在丙型肝炎病毒感染中起重要作用。在这项研究中，我们首先利用成簇的规则间隔短回文重复序列(CRISPR)/CRISPR相关蛋白9系统建立了一个来源于人肝细胞Huh7.5.1-8的OCLN基因敲除细胞系，其中使用了两个表达单引导RNA的独立靶向质粒。一个已建立的细胞克隆命名为OKH-4，在N-末端截断了OCLN基因，免疫印迹分析显示OCLN蛋白完全缺陷。OKH-4细胞对不同基因型丙型肝炎病毒的感染被消除，OCLN蛋白在OKH-4细胞中的外源表达完全逆转了对丙型肝炎病毒感染的容许性。此外，利用感染了丙型肝炎病毒的Huh7.5.1-8细胞和OKH-4细胞的共培养系统，我们发现OCLN在丙型肝炎病毒的细胞间传播中也是至关重要的。因此，我们认为OCLN是丙型肝炎病毒感染人肝细胞所必需的。进一步用丙型肝炎病毒基因组RNA转染的OKH-4细胞或携带丙型肝炎病毒亚基因组复制子的OKH-4细胞进行的实验表明，OCLN主要参与了丙型肝炎病毒生命周期的进入步骤。OCLN的第二个胞外环，尤其是两个半胱氨酸残基，是丙型肝炎病毒感染肝细胞的关键。OKH-4细胞可能不仅是了解丙型肝炎病毒进入的整个机制的有用工具，而且可能是了解OCLN的生物学功能的有用工具。
It is well known that occludin (OCLN) is involved in hepatitis C virus (HCV) entry into hepatocytes, but there has been no conclusive evidence that OCLN is essential for HCV infection. In this study, we first established an OCLN-knockout cell line derived from human hepatic Huh7.5.1-8 cells using the clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9 system, in which two independent targeting plasmids expressing single-guide RNAs were used. One established cell clone, named OKH-4, had the OCLN gene truncated in the N-terminal region, and a complete defect of the OCLN protein was shown using immunoblot analysis. Infection of OKH-4 cells with various genotypes of HCV was abolished, and exogenous expression of the OCLN protein in OKH-4 cells completely reversed permissiveness to HCV infection. In addition, using a co-culture system of HCV-infected Huh7.5.1-8 cells with OKH-4 cells, we showed that OCLN is also critical for cell-to-cell HCV transmission. Thus, we concluded that OCLN is essential for HCV infection of human hepatic cells. Further experiments using HCV genomic RNA-transfected OKH-4 cells or HCV subgenomic replicon-harboring OKH-4 cells suggested that OCLN is mainly involved in the entry step of the HCV life cycle. It was also demonstrated that the second extracellular loop of OCLN, especially the two cysteine residues, is critical for HCV infection of hepatic cells. OKH-4 cells may be a useful tool for understanding not only the entire mechanism of HCV entry, but also the biological functions of OCLN.