Cell Entry, Efficient RNA Replication, and Production of Infectious Hepatitis C Virus Progeny in Mouse Liver-Derived Cells
Cell Entry, Efficient RNA Replication, and Production of Infectious Hepatitis C Virus Progeny in Mouse Liver-Derived Cells
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DOI：
10.1002/hep.26626
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发表时间：
2014-01-01
期刊：
影响因子：
13.5
通讯作者：
Pietschmann, Thomas
中科院分区：
文献类型：
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作者：
Frentzen, Anne;Anggakusuma;Pietschmann, Thomas
只有人类和黑猩猩容易受到丙型肝炎病毒（HCV）的慢性感染。HCV的限制性种属嗜性由病毒生命周期不同阶段的不同宿主因子需求决定。此外，有效的先天免疫靶向排除了HCV在非人细胞中的有效繁殖。已经探索了HCV宿主因子用于细胞进入和病毒释放的种属特异性。然而，小鼠肝细胞中HCV RNA复制效率低下的原因仍然难以捉摸。为了解决这一问题，我们通过体内永生化产生了在线粒体抗病毒信号蛋白（MAVS）、干扰素调节因子3（IRF 3）或干扰素-/受体（IFNAR）中具有特异性病变的新型小鼠肝源性细胞系。在这些细胞中减弱的先天免疫应答适度地增加了HCV RNA的复制。然而，肝脏特异性人microRNA 122（miR-122）的异位表达进一步促进了所有敲除细胞系中的RNA复制。值得注意的是，MAVS（-/-）miR-122细胞维持了活跃的HCV RNA复制，达到了与高度容许的人肝癌细胞系Huh-7.5相当的水平。RNA复制依赖于小鼠亲环蛋白和磷脂酰肌醇-4激酶III α（PI 4KIII），转染全长病毒RNA后也观察到。此外，人或小鼠载脂蛋白E（ApoE）的异位表达足以释放感染性颗粒。最后，人进入辅因子的表达使这些细胞允许HCV感染，从而证实了HCV复制周期的所有步骤都可以在小鼠肝源性细胞中重建。结论：先天免疫功能减弱、miR-122和HCV进入因子的表达增加了HCV在小鼠肝源性细胞系中的增殖。（肝病学2014;58：78-88）
Only humans and chimpanzees are susceptible to chronic infection by hepatitis C virus (HCV). The restricted species tropism of HCV is determined by distinct host factor requirements at different steps of the viral life cycle. In addition, effective innate immune targeting precludes efficient propagation of HCV in nonhuman cells. Species-specificity of HCV host factor usage for cell entry and virus release has been explored. However, the reason for inefficient HCV RNA replication efficiency in mouse liver cells remains elusive. To address this, we generated novel mouse liver-derived cell lines with specific lesions in mitochondrial antiviral signaling protein (MAVS), interferon regulatory factor 3 (IRF3), or Interferon-/ receptor (IFNAR) by in vivo immortalization. Blunted innate immune responses in these cells modestly increased HCV RNA replication. However, ectopic expression of liver-specific human microRNA 122 (miR-122) further boosted RNA replication in all knockout cell lines. Remarkably, MAVS(-/-)miR-122 cells sustained vigorous HCV RNA replication, attaining levels comparable to the highly permissive human hepatoma cell line Huh-7.5. RNA replication was dependent on mouse cyclophilin and phosphatidylinositol-4 kinase III alpha (PI4KIII) and was also observed after transfection of full-length viral RNA. Additionally, ectopic expression of either human or mouse apolipoprotein E (ApoE) was sufficient to permit release of infectious particles. Finally, expression of human entry cofactors rendered these cells permissive to HCV infection, thus confirming that all steps of the HCV replication cycle can be reconstituted in mouse liver-derived cells. Conclusion: Blunted innate immunity, abundant miR-122, and HCV entry factor expression permits propagation of HCV in mouse liver-derived cell lines. (Hepatology 2014;58:78-88)