Institute of Experimental Virology, TWINCORE, Centre for Experimental and Clinical Infection Research, a joint venture between the Helmholtz Centre for Infection Research and the Hannover Medical School, Hannover, Germany.
Hepatology. 2014 Jan;59(1):78-88. doi: 10.1002/hep.26626. Epub 2013 Nov 18.
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-α/β 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 (PI4KIIIα) 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.
Blunted innate immunity, abundant miR-122, and HCV entry factor expression permits propagation of HCV in mouse liver-derived cell lines.
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 的受限物种嗜性由病毒生命周期不同步骤中独特的宿主因素要求决定。此外，有效的先天免疫靶向作用阻止了 HCV 在非人类细胞中的有效繁殖。已经探索了 HCV 宿主因子用于细胞进入和病毒释放的物种特异性。然而，HCV 在小鼠肝细胞中复制效率低的原因仍不清楚。为了解决这个问题，我们通过体内永生化生成了具有特定线粒体抗病毒信号蛋白（MAVS）、干扰素调节因子 3（IRF3）或干扰素-α/β受体（IFNAR）损伤的新型小鼠肝源性细胞系。这些细胞中先天免疫反应迟钝适度增加了 HCV RNA 复制。然而，肝特异性人 microRNA 122（miR-122）的异位表达进一步提高了所有敲除细胞系中的 RNA 复制。值得注意的是，MAVS(-/-)miR-122 细胞维持了强烈的 HCV RNA 复制，达到与高度允许的人肝癌细胞系 Huh-7.5 相当的水平。RNA 复制依赖于小鼠环孢菌素和磷脂酰肌醇-4 激酶 IIIα（PI4KIIIα），并且在用全长病毒 RNA 转染后也观察到。此外，人载脂蛋白 E（ApoE）的异位表达足以允许释放感染性颗粒。最后，表达人进入共因子使这些细胞对 HCV 感染具有允许性，从而证实 HCV 复制周期的所有步骤都可以在小鼠肝源性细胞中重建。
先天免疫迟钝、丰富的 miR-122 和 HCV 进入因子的表达允许 HCV 在小鼠肝源性细胞系中繁殖。
