Replication of hepatitis C virus (HCV) RNA in mouse embryonic fibroblasts: Protein kinase R (PKR)-dependent and PKR-independent mechanisms for controlling HCV RNA replication and mediating interferon activities
Replication of hepatitis C virus (HCV) RNA in mouse embryonic fibroblasts: Protein kinase R (PKR)-dependent and PKR-independent mechanisms for controlling HCV RNA replication and mediating interferon activities
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DOI：
10.1128/jvi.00586-06
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发表时间：
2006-08-01
影响因子：
5.4
通讯作者：
Luo, Guangxiang
中科院分区：
文献类型：
--
作者：
Chang, Kyung-Soo;Cai, Zhaohui;Luo, Guangxiang
丙型肝炎病毒（HCV）感染导致慢性肝炎，目前用α干扰素（IFN-α）为基础的疗法治疗。然而，慢性HCV感染和基于IFN的治疗的潜在机制尚未确定。蛋白激酶R（PKR）参与控制HCV复制和介导IFN诱导的抗病毒反应。在这份报告中，我们证明了基因型2a HCV的亚基因组RNA复制子在小鼠胚胎成纤维细胞（MEFs）中有效地复制，通过细胞集落形成效率和HCV蛋白质和正链和负链RNA的检测来确定。此外，发现亚基因组HCV RNA在PKR敲除（PKR-/-）MEF中比在野生型（PKR+/+）MEF中更有效地复制。通过特异性小干扰RNA敲低PKR的表达显著增强HCV RNA的复制水平，表明PKR参与HCV RNA复制的控制。ISG 56（p56）的水平由HCV RNA复制诱导，表明PKR非依赖性抗病毒途径的激活。此外，IFN-α/β在PKR-/- MEFs中与在PKR+/+ MEFs中一样有效地抑制HCV RNA复制。这些发现表明PKR非依赖性抗病毒途径在控制HCV复制和介导IFN诱导的抗病毒效应中起重要作用。我们的研究结果也为HCV复制转基因小鼠模型的开发提供了基础，并为进一步确定细胞基因在建立慢性HCV感染和介导细胞内先天性抗病毒反应中的作用奠定了基础。
Hepatitis C virus (HCV) infection causes chronic hepatitis and is currently treated with alpha interferon (IFN-alpha)-based therapies. The underlying mechanisms of chronic HCV infection and IFN-based therapies, however, have not been defined. Protein kinase R (PKR) was implicated in the control of HCV replication and mediation of IFN-induced antiviral response. In this report, we demonstrate that a subgenomic RNA replicon of genotype 2a HCV replicated efficiently in mouse embryonic fibroblasts (MEFs), as determined by cell colony formation efficiency and the detection of HCV proteins and both positive- and negative-strand RNAs. Additionally, the subgenomic HCV RNA was found to replicate more efficiently in the PKR knockout (PKR-/-) MEF than in the wild-type (PKR+/+) MEF. The knockdown expression of PKR by specific small interfering RNAs significantly enhanced the level of HCV RNA replication, suggesting that PKR is involved in the control of HCV RNA replication. The level of ISG56 (p56) was induced by HCV RNA replication, indicating the activation of PKR-independent antiviral pathways. Furthermore, IFN-alpha/beta inhibited HCV RNA replication in PKR-/- MEFs as efficiently as in PKR+/+ MEFs. These findings demonstrate that PKR-independent antiviral pathways play important roles in controlling HCV replication and mediating IFN-induced antiviral effect. Our findings also provide a foundation for the development of transgenic mouse models of HCV replication and set a stage to further define the roles of cellular genes in the establishment of chronic HCV infection and the mediation of intracellular innate antiviral response by using MEFs derived from diverse gene knockout animals.