Department of Gastroenterology, Graduate School of Medicine, University of Tokyo, Tokyo, Japan.
Liver Int. 2010 Feb;30(2):311-8. doi: 10.1111/j.1478-3231.2009.02144.x. Epub 2009 Oct 16.
Double-stranded RNA-activated protein kinase (PKR), an interferon (IFN)-stimulated gene, is activated by binding with double-stranded RNA, a putative replicative intermediate of the hepatitis C virus (HCV). Activated PKR phosphorylates the alpha subunit of eukaryotic initiation factor-2 to inhibit the translation of viral protein.
AIMS/METHODS: We established stable PKR knockdown Huh7 cells using RNA interference and investigated the effect of PKR against HCV replication using a subgenomic replicon that expressed luciferase reporter protein and the JFH1 full-length HCV genome.
In stable PKR knockdown cells that harboured a subgenomic replicon, luciferase activity was approximately three times higher than that of control cells, indicating that the subgenomic replicon replicated with a higher efficiency in stable PKR knockdown cells than that in control cells. Furthermore, stable PKR knockdown cells secreted significantly more HCV particles than did control cells after transfection with the full-length HCV genome. The replication of the subgenomic replicon was suppressed by the addition of IFN-alpha in both cells. Although the extent of suppression was significantly lower in stable PKR knockdown than control cells using a low concentration (2.5-5 U/ml) of IFN-alpha, even 10 U/ml IFN-alpha suppressed the replication of subgenomic replicon by >98% in both cells.
Double-stranded RNA-activated protein kinase plays an important role in suppressing HCV replication in an innate state, but may not be essential in IFN therapy.
双链 RNA 激活的蛋白激酶（PKR）是一种干扰素（IFN）刺激基因，通过与双链 RNA 结合而被激活，双链 RNA 是丙型肝炎病毒（HCV）的一种潜在复制中间体。激活的 PKR 磷酸化真核起始因子-2 的α亚基，从而抑制病毒蛋白的翻译。
目的/方法：我们使用 RNA 干扰技术建立了稳定的 PKR 敲低 Huh7 细胞，并使用表达荧光素酶报告蛋白的亚基因组复制子和 JFH1 全长 HCV 基因组来研究 PKR 对 HCV 复制的影响。
在稳定的 PKR 敲低细胞中，亚基因组复制子的荧光素酶活性比对照细胞高约 3 倍，表明亚基因组复制子在稳定的 PKR 敲低细胞中的复制效率高于对照细胞。此外，在转染全长 HCV 基因组后，稳定的 PKR 敲低细胞分泌的 HCV 颗粒明显多于对照细胞。IFN-α的加入抑制了两种细胞中亚基因组复制子的复制。虽然用低浓度（2.5-5 U/ml）IFN-α时，稳定的 PKR 敲低细胞中的抑制程度明显低于对照细胞，但即使使用 10 U/ml IFN-α，两种细胞中亚基因组复制子的复制也被抑制了>98%。
双链 RNA 激活的蛋白激酶在先天状态下抑制 HCV 复制中发挥重要作用，但在 IFN 治疗中可能不是必需的。
