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Effect of the core protein of Hepatitis C virus (HCV) on RNA interference

丙型肝炎病毒（HCV）核心蛋白对RNA干扰的影响

基本信息

批准号：
16590643
负责人：
DATE Takayasu
金额：
$ 2.24万
依托单位：
KANAZAWA MEDICAL UNIVERSITY
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2004
资助国家：
日本
起止时间：
2004 至 2005
项目状态：
已结题

项目摘要

1)Recombinant His-tagged PKR was prepared as a non-phosphorylated form and characterized. PKR bound double-stranded (ds) RNA with less than 26 nucleotides in length. However, PER was not autophosphorylated (activated) by such short dsRNA in vitro. Autophosphorylation of PKR requires dsRNA with longer than 31 nucleotides in length in vitro. Activation of PKR by long dsRNA was inhibited by short dsRNA (21.23 nucleotides in length)2)Partial activation of cellular PKR in HepG2 cells was observed by Western blot analysis using antibody raised to phosphothreonine at 446. It may be induced by intrinsic dsRNA. When shRNA-expression plasmid was introduced, PKR was strongly suppressed, thereby, resulting in the inhibition of phosphorylation of the alpha subunit of eukaryotic translation factor. (eIF2α). The sequence of short dsRNA generated from shRNA had a random sequence and no target for cellular mRNA.The rate of protein synthesis was increased several times by luciferase reporter assay.3)In order to investigate the effect of short dsRNA on RNA virus production, we examined whether Japanese encephalitis virus (JEV), a model HCV virus, was suppressed in shRNA-expressing cells. Activation of PKR was observed in shRNA expressing cells in the same extent as observed in control cells. Levels of phosphorylation of eIF2α and expression of viral envelope protein were almost the same as that in control cells. Therefore, inactivation of PKR by short dsRNA was not found in the virulent conditions.4)The effect of the hepatitis C virus (HCV)-core protein on RNA interference was examined in HepG2 cells using luciferase-expression plasmid and its siRNAs. The core protein slightly enhanced the RNAi effect compared with other HCV RNA-binding proteins, NS3 and NS5A proteins. However, the effect was very small and was independent of RNA binding activity of the core protein.

1)以非磷酸化形式制备了His标记的重组PKR，并对其进行了表征。PKR结合的双链RNA，长度小于26个核苷酸。然而，在体外，PER不能被如此短的dsRNA自动磷酸化(激活)。在体外，PKR的自磷酸化需要长度超过31个核苷酸的dsRNA。长dsRNA(全长21.23个核苷酸)可抑制长dsRNA对PKR的激活作用。2)用抗446位磷酸苏氨酸的抗体进行Western印迹分析，发现细胞内PKR部分激活。它可能是由内源性dsRNA诱导的。当引入shRNA表达载体时，PKR被强烈抑制，从而导致真核翻译因子α亚基的磷酸化被抑制。(eif2α)。由shRNA产生的短dsRNA序列具有随机序列，没有针对细胞mRNA的靶点，通过荧光素酶报告基因分析，蛋白质合成速度增加了几倍。3)为了研究短dsRNA对RNA病毒产生的影响，我们在shRNA表达细胞中检测了模型丙型肝炎病毒--日本脑炎病毒(JEV)是否受到抑制。在表达shRNA的细胞中观察到与对照细胞相同程度的PKR激活。EIF2α的磷酸化水平和病毒包膜蛋白的表达水平与对照细胞基本相同。4)利用荧光素酶表达载体及其siRNAs，检测了丙型肝炎病毒核心蛋白对HepG2细胞RNA干扰的影响。与其他丙型肝炎病毒RNA结合蛋白NS3和NS5A蛋白相比，核心蛋白略有增强RNAi效应。然而，这种影响很小，并且不依赖于核心蛋白的RNA结合活性。