Identification of human kinases involved in hepatitis C virus replication by small interference RNA library screening
Identification of human kinases involved in hepatitis C virus replication by small interference RNA library screening
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DOI：
10.1074/jbc.m703988200
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发表时间：
2008-01-04
影响因子：
4.8
通讯作者：
Schultz, Peter G.
中科院分区：
文献类型：
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作者：
Supekova, Lubica;Supek, Frantisek;Schultz, Peter G.
丙型肝炎病毒（HCV）的繁殖是一个复杂的过程，需要宿主和病毒蛋白。为了便于鉴定HCV复制所需的宿主细胞因子，我们筛选了一组小干扰rna，这些干扰rna优先靶向人类蛋白激酶，使用表达萤火虫荧光素酶基因的HCV复制子作为遗传报告基因。在携带HCV复制子的细胞中，研究人员发现了三种人类激酶（Csk、Jak1和Vrk1）特异性的小干扰RNA，它们可重复性地降低病毒RNA和病毒蛋白水平。用Csk小分子抑制剂处理复制子细胞也导致HCV RNA和蛋白质的显著减少，进一步支持Csk在HCV复制中的作用。然后研究了靶向8种已知受Csk负调控的激酶的sirna的作用；敲低其中一种激酶Fyn会导致HCV复制子的上调，这表明Csk通过Fyn介导其对HCV复制的影响。用Csk抑制剂处理的复制子细胞比对照细胞含有更低水平的Fyn磷酸化形式，进一步证实了这一结论。
The propagation of the hepatitis C virus (HCV) is a complex process that requires both host and viral proteins. To facilitate identification of host cell factors that are required for HCV replication, we screened a panel of small interference RNAs that preferentially target human protein kinases using an HCV replicon expressing the firefly luciferase gene as a genetic reporter. Small interference RNAs specific for three human kinases, Csk, Jak1, and Vrk1, were identified that reproducibly reduce viral RNA and viral protein levels in HCV replicon-bearing cells. Treatment of replicon cells with a small molecule inhibitor of Csk also resulted in a significant reduction in HCV RNA and proteins, further supporting a role for Csk in HCV replication. The effects of siRNAs targeting eight kinases known to be negatively regulated by Csk were then examined; knock down of one of these kinases, Fyn, resulted in up-regulation of the HCV replicon, suggesting that Csk mediates its effect on HCV replication through Fyn. This conclusion was further corroborated by demonstration that replicon cells treated with Csk inhibitor contained lower levels of the phosphorylated form of Fyn than control cells.