The heat shock protein inhibitor Quercetin attenuates hepatitis C virus production.
The heat shock protein inhibitor Quercetin attenuates hepatitis C virus production.
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DOI：
10.1002/hep.23232
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发表时间：
2009-12
期刊：
影响因子：
13.5
通讯作者：
French, Samuel W.
中科院分区：
文献类型：
--
作者：
Gonzalez, Oscar;Fontanes, Vanessa;Raychaudhuri, Santanu;Loo, Rachel;Loo, Joseph;Arumugaswami, Vaithilingaraja;Sun, Ren;Dasgupta, Asim;French, Samuel W.
丙型肝炎病毒（HCV）基因组通过内部核糖体进入位点（IRES）翻译为单个多蛋白前体，随后切割成单个成熟病毒蛋白。非结构蛋白5A（NS 5A）是这些蛋白质中的一种，其涉及调节病毒基因组复制、从病毒IRES的翻译和病毒包装。我们试图鉴定与NS 5A相互作用的细胞蛋白，并确定这些相互作用是否可能在病毒产生中发挥作用。共免疫沉淀的NS 5A复合物经质谱分析鉴定为热休克蛋白（HSP）40和70。共聚焦显微镜证实了NS 5A与HSP 40和HSP 70的共定位。免疫共沉淀NS 5A复合物的Western分析进一步证实了HSP 40和HSP 70与NS 5A的相互作用。非瞬时转染、基于荧光素酶的组织培养IRES试验证明NS 5A增强了HCV IRES介导的翻译，而小干扰RNA（SiRNA）介导的HSP 70敲减减少了这种增强。用HSP合成抑制剂槲皮素治疗显著降低了基线IRES活性及其由NS 5A引起的增强。HSP 70敲除也适度减少病毒蛋白质积累，而HSP 40和HSP 70敲除均减少使用与海肾荧光素酶报告基因融合的J6/JFH病毒的HCV细胞培养系统中的感染性病毒颗粒产生。在无毒浓度下，用槲皮素处理可减少感染性颗粒的产生。槲皮素对病毒产生的显著抑制可能部分与HSP 40和HSP 70的减少及其参与IRES翻译以及病毒形态发生或分泌的可能性有关。槲皮素可以允许病毒生命周期的解剖，并具有潜在的治疗用途，以减少病毒的产生，具有低相关毒性。
The hepatitis C viral (HCV) genome is translated through an internal ribosome entry site (IRES) as a single polyprotein precursor that is subsequently cleaved into individual mature viral proteins. Non-structural protein 5A (NS5A) is one of these proteins that has been implicated in regulation of viral genome replication, translation from the viral IRES and viral packaging. We sought to identify cellular proteins that interact with NS5A and determine whether these interactions may play a role in viral production. Mass spectrometric analysis of coimmunoprecipitated NS5A complexes from cell extracts identified heat shock proteins (HSPs) 40 and 70.Weconfirmed anNS5A/HSPinteraction by confocal microscopy demonstrating colocalization of NS5A with HSP40 and with HSP70. Western analysis of coimmunoprecipitated NS5A complexes further confirmed interaction of HSP40 and HSP70 with NS5A.Atransient transfection, luciferase-based, tissue culture IRES assay demonstrated NS5A augmentation of HCV IRES-mediated translation, and small interfering RNA (siRNA)-mediated knockdown of HSP70 reduced this augmentation. Treatment with an inhibitor of HSP synthesis, Quercetin, markedly reduced baseline IRES activity and its augmentation by NS5A. HSP70 knockdown also modestly reduced viral protein accumulation, whereas HSP40 and HSP70 knockdown both reduced infectious viral particle production in an HCV cell culture system using the J6/JFH virus fused to the Renilla luciferase reporter. Treatment with Quercetin reduced infectious particle production at nontoxic concentrations. The marked inhibition of virus production by Quercetin may partially be related to reduction of HSP40 and HSP70 and their potential involvement in IRES translation, as well as viral morphogenesis or secretion. Quercetin may allow for dissection of the viral life cycle and has potential therapeutic use to reduce virus production with low associated toxicity.
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