Gonzalez Oscar, Fontanes Vanessa, Raychaudhuri Santanu, Loo Rachel, Loo Joseph, Arumugaswami Vaithilingaraja, Sun Ren, Dasgupta Asim, French Samuel W
Department of Pathology and Laboratory Medicine, David Geffen School of Medicine at University of California, Los Angeles, CA 90095-1732, USA.
Hepatology. 2009 Dec;50(6):1756-64. doi: 10.1002/hep.23232.
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. Nonstructural 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. We confirmed an NS5A/HSP interaction 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. A transient 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.
丙型肝炎病毒（HCV）基因组通过内部核糖体进入位点（IRES）翻译为单一多蛋白前体，随后被切割成单个成熟病毒蛋白。非结构蛋白5A（NS5A）是其中一种与病毒基因组复制调控、从病毒IRES翻译以及病毒包装有关的蛋白。我们试图鉴定与NS5A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。对细胞提取物中共免疫沉淀的NS5A复合物进行质谱分析，鉴定出热休克蛋白（HSP）40和70。我们通过共聚焦显微镜证实了NS5A与HSP的相互作用，显示NS5A与HSP40和HSP70共定位。对共免疫沉淀的NS5A复合物进行的蛋白质印迹分析进一步证实了HSP40和HSP70与NS5A的相互作用。基于荧光素酶的瞬时转染组织培养IRES试验表明，NS5A增强了HCV IRES介导的翻译，而小干扰RNA（siRNA）介导的HSP70敲低降低了这种增强作用。用HSP合成抑制剂槲皮素处理显著降低了基线IRES活性及其被NS5A增强的作用。在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，HSP70敲低也适度降低了病毒蛋白积累，而HSP40和HSP70敲低均降低了感染性病毒颗粒的产生。用槲皮素处理在无毒浓度下降低了感染性颗粒的产生。槲皮素对病毒产生的显著抑制作用可能部分与HSP40和HSP70的减少及其在IRES翻译以及病毒形态发生或分泌中的潜在参与有关。
槲皮素可能有助于剖析病毒生命周期，并具有潜在的治疗用途，可在低相关毒性的情况下减少病毒产生。
