Department of Medical Microbiology and Immunology, University of Alberta, Edmonton, Alberta, Canada.
Li Ka Shing Institute of Virology, University of Alberta, Edmonton, Alberta, Canada.
Sci Rep. 2017 May 31;7(1):2495. doi: 10.1038/s41598-017-02648-6.
Direct acting antivirals against hepatitis C virus (HCV) have markedly improved cure rates in the past few years. However, they are expensive, with only few targeting host cell factors, and affecting virus assembly and release. Huh7.5 cells infected with a JFH-1 clone of HCV were treated with two different glycogen synthase kinase (GSK3)-β inhibitors; AR-A014418 and lithium chloride. Intra- and extracellular HCV virions and specific infectivity was determined using real-time RT-PCR and TCID50, and changes in lipid production were identified by enzyme-linked immunoassay and mass spectrometry analyses. Similarly, effect on two HCV replicon cells were identified by the luciferase activity. Although there was limited effect on virus replication in Huh7.5 cells and replicons, Huh7.5 cells treated with GSK3β inhibitors produced significantly less viral particles in comparison to untreated cells. In addition, the treated cells synthesized significantly lower amounts of ApoB and trapped the ApoE lipoproteins in the cells. In conclusion, our study suggests that GSK3β plays a pivotal role in HCV virion assembly and release mediated in part through inhibition of apolipoprotein synthesis.
近年来，直接作用的抗病毒药物（DAAs）显著提高了丙型肝炎病毒（HCV）的治愈率。然而，这些药物价格昂贵，只有少数靶向宿主细胞因子，影响病毒的组装和释放。用两种不同的糖原合酶激酶（GSK3）-β抑制剂 AR-A014418 和氯化锂处理感染 JFH-1 克隆 HCV 的 Huh7.5 细胞。通过实时 RT-PCR 和 TCID50 测定细胞内外 HCV 病毒粒子和特异性感染力，并通过酶联免疫吸附试验和质谱分析鉴定脂质产生的变化。同样，通过荧光素酶活性鉴定对两种 HCV 复制子细胞的作用。尽管 GSK3β 抑制剂对 Huh7.5 细胞和复制子中的病毒复制的影响有限，但与未处理的细胞相比，用 GSK3β 抑制剂处理的 Huh7.5 细胞产生的病毒颗粒明显减少。此外，处理过的细胞合成的 ApoB 明显减少，并将 ApoE 脂蛋白滞留在细胞内。总之，我们的研究表明，GSK3β 在 HCV 病毒粒子的组装和释放中发挥关键作用，部分通过抑制载脂蛋白的合成来实现。
