Liu Chun-Chun, Zhang Yun-Na, Li Zhao-Yao, Hou Jin-Xiu, Zhou Jing, Kan Lin, Zhou Bin, Chen Pu-Yan
College of Veterinary Medicine, Nanjing Agricultural University, Nanjing, China.
College of Veterinary Medicine, Nanjing Agricultural University, Nanjing, China
J Virol. 2017 Sep 12;91(19). doi: 10.1128/JVI.01113-17. Print 2017 Oct 1.
During infection Japanese encephalitis virus (JEV) generally enters host cells via receptor-mediated clathrin-dependent endocytosis. The trafficking of JEV within endosomes is controlled by Rab GTPases, but which Rab proteins are involved in JEV entry into BHK-21 cells is unknown. In this study, entry and postinternalization of JEV were analyzed using biochemical inhibitors, RNA interference, and dominant negative (DN) mutants. Our data demonstrate that JEV entry into BHK-21 cells depends on clathrin, dynamin, and cholesterol but not on caveolae or macropinocytosis. The effect on JEV infection of dominant negative (DN) mutants of four Rab proteins that regulate endosomal trafficking was examined. Expression of DN Rab5 and DN Rab11, but not DN Rab7 and DN Rab9, significantly inhibited JEV replication. These results were further tested by silencing Rab5 or Rab11 expression before viral infection. Confocal microscopy showed that virus particles colocalized with Rab5 or Rab11 within 15 min after virus entry, suggesting that after internalization JEV moves to early and recycling endosomes before the release of the viral genome. Our findings demonstrate the roles of Rab5 and Rab11 on JEV infection of BHK-21 cells through the endocytic pathway, providing new insights into the life cycle of flaviviruses. Although Japanese encephalitis virus (JEV) utilizes different endocytic pathways depending on the cell type being infected, the detailed mechanism of its entry into BHK-21 cells is unknown. Understanding the process of JEV endocytosis and postinternalization will advance our knowledge of JEV infection and pathogenesis as well as provide potential novel drug targets for antiviral intervention. With this objective, we used systematic approaches to dissect this process. The results show that entry of JEV into BHK-21 cells requires a low-pH environment and that the process occurs through dynamin-, actin-, and cholesterol-dependent clathrin-mediated endocytosis that requires Rab5 and Rab11. Our work provides a detailed picture of the entry of JEV into BHK-21 cells and the cellular events that follow.
在感染过程中，日本脑炎病毒（JEV）通常通过受体介导的网格蛋白依赖性内吞作用进入宿主细胞。JEV在内体中的运输受Rab GTP酶控制，但尚不清楚哪些Rab蛋白参与JEV进入BHK-21细胞的过程。在本研究中，我们使用生化抑制剂、RNA干扰和显性负性（DN）突变体分析了JEV的进入和内化后过程。我们的数据表明，JEV进入BHK-21细胞依赖于网格蛋白、发动蛋白和胆固醇，而不依赖于小窝或巨胞饮作用。我们检测了调节内体运输的四种Rab蛋白的显性负性（DN）突变体对JEV感染的影响。DN Rab5和DN Rab11的表达显著抑制了JEV复制，而DN Rab7和DN Rab9则没有。在病毒感染前通过沉默Rab5或Rab11的表达对这些结果进行了进一步验证。共聚焦显微镜显示，病毒进入后15分钟内，病毒颗粒与Rab5或Rab11共定位，这表明内化后JEV在病毒基因组释放之前转移至早期内体和循环内体。我们的数据证明了Rab5和Rab11在JEV通过内吞途径感染BHK-21细胞过程中的作用，为黄病毒的生命周期提供了新的见解。尽管日本脑炎病毒（JEV）根据被感染的细胞类型利用不同的内吞途径，但其进入BHK-21细胞的详细机制尚不清楚。了解JEV内吞作用和内化后过程将增进我们对JEV感染和发病机制的认识，并为抗病毒干预提供潜在的新型药物靶点。基于这一目标，我们采用系统方法剖析这一过程。结果表明，JEV进入BHK-21细胞需要低pH环境，且该过程通过发动蛋白、肌动蛋白和胆固醇依赖性的网格蛋白介导的内吞作用发生，这一过程需要Rab5和Rab11参与。我们的工作详细描绘了JEV进入BHK-21细胞的过程以及随后的细胞事件。
