Division of Virology, Department of Infection and Immunity, Jichi Medical University School of Medicine, Tochigi-Ken 329-0498, Japan.
Division of Histology and Cell Biology, Department of Anatomy, Jichi Medical University School of Medicine, Tochigi-Ken 329-0498, Japan.
J Gen Virol. 2014 Oct;95(Pt 10):2166-2175. doi: 10.1099/vir.0.066910-0. Epub 2014 Jun 26.
Our previous studies indicated that hepatitis E virus (HEV) forms membrane-associated particles in the cytoplasm, most likely by budding into intracellular vesicles, and requires the multivesicular body (MVB) pathway to release virus particles, and the released HEV particles with a lipid membrane retain the trans-Golgi network protein 2 on their surface. To examine whether HEV utilizes the exosomal pathway to release the virus particles, we analysed whether the virion release from PLC/PRF/5 cells infected with genotype 3 HEV (strain JE03-1760F) is affected by treatment with bafilomycin A1 or GW4869, or by the introduction of a small interfering RNA (siRNA) against Rab27A or Hrs. The extracellular HEV RNA titre was increased by treatment with bafilomycin A1, but was decreased by treatment with GW4869. The relative levels of virus particles released from cells depleted of Rab27A or Hrs were decreased to 16.1 and 11.5 %, respectively, of that released from cells transfected with negative control siRNA. Electron microscopic observations revealed the presence of membrane-associated virus-like particles with a diameter of approximately 50 nm within the MVB, which possessed internal vesicles in infected cells. Immunoelectron microscopy showed positive immunogold staining for the HEV ORF2 protein on the intraluminal vesicles within the MVB. Additionally, immunofluorescence analysis indicated the triple co-localization of the ORF2, ORF3 and CD63 proteins in the cytoplasm, as specific loculated signals, supporting the presence of membrane-associated HEV particles within the MVB. These findings indicate that membrane-associated HEV particles are released together with internal vesicles through MVBs by the cellular exosomal pathway.
我们之前的研究表明，戊型肝炎病毒（HEV）在细胞质中形成膜相关颗粒，很可能通过出芽进入细胞内囊泡，并需要多泡体（MVB）途径释放病毒颗粒，并且带有脂质膜的释放的 HEV 颗粒在其表面保留跨高尔基网络蛋白 2。为了检查 HEV 是否利用细胞外体途径释放病毒颗粒，我们分析了感染基因型 3 HEV（株 JE03-1760F）的 PLC/PRF/5 细胞中病毒粒子的释放是否受 bafilomycin A1 或 GW4869 的处理、Rab27A 或 Hrs 的小干扰 RNA（siRNA）的引入的影响。用 bafilomycin A1 处理可增加细胞外 HEV RNA 滴度，但用 GW4869 处理可降低病毒粒子的释放。耗尽 Rab27A 或 Hrs 的细胞中释放的病毒粒子的相对水平分别降低至阴性对照 siRNA 转染的细胞中释放的病毒粒子的 16.1%和 11.5%。电子显微镜观察显示，在感染细胞中，MVB 内存在直径约为 50nm 的膜相关病毒样颗粒，其内具有内部囊泡。免疫电子显微镜显示，在 MVB 内的腔内小泡上，针对 HEV ORF2 蛋白的免疫金染色呈阳性。此外，免疫荧光分析表明，ORF2、ORF3 和 CD63 蛋白在细胞质中三重共定位，作为特异性局灶信号，支持 MVB 内存在膜相关的 HEV 颗粒。这些发现表明，膜相关的 HEV 颗粒通过细胞外体途径与 MVB 中的内部囊泡一起释放。
