Hepatitis E virus egress depends on the exosomal pathway, with secretory exosomes derived from multivesicular bodies
Hepatitis E virus egress depends on the exosomal pathway, with secretory exosomes derived from multivesicular bodies
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DOI：
10.1099/vir.0.066910-0
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发表时间：
2014-10-01
影响因子：
3.8
通讯作者：
Okamoto, Hiroaki
中科院分区：
文献类型：
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作者：
Nagashima, Shigeo;Jirintai, Suljid;Okamoto, Hiroaki
我们的前期研究表明，戊型肝炎病毒（HEV）在细胞质中形成膜相关颗粒，最可能是通过出芽形成胞内囊泡，并需要多泡体（MVB）途径释放病毒颗粒，释放的HEV颗粒具有脂膜，其表面保留有trans-Golgi网络蛋白2。为了检查HEV是否利用外泌体途径来释放病毒颗粒，我们分析了感染基因型3 HEV（株JE 03 - 1760 F）的PLC/PRF/5细胞的病毒体释放是否受到巴弗洛霉素A1或GW 4869处理的影响，或者受到针对Rab 27 A或Hrs的小干扰RNA（siRNA）的引入的影响。细胞外HEV RNA滴度增加与巴夫洛霉素A1的治疗，但与GW 4869治疗下降。从消耗Rab 27 A或Hrs的细胞释放的病毒颗粒的相对水平分别降低至从用阴性对照siRNA转染的细胞释放的病毒颗粒的相对水平的16.1%和11.5%。电子显微镜观察显示MVB内存在直径约50 nm的膜相关病毒样颗粒，其在感染细胞中具有内部囊泡。免疫电镜显示MVB内管腔内囊泡上HEV ORF 2蛋白免疫金染色阳性。此外，免疫荧光分析表明ORF 2，ORF 3和CD 63蛋白在细胞质中的三重共定位，作为特定的定位信号，支持MVB内存在膜相关的HEV颗粒。这些发现表明，膜相关的HEV颗粒与内部囊泡一起通过细胞外泌体途径通过MVB释放。
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.