Nagashima Shigeo, Takahashi Masaharu, Kobayashi Tominari, Nishizawa Tsutomu, Nishiyama Takashi, Primadharsini Putu Prathiwi, Okamoto Hiroaki
Division of Virology, Department of Infection and Immunity, Jichi Medical University School of Medicine, Tochigi, Japan.
Division of Virology, Department of Infection and Immunity, Jichi Medical University School of Medicine, Tochigi, Japan
J Virol. 2017 Oct 27;91(22). doi: 10.1128/JVI.00822-17. Print 2017 Nov 15.
Our previous studies demonstrated that membrane-associated hepatitis E virus (HEV) particles-now considered "quasi-enveloped particles"-are present in the multivesicular body with intraluminal vesicles (exosomes) in infected cells and that the release of HEV virions is related to the exosomal pathway. In this study, we characterized exosomes purified from the culture supernatants of HEV-infected PLC/PRF/5 cells. Purified CD63-, CD9-, or CD81-positive exosomes derived from the culture supernatants of HEV-infected cells that had been cultivated in serum-free medium were found to contain HEV RNA and the viral capsid (ORF2) and ORF3 proteins, as determined by reverse transcription-PCR (RT-PCR) and Western blotting, respectively. Furthermore, immunoelectron microscopy, with or without prior detergent and protease treatment, revealed the presence of virus-like particles in the exosome fraction. These particles were 39.6 ± 1.0 nm in diameter and were covered with a lipid membrane. After treatment with detergent and protease, the diameter of these virus-like particles was 26.9 ± 0.9 nm, and the treated particles became accessible with an anti-HEV ORF2 monoclonal antibody (MAb). The HEV particles in the exosome fraction were capable of infecting naive PLC/PRF/5 cells but were not neutralized by an anti-HEV ORF2 MAb which efficiently neutralizes nonenveloped HEV particles in cell culture. These results indicate that the membrane-wrapped HEV particles released by the exosomal pathway are copurified with the exosomes in the exosome fraction and suggest that the capsids of HEV particles are individually covered by lipid membranes resembling those of exosomes, similar to enveloped viruses. Hepatitis E, caused by HEV, is an important infectious disease that is spreading worldwide. HEV infection can cause acute or fulminant hepatitis and can become chronic in immunocompromised hosts, including patients after organ transplantation. The HEV particles present in feces and bile are nonenveloped, while those in circulating blood and culture supernatants are covered with a cellular membrane, similar to enveloped viruses. Furthermore, these membrane-associated and -unassociated HEV particles can be propagated in cultured cells. The significance of our research is that the capsids of HEV particles are individually covered by a lipid membrane that resembles the membrane of exosomes, similar to enveloped viruses, and are released from infected cells via the exosomal pathway. These data will help to elucidate the entry mechanisms and receptors for HEV infection in the future. This is the first report to characterize the detailed morphological features of membrane-associated HEV particles.
我们之前的研究表明，膜相关戊型肝炎病毒（HEV）颗粒——现被认为是“准包膜颗粒”——存在于受感染细胞的多泡体及腔内小泡（外泌体）中，且HEV病毒粒子的释放与外泌体途径有关。在本研究中，我们对从HEV感染的PLC/PRF/5细胞培养上清中纯化的外泌体进行了表征。通过无血清培养基培养HEV感染细胞，从其培养上清中纯化得到的CD63、CD9或CD81阳性外泌体，经逆转录聚合酶链反应（RT-PCR）和蛋白质免疫印迹法分别测定，发现其含有HEV RNA以及病毒衣壳（ORF2）和ORF3蛋白。此外，免疫电子显微镜检查显示，无论有无事先用去污剂和蛋白酶处理，在外泌体组分中均存在病毒样颗粒。这些颗粒直径为39.6±1.0nm，表面覆盖有脂质膜。用去污剂和蛋白酶处理后，这些病毒样颗粒的直径为26.9±0.9nm，且处理后的颗粒可与抗HEV ORF2单克隆抗体（MAb）结合。外泌体组分中的HEV颗粒能够感染未感染的PLC/PRF/5细胞，但不能被在细胞培养中有效中和非包膜HEV颗粒的抗HEV ORF2 MAb中和。这些结果表明，通过外泌体途径释放的膜包裹HEV颗粒与外泌体组分中的外泌体共纯化，提示HEV颗粒的衣壳被类似于外泌体的脂质膜单独包裹，类似于包膜病毒。戊型肝炎由HEV引起，是一种在全球范围内传播的重要传染病。HEV感染可导致急性或暴发性肝炎，在免疫功能低下的宿主（包括器官移植后的患者）中可转为慢性。粪便和胆汁中的HEV颗粒是非包膜的，而循环血液和培养上清中的HEV颗粒则被细胞膜覆盖，类似于包膜病毒。此外，这些与膜相关和不相关的HEV颗粒均可在培养细胞中增殖。我们研究的意义在于，HEV颗粒的衣壳被类似于外泌体膜的脂质膜单独包裹，类似于包膜病毒，并通过外泌体途径从受感染细胞中释放。这些数据将有助于阐明未来HEV感染的进入机制和受体。这是首篇对膜相关HEV颗粒详细形态特征进行表征的报告。
