Characterization of the Quasi-Enveloped Hepatitis E Virus Particles Released by the Cellular Exosomal Pathway
Characterization of the Quasi-Enveloped Hepatitis E Virus Particles Released by the Cellular Exosomal Pathway
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DOI：
10.1128/jvi.00822-17
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发表时间：
2017-11-01
影响因子：
5.4
通讯作者：
Okamoto, Hiroaki
中科院分区：
文献类型：
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作者：
Nagashima, Shigeo;Takahashi, Masaharu;Okamoto, Hiroaki
我们之前的研究表明，膜相关戊型肝炎病毒（HEV）颗粒（现在被认为是“准包膜颗粒”）存在于受感染细胞中具有管腔内囊泡（外泌体）的多泡体中，并且HEV病毒粒子的释放与外泌体途径有关。在这项研究中，我们表征了从 HEV 感染的 PLC/PRF/5 细胞的培养上清液中纯化的外泌体。通过逆转录 PCR (RT-PCR) 和蛋白质印迹法分别测定，来自在无血清培养基中培养的 HEV 感染细胞的培养上清液的纯化 CD63、CD9 或 CD81 阳性外泌体含有 HEV RNA 以及病毒衣壳 (ORF2) 和 ORF3 蛋白。此外，免疫电子显微镜，无论是否经过事先的去污剂和蛋白酶处理，都揭示了外泌体部分中存在病毒样颗粒。这些颗粒直径为 39.6 +/- 1.0 nm，并覆盖有脂质膜。用去污剂和蛋白酶处理后，这些病毒样颗粒的直径为 26.9 +/- 0.9 nm，并且处理过的颗粒可与抗 HEV ORF2 单克隆抗体 (MAb) 接触。外泌体部分中的 HEV 颗粒能够感染初始 PLC/PRF/5 细胞，但不能被抗 HEV ORF2 MAb 中和，而抗 HEV ORF2 MAb 可有效中和细胞培养物中的无包膜 HEV 颗粒。这些结果表明，外泌体途径释放的膜包裹的HEV颗粒与外泌体部分中的外泌体共纯化，并表明HEV颗粒的衣壳单独被类似于外泌体的脂质膜覆盖，与包膜病毒相似。 重要性 由HEV引起的戊型肝炎是一种在世界范围内传播的重要传染病。 HEV 感染可引起急性或暴发性肝炎，并可在免疫功能低下的宿主（包括器官移植后的患者）中转为慢性肝炎。粪便和胆汁中存在的 HEV 颗粒是无包膜的，而循环血液和培养物上清液中的 HEV 颗粒则被细胞膜覆盖，类似于有包膜病毒。此外，这些膜结合和非结合的HEV颗粒可以在培养的细胞中繁殖。我们研究的意义在于，HEV颗粒的衣壳单独被一层类似于外泌体膜的脂质膜覆盖，类似于包膜病毒，并通过外泌体途径从感染细胞中释放出来。这些数据将有助于阐明未来HEV感染的进入机制和受体。这是第一份描述膜相关 HEV 颗粒详细形态特征的报告。
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.IMPORTANCE 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.