Division of Virology, Department of Infection and Immunity, Jichi Medical University School of Medicine, 3311-1 Yakushiji, Shimotsuke-shi, Tochigi-ken, 329-0498, Japan.
Department of Emerging Infectious Diseases, Institute of Tropical Medicine, Nagasaki University, 1-12-4 Sakamoto, Nagasaki-shi, Nagasaki-ken, 852-8523, Japan.
Virus Res. 2020 Mar;278:197868. doi: 10.1016/j.virusres.2020.197868. Epub 2020 Jan 18.
Recent reports have shown that rat hepatitis E virus (HEV) is capable of infecting humans. We also successfully propagated rat HEV into human PLC/PRF/5 cells, raising the possibility of a similar mechanism shared by human HEV and rat HEV. Rat HEV has the proline-rich sequence, PxYPMP, in the open reading frame 3 (ORF3) protein that is indispensable for its release. However, the release mechanism remains unclear. The overexpression of dominant-negative (DN) mutant of vacuolar protein sorting (Vps)4A or Vps4B decreased rat HEV release to 23.9 % and 18.0 %, respectively. The release of rat HEV was decreased to 8.3 % in tumor susceptibility gene 101 (Tsg101)-depleted cells and to 31.5 % in apoptosis-linked gene 2-interacting protein X (Alix)-depleted cells. Although rat HEV ORF3 protein did not bind to Tsg101, we found a 90-kDa protein capable of binding to wild-type rat HEV ORF3 protein but not to ORF3 mutant with proline to leucine mutations in the PxYPMP motif. Rat HEV release was also decreased in Ras-associated binding 27A (Rab27A)- or hepatocyte growth factor-regulated tyrosine kinase substrate (Hrs)-depleted cells (to 20.1 % and 18.5 %, respectively). In addition, the extracellular rat HEV levels in the infected PLC/PRF/5 cells were increased after treatment with Bafilomycin A1 and decreased after treatment with GW4869. These results indicate that rat HEV utilizes multivesicular body (MVB) sorting for its release and that the exosomal pathway is required for rat HEV egress. A host protein alternative to Tsg101 that can bind to rat HEV ORF3 should be explored in further study.
最近的报告表明，大鼠肝炎病毒（HEV）能够感染人类。我们还成功地将大鼠 HEV 繁殖到人类 PLC/PRF/5 细胞中，这增加了人类 HEV 和大鼠 HEV 可能具有相似机制的可能性。大鼠 HEV 的开放阅读框 3（ORF3）蛋白中存在脯氨酸丰富序列 PxYPMP，这对于其释放是必不可少的。然而，释放机制仍不清楚。过表达液泡蛋白分选（Vps）4A 或 Vps4B 的显性负（DN）突变体分别将大鼠 HEV 的释放降低到 23.9％和 18.0％。肿瘤易感性基因 101（Tsg101）耗竭细胞中大鼠 HEV 的释放降低到 8.3％，凋亡相关基因 2 相互作用蛋白 X（Alix）耗竭细胞中大鼠 HEV 的释放降低到 31.5％。尽管大鼠 HEV ORF3 蛋白不与 Tsg101 结合，但我们发现了一种 90kDa 的蛋白质，能够与野生型大鼠 HEV ORF3 蛋白结合，但不能与 PxYPMP 基序中的脯氨酸突变为亮氨酸的 ORF3 突变体结合。Rab27A 或肝细胞生长因子调节的酪氨酸激酶底物（Hrs）耗竭细胞中大鼠 HEV 的释放也分别降低至 20.1％和 18.5％。此外，感染 PLC/PRF/5 细胞中的大鼠 HEV 细胞外水平在用巴佛洛霉素 A1 处理后增加，在用 GW4869 处理后降低。这些结果表明，大鼠 HEV 利用多泡体（MVB）分选进行释放，并且外泌体途径是大鼠 HEV 出芽所必需的。在进一步的研究中，应该探索可以结合大鼠 HEV ORF3 的 Tsg101 的替代宿主蛋白。
