Division of Gastroenterology and Hepatology, Centre Hospitalier Universitaire Vaudois, University of Lausanne, Lausanne, Switzerland.
Division of Gastroenterology and Hepatology, Centre Hospitalier Universitaire Vaudois, University of Lausanne, Lausanne, Switzerland
J Virol. 2019 Sep 12;93(19). doi: 10.1128/JVI.00459-19. Print 2019 Oct 1.
Hepatitis E virus (HEV) is one of the most common causes of acute hepatitis and jaundice in the world. Current understanding of the molecular virology and pathogenesis of hepatitis E is incomplete, due particularly to the limited availability of functional tools. Here, we report the development of tagged HEV genomes as a novel tool to investigate the viral life cycle. A selectable subgenomic HEV replicon was subjected to random 15-nucleotide sequence insertion using transposon-based technology. Viable insertions in the open reading frame 1 (ORF1) protein were selected in a hepatoblastoma cell line. Functional insertion sites were identified downstream of the methyltransferase domain, in the hypervariable region (HVR), and between the helicase and RNA-dependent RNA polymerase domains. HEV genomes harboring a hemagglutinin (HA) epitope tag or a small luciferase (NanoLuc) in the HVR were found to be fully functional and to allow the production of infectious virus. NanoLuc allowed quantitative monitoring of HEV infection and replication by luciferase assay. The use of HA-tagged replicons and full-length genomes allowed localization of putative sites of HEV RNA replication by the simultaneous detection of viral RNA by fluorescence hybridization and of ORF1 protein by immunofluorescence. Candidate HEV replication complexes were found in cytoplasmic dot-like structures which partially overlapped ORF2 and ORF3 proteins as well as exosomal markers. Hence, tagged HEV genomes yield new insights into the viral life cycle and should allow further investigation of the structure and composition of the viral replication complex. Hepatitis E virus (HEV) infection is an important cause of acute hepatitis and may lead to chronic infection in immunocompromised patients. Knowledge of the viral life cycle is incomplete due to the limited availability of functional tools. In particular, low levels of expression of the ORF1 protein or limited sensitivity of currently available antibodies or both limit our understanding of the viral replicase. Here, we report the successful establishment of subgenomic HEV replicons and full-length genomes harboring an epitope tag or a functional reporter in the ORF1 protein. These novel tools should allow further characterization of the HEV replication complex and to improve our understanding of the viral life cycle.
戊型肝炎病毒（HEV）是世界上导致急性肝炎和黄疸的最常见原因之一。目前对戊型肝炎的分子病毒学和发病机制的了解并不完整，这主要是由于功能工具的有限可用性。在这里，我们报告了标记的 HEV 基因组的开发，作为研究病毒生命周期的新工具。选择的亚基因组 HEV 复制子通过转座子技术进行随机 15 个核苷酸序列插入。在肝癌细胞系中选择了在 ORF1 蛋白中具有选择性功能的插入物。功能插入位点位于甲基转移酶结构域、高度可变区（HVR）和螺旋酶与 RNA 依赖性 RNA 聚合酶结构域之间。在 HVR 中带有血凝素（HA）表位标签或小萤光素酶（NanoLuc）的 HEV 基因组被发现具有完全功能，并能够产生感染性病毒。NanoLuc 通过萤光素酶测定允许定量监测 HEV 感染和复制。使用 HA 标记的复制子和全长基因组允许通过荧光杂交同时检测病毒 RNA 和 ORF1 蛋白来定位 HEV RNA 复制的假定部位。在细胞质点状结构中发现了候选 HEV 复制复合物，这些结构与 ORF2 和 ORF3 蛋白以及外泌体标记部分重叠。因此，标记的 HEV 基因组为病毒生命周期提供了新的见解，并且应该允许进一步研究病毒复制复合物的结构和组成。戊型肝炎病毒（HEV）感染是急性肝炎的重要原因，并且可能导致免疫功能低下的患者发生慢性感染。由于功能工具的有限可用性，对病毒生命周期的了解并不完整。特别是，ORF1 蛋白的低表达水平或当前可用抗体的有限敏感性或两者都限制了我们对病毒复制酶的理解。在这里，我们报告了成功建立了带有 ORF1 蛋白中的表位标签或功能性报告基因的亚基因组 HEV 复制子和全长基因组。这些新工具应该允许进一步表征 HEV 复制复合物，并提高我们对病毒生命周期的理解。
