Division of Gastroenterology and Hepatology, Lausanne University Hospital and University of Lausanne, Lausanne 1011, Switzerland.
Proc Natl Acad Sci U S A. 2023 Dec 26;120(52):e2307423120. doi: 10.1073/pnas.2307423120. Epub 2023 Dec 18.
Hepatitis E virus (HEV) is a major cause of acute hepatitis worldwide. As the other positive-strand RNA viruses, it is believed to replicate its genome in a membrane-associated replication complex. However, current understanding of the host factors required for productive HEV infection is limited and the site as well as the composition of the HEV replication complex are still poorly characterized. To identify host factors required for HEV RNA replication, we performed a genome-wide CRISPR/Cas9 screen in permissive human cell lines harboring subgenomic HEV replicons allowing for positive and negative selection. Among the validated candidates, Ras-related early endosomal protein Rab5A was selected for further characterization. siRNA-mediated silencing of Rab5A and its effectors APPL1 and EEA1, but not of the late and recycling endosome components Rab7A and Rab11A, respectively, significantly reduced HEV RNA replication. Furthermore, pharmacological inhibition of Rab5A and of dynamin-2, required for the formation of early endosomes, resulted in a dose-dependent decrease of HEV RNA replication. Colocalization studies revealed close proximity of Rab5A, the HEV ORF1 protein, corresponding to the viral replicase, as well as HEV positive- and negative-strand RNA. In conclusion, we successfully exploited CRISPR/Cas9 and selectable subgenomic replicons to identify host factors of a noncytolytic virus. This approach revealed a role for Rab5A and early endosomes in HEV RNA replication, likely by serving as a scaffold for the establishment of functional replication complexes. Our findings yield insights into the HEV life cycle and the virus-host interactions required for productive infection.
戊型肝炎病毒（HEV）是全球急性肝炎的主要病因。与其他正链 RNA 病毒一样，它被认为在膜相关的复制复合物中复制其基因组。然而，目前对产生活跃 HEV 感染所需的宿主因子的了解有限，HEV 复制复合物的位置和组成仍未得到很好的描述。为了鉴定产生活跃 HEV RNA 复制所需的宿主因子，我们在含有允许正选择和负选择的亚基因组 HEV 复制子的允许性人细胞系中进行了全基因组 CRISPR/Cas9 筛选。在验证的候选物中，选择 Ras 相关早期内体蛋白 Rab5A 进行进一步表征。Rab5A 及其效应物 APPL1 和 EEA1 的 siRNA 介导的沉默，但不是晚期和再循环内体成分 Rab7A 和 Rab11A 的沉默，分别显著降低了 HEV RNA 复制。此外，Rab5A 和 dynamin-2 的药理学抑制，这是早期内体形成所必需的，导致 HEV RNA 复制呈剂量依赖性下降。共定位研究显示，Rab5A 与 HEV ORF1 蛋白（对应于病毒复制酶）以及 HEV 正链和负链 RNA 非常接近。总之，我们成功地利用 CRISPR/Cas9 和可选择的亚基因组复制子来鉴定非细胞毒性病毒的宿主因子。这种方法揭示了 Rab5A 和早期内体在 HEV RNA 复制中的作用，可能通过充当功能性复制复合物建立的支架。我们的发现深入了解了 HEV 生命周期和产生活跃感染所需的病毒-宿主相互作用。
