1. Academic Validation
  2. A genome-wide CRISPR/Cas9 screen identifies a role for Rab5A and early endosomes in hepatitis E virus replication

A genome-wide CRISPR/Cas9 screen identifies a role for Rab5A and early endosomes in hepatitis E virus replication

  • Proc Natl Acad Sci U S A. 2023 Dec 26;120(52):e2307423120. doi: 10.1073/pnas.2307423120.
Noémie Oechslin 1 Nathalie Da Silva 1 Maliki Ankavay 1 Darius Moradpour 1 Jérôme Gouttenoire 1
Affiliations

Affiliation

  • 1 Division of Gastroenterology and Hepatology, Lausanne University Hospital and University of Lausanne, Lausanne 1011, Switzerland.
Abstract

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.

Keywords

Rab5A; early endosome; hepatitis E virus; positive-strand RNA virus; replication complex.

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