Hepatitis E virus enters liver cells through receptor-dependent clathrin-mediated endocytosis

Summary.  We investigated the virus–host interaction for hepatitis E virus (HEV) by performing competitive binding assays using in vitro assembled virus-like particles (VLPs). We used Escherichia coli expressed native capsid protein (pORF2) and its mutants with an attached Gly₍₅₎-Ala (linker) reporter [enhanced green fluorescent protein (EGFP)/firefly luciferase (Fluc)]. Transmission electron microscopy and nanoparticle tracking showed near uniform particles of approximately 30–35 nm in diameter for pORF2 VLPs and 60–100 nm for reporter-linked VLPs. Binding of reporter-linked full-length (1–660aa) and N-terminal truncated (Δ1–112aa) pORF2 VLPs to Huh7 cell surfaces was found to be specific with 1.92 ± 0.065 × 10⁵ sites per cell. Saturation binding indicated an equilibrium dissociation constant (K_d) of 121.1 ± 23.83 and 123.8 ± 16.15 nm for pORF2-linker-EGFP and pORF2-linker-Fluc VLPs respectively. A similar binding pattern was observed for Δ1–112aa pORF2-linker-EGFP and Δ1–112aa pORF2-linker-Fluc VLPs with K_d values of 123.6 ± 10.60 and 135.6 ± 16.19 nm respectively. The affinity (log K_i) of pORF2 binding on Huh7 cells in the presence of EGFP-tagged and Fluc-tagged pORF2 VLPs was found to be approximately 2.0. However, no VLP formation or binding was observed with refolded C-terminal truncated (Δ458–660aa) pORF2. We investigated HEV internalization using fluorescent VLPs (EGFP-VLPs), which showed vesicle-mediated uptake starting at 5 min post-incubation. The uptake of VLPs could be stopped by inhibitors for clathrin-dependent endocytosis, but not by caveosome inhibitors. No binding and uptake of EGFP-VLPs were observed on non-hepatic cell lines (HeLa and SiHa). These findings suggest that HEV attaches to the host cell via a specific high affinity receptor and enters the cytoplasm by clathrin-mediated endocytosis.