title: Hepatitis E virus enters liver cells through receptor-dependent clathrin-mediated endocytosis.
abstract: 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 × 10(5) 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.
authors.affiliation: Department of Pathology, All India Institute of Medical Sciences, Ansari Nagar, New Delhi, India.
authors.forename: N
authors.initials: N
authors.lastname: Kapur
authors.forename: D
authors.initials: D
authors.lastname: Thakral
authors.forename: H
authors.initials: H
authors.lastname: Durgapal
authors.forename: S K
authors.initials: SK
authors.lastname: Panda
mesh_terms: D019677:Artificial Gene Fusion; D002460:Cell Line; D002966:Clathrin / Q000378:metabolism*; D004705:Endocytosis; D004926:Escherichia coli / Q000235:genetics; D017930:Genes, Reporter; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D016752:Hepatitis E virus / Q000472:pathogenicity*; D022781:Hepatocytes / Q000502:physiology / Q000821:virology*; D006801:Humans; D049409:Luciferases, Firefly / Q000235:genetics / Q000378:metabolism; D011991:Receptors, Virus / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics; D013194:Staining and Labeling / Q000379:methods; D014764:Viral Proteins / Q000235:genetics; D022701:Virosomes / Q000378:metabolism; D053586:Virus Internalization
chemical_list: D002966:Clathrin; D011991:Receptors, Virus; D011993:Recombinant Fusion Proteins; D014764:Viral Proteins; D022701:Virosomes; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; D049409:Luciferases, Firefly