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.

Links

Publisher Full Text

Authors

Kapur N, Thakral D, Durgapal H, Panda SK

Institution

Department of Pathology, All India Institute of Medical Sciences, Ansari Nagar, New Delhi, India.

Source

Journal of viral hepatitis 19:6 2012 Jun pg 436-48

MeSH

Artificial Gene Fusion
Cell Line
Clathrin
Endocytosis
Escherichia coli
Genes, Reporter
Green Fluorescent Proteins
Hepatitis E virus
Hepatocytes
Humans
Luciferases, Firefly
Receptors, Virus
Recombinant Fusion Proteins
Staining and Labeling
Viral Proteins
Virosomes
Virus Internalization

Pub Type(s)

Journal Article
Research Support, Non-U.S. Gov't

Language

eng

PubMed ID

22571906