Hepatitis E virus enters liver cells through receptor‐dependent clathrin‐mediated endocytosis
Identifieur interne : 000C07 ( Main/Exploration ); précédent : 000C06; suivant : 000C08Hepatitis E virus enters liver cells through receptor‐dependent clathrin‐mediated endocytosis
Auteurs : N. Kapur [Inde] ; D. Thakral [Inde] ; H. Durgapal [Inde] ; S. K. Panda [Inde]Source :
- Journal of Viral Hepatitis [ 1352-0504 ] ; 2012-06.
Abstract
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 (Kd) 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 Kd values of 123.6 ± 10.60 and 135.6 ± 16.19 nm respectively. The affinity (log Ki) 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.
Url:
DOI: 10.1111/j.1365-2893.2011.01559.x
Affiliations:
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Kapur</name><affiliation wicri:level="1"><country xml:lang="fr">Inde</country><wicri:regionArea>Department of Pathology, All India Institute of Medical Sciences, Ansari Nagar, New Delhi</wicri:regionArea><wicri:noRegion>New Delhi</wicri:noRegion></affiliation></author><author><name sortKey="Thakral, D" sort="Thakral, D" uniqKey="Thakral D" first="D." last="Thakral">D. Thakral</name><affiliation wicri:level="1"><country xml:lang="fr">Inde</country><wicri:regionArea>Department of Pathology, All India Institute of Medical Sciences, Ansari Nagar, New Delhi</wicri:regionArea><wicri:noRegion>New Delhi</wicri:noRegion></affiliation></author><author><name sortKey="Durgapal, H" sort="Durgapal, H" uniqKey="Durgapal H" first="H." last="Durgapal">H. Durgapal</name><affiliation wicri:level="1"><country xml:lang="fr">Inde</country><wicri:regionArea>Department of Pathology, All India Institute of Medical Sciences, Ansari Nagar, New Delhi</wicri:regionArea><wicri:noRegion>New Delhi</wicri:noRegion></affiliation></author><author><name sortKey="Panda, S K" sort="Panda, S K" uniqKey="Panda S" first="S. K." last="Panda">S. K. Panda</name><affiliation wicri:level="1"><country xml:lang="fr">Inde</country><wicri:regionArea>Department of Pathology, All India Institute of Medical Sciences, Ansari Nagar, New Delhi</wicri:regionArea><wicri:noRegion>New Delhi</wicri:noRegion></affiliation></author></analytic><monogr></monogr><series><title level="j" type="main">Journal of Viral Hepatitis</title><title level="j" type="alt">JOURNAL OF VIRAL HEPATITIS</title><idno type="ISSN">1352-0504</idno><idno type="eISSN">1365-2893</idno><imprint><biblScope unit="vol">19</biblScope><biblScope unit="issue">6</biblScope><biblScope unit="page" from="436">436</biblScope><biblScope unit="page" to="448">448</biblScope><biblScope unit="page-count">13</biblScope><publisher>Blackwell Publishing Ltd</publisher><pubPlace>Oxford, UK</pubPlace><date type="published" when="2012-06">2012-06</date></imprint><idno type="ISSN">1352-0504</idno></series></biblStruct></sourceDesc><seriesStmt><idno type="ISSN">1352-0504</idno></seriesStmt></fileDesc><profileDesc><textClass></textClass></profileDesc></teiHeader><front><div type="abstract" xml:lang="en">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 (Kd) 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 Kd values of 123.6 ± 10.60 and 135.6 ± 16.19 nm respectively. The affinity (log Ki) 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.</div></front></TEI><affiliations><list><country><li>Inde</li></country></list><tree><country name="Inde"><noRegion><name sortKey="Kapur, N" sort="Kapur, N" uniqKey="Kapur N" first="N." last="Kapur">N. Kapur</name></noRegion><name sortKey="Durgapal, H" sort="Durgapal, H" uniqKey="Durgapal H" first="H." last="Durgapal">H. Durgapal</name><name sortKey="Panda, S K" sort="Panda, S K" uniqKey="Panda S" first="S. K." last="Panda">S. K. Panda</name><name sortKey="Thakral, D" sort="Thakral, D" uniqKey="Thakral D" first="D." last="Thakral">D. Thakral</name></country></tree></affiliations></record>Pour manipuler ce document sous Unix (Dilib)
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