Open AccessHepatitis B virus entry into HepG2‐NTCP cells requires clathrin‐mediated endocytosis
Author(s) -
Herrscher Charline,
Pastor Florentin,
BurlaudGaillard Julien,
Dumans Amélie,
Seigneuret Florian,
Moreau Alain,
Patient Romuald,
Eymieux Sebastien,
Rocquigny Hugues,
Hourioux Christophe,
Roingeard Philippe,
Blanchard Emmanuelle
Publication year - 2020
Publication title -
cellular microbiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.542
H-Index - 138
eISSN - 1462-5822
pISSN - 1462-5814
DOI - 10.1111/cmi.13205
Subject(s) - endocytosis , clathrin , biology , hepatitis b virus , pinocytosis , viral entry , microbiology and biotechnology , receptor mediated endocytosis , caveolae , actin cytoskeleton , small interfering rna , virology , cell , cell culture , cytoskeleton , virus , signal transduction , viral replication , transfection , biochemistry , genetics
Abstract Hepatitis B virus (HBV) is a leading cause of cirrhosis and hepatocellular carcinoma worldwide, with 250 million individuals chronically infected. Many stages of the HBV infectious cycle have been elucidated, but the mechanisms of HBV entry remain poorly understood. The identification of the sodium taurocholate cotransporting polypeptide (NTCP) as an HBV receptor and the establishment of NTCP‐overexpressing hepatoma cell lines susceptible to HBV infection opens up new possibilities for investigating these mechanisms. We used HepG2‐NTCP cells, and various chemical inhibitors and RNA interference (RNAi) approaches to investigate the host cell factors involved in HBV entry. We found that HBV uptake into these cells was dependent on the actin cytoskeleton and did not involve macropinocytosis or caveolae‐mediated endocytosis. Instead, entry occurred via the clathrin‐mediated endocytosis pathway. HBV internalisation was inhibited by pitstop‐2 treatment and RNA‐mediated silencing (siRNA) of the clathrin heavy chain, adaptor protein AP‐2 and dynamin‐2. We were able to visualise HBV entry in clathrin‐coated pits and vesicles by electron microscopy (EM) and cryo‐EM with immunogold labelling. These data demonstrating that HBV uses a clathrin‐mediated endocytosis pathway to enter HepG2‐NTCP cells increase our understanding of the complete HBV life cycle.