Vectorial Release of Hepatitis E Virus in Polarized Human Hepatocytes

Abstract

Hepatitis E virus (HEV) is a common cause of acute viral hepatitis worldwide. Most HEV infections are asymptomatic but immunocompromised patients infected with HEV3, HEV4 or HEV7 may develop chronic infections. The HEV particles in stools are naked (nHEV) while those in the serum and culture supernatants (eHEV) are associated with lipids. Hepatocytes are polarized epithelial cells that have basolateral (to blood) and apical (to bile) exosomal pathways. We isolated a subclone, F2, from the human hepatocarcinoma cell line HepG2/C3A that grew as a polarized monolayer culture and had better HEV production than HepG2/C3A. F2 cells cultured on semi-permeable collagen inserts and infected basolaterally with nHEV3 released 94.6% of virus particles apically, those infected with eHEV3 released 96.8% apically, and eHEV1-infected cells released 99.3% apically. Transcytosis was not involved. Density gradient centrifugation and NP40 treatment showed that HEV particles released both apically and basolaterally were lipid-associated. The apically-released HEV3 and HEV1 particles were six- and nine-times more infectious than those released basolaterally, respectively. Confocal microscopy indicated that the ORF2 capsid protein colocalized apically with ORF3 virus protein, the apical marker DPP4, and the recycling endosome GTPase Rab27a. The amounts of soluble glycosylated ORF2 secreted apically and basolaterally were similar. These polarized hepatocyte data suggest that infectious HEV particles are mainly released to bile, while the small fraction released to blood could spread HEV throughout the host. Importance The hepatitis E virus (HEV) in stools is naked while those in culture supernatants and patients’ blood are lipid-associated. Its life cycle in hepatocytes, polarized cells with basolateral side communicating with blood and apical side connected with bile, is incompletely understood. We have developed a polarized hepatocyte model, and used these cells to analyze the supernatants bathing the apical and basolateral sides and HEV subcellular distribution. HEV viral particles from both sides were lipid-associated and most infectious HEV particles left the cell via its apical side. Similar amounts of the soluble capsid protein ORF2 were secreted from both sides of the hepatocytes. This model mimicking physiological conditions should help clarify the HEV cell cycle in polarized hepatocytes.