Vectorial entry and release of hepatitis A virus in polarized human hepatocytes

Abstract

Hepatitis A virus (HAV) is an enterically transmitted virus that replicates predominantly in hepatocytes within the liver before excretion via bile through feces. Hepatocytes are polarized epithelial cells, and it has been assumed that the virus load in bile results from direct export of HAV via the apical domain of polarized hepatocytes. We have developed a subclone of hepatocyte-derived HepG2 cells (clone N6) that maintains functional characteristics of polarized hepatocytes but displays morphology typical of columnar epithelial cells, rather than the complex morphology that is typical of hepatocytes. N6 cells form microcolonies of polarized cells when grown on glass and confluent monolayers of polarized cells on semipermeable membranes. When N6 microcolonies were exposed to HAV, infection was restricted to peripheral cells of polarized colonies, whereas all cells could be infected in colonies of nonpolarized HepG2 cells (clone C11) or following disruption of tight junctions in N6 colonies with EGTA. This suggests that viral entry occurs predominantly via the basolateral plasma membrane, consistent with uptake of virus from the bloodstream after enteric exposure, as expected. Viral export was also found to be markedly vectorial in N6 but not C11 cells. However, rather than being exported from the apical domain as expected, more than 95% of HAV was exported via the basolateral domain of N6 cells, suggesting that virus is first excreted from infected hepatocytes into the bloodstream rather than to the biliary tree. Enteric excretion of HAV may therefore rely on reuptake and transcytosis of progeny HAV across hepatocytes into the bile. These studies provide the first example of the interactions between viruses and polarized hepatocytes.

FIG. 1.

Epithelial cell polarity. A. Hepatocytes in vivo and in vitro develop complex polarity, such that adjacent apical (Ap) surfaces will form bile canaliculae (BC). B. Other polarized epithelial cells grow with a simple (columnar) orientation of polarity. C. The apical and basolateral domains are both accessible when cells, such as those in panel B, are grown as a confluent monolayer on semipermeable membranes, with tight junctions between cells preventing lateral diffusion of substrates.

FIG. 2.

Polarized HepG2 cells. A. ZO-1 (green) staining of HepG2 cells shows a population of cells (nuclei, red) with mixed polarity, with areas of bile cyst formation (arrowhead) and areas of intracellular tight junction formation (arrow). Subcloning yielded populations of cells with a nonpolarized phenotype (C11) and polarized phenotype (N6), showing the extensive honeycomb distribution of ZO-1 (green) in the latter. B. x-z section of cells stained for the apically distributed protein CD26 (red) and the tight junction protein ZO-1 (green). The N6 cells show predominantly apical localization of the CD26 protein at the same level as the intracellular tight junctions, while the C11 cells have no detectable ZO-1 staining and a widespread intracellular CD26 distribution. C. The ultrastructure of N6 cells was examined by electron microscopy. These cells display many of the basic features of polarized epithelial cells. The apical surfaces show extensive villi (arrowheads), and intracellular tight junction complexes (arrow) can be seen.

FIG. 3.

Vectorial distribution of CD26 in polarized hepatocytes. The images are a Z-series of 2-μm sections from basolateral (A) to apical (H) through a colony of N6 cells. Polarized central cells of the colony show ZO-1 staining (green). Nonpolarized peripheral cells display intracellular CD26 staining (red) throughout the cytoplasm, while the central cells show marked apical distribution of protein, confirming that these cells are functionally polarized. Bar, 30 μm.

FIG. 4.

Export of albumin from subcloned cells. Cells grown on semipermeable membranes for 30 days were examined for albumin export into the apical and basolateral domains. (A and B) C11 (A) and N6 (B) cells export similar amounts of total albumin (triangles) but differ significantly in amounts of basolateral albumin (bars) secreted per hour. (C) Percent basolateral albumin export (of total export) is shown. C11 cells (gray bars) exported albumin equally into the apical and basolateral domains. Polarized N6 cells (black bars) show a gradual increase in percent basolateral albumin export, which is maximal at over 75% between days 14 and 24.

FIG. 5.

HAV infects hepatocytes from the basolateral domain. Cells grown in microcolonies were infected with HAV and examined for HAV (red) and ZO-1(green) expression by immunofluorescence. (A to C) Uncloned HepG2 cells show occasional bile cyst formation (arrows) at the apical domain of adjacent cells, indicating these cells are polarized, but as basolateral domains are facing both the glass and the media, cells throughout the colony are infected. (D to F) Similarly, C11 cells demonstrate that infection occurs readily throughout the colonies of cells that are not polarized. (G to I) The basolateral domains of N6 cells are adherent to the glass except at the periphery of the colony. Central polarized cells, with extensive tight junction formation as shown by ZO-1 staining, are unable to be infected with HAV, while peripheral cells are readily infected. Bar, 30 μm.

FIG. 6.

Disruption of tight junctions of polarized hepatocytes permits infection. (A) N6 and C11 cells grown in microcolonies were treated with EGTA for 10 min to disrupt the tight junctions and then infected with HAV. Cells were stained for ZO-1 (green), HAV (red), and nuclei (blue). Before EGTA treatment, only peripheral cells of the N6 colony (panel i), but all cells of the C11 colony (ii), were infectible with virus. Once tight junctions were disrupted, N6 cells also showed extensive viral infection throughout the colony despite reformation of tight junctions postinfection (iii). (B) N6 cells were untreated (pre-EGTA) or treated with EGTA for 10 min (EGTA) or incubated with normal medium for a further hour after EGTA treatment (post-EGTA) and then fixed and stained for ZO-1. Tight junctions were disrupted by the EGTA treatment but reformed within 1 h. Bar, 20 μm.

FIG. 7.

Vectorial export of hepatitis A virus. N6 and C11 cells grown on semipermeable membranes were assayed for viral export into the apical and basolateral domains. Both albumin (black) and HAV (gray) are exported predominantly into the basolateral domain by polarized N6 cells, indicating vectorial export of these substrates in polarized hepatocytes. Nonpolarized C11 cells exported about 50% of both the albumin and virus into the basolateral domain.