Hepatocyte polarization is essential for the productive entry of the hepatitis B virus
Creators: name: Schulze, Andreas
name: Mills, Kerry
name: Weiss, Thomas S.
name: Urban, Stephan
Publisher: name: Universität Regensburg
Year: 2011
Subjects: subject: 610 Medizin

description: Abstract

Human hepatitis B virus (HBV) is characterized by a high species specificity and a distinct liver tropism. Within the liver, HBV replication occurs in differentiated and polarized hepatocytes. Accordingly, the in vitro HBV infection of primary human hepatocytes (PHHs) and the human hepatoma cell line, HepaRG, is restricted to differentiated, hepatocyte-like cells. Though preparations of PHH contain up to 100% hepatic cells, cultures of differentiated HepaRG cells are a mixture of hepatocyte-like and biliary-like epithelial cells. We used PHH and HepaRG cells and compared the influence of virus inoculation dose, cell differentiation, and polarization on productive HBV infection. At multiplicities of genome equivalents (mge) &gt;8,000, almost 100% of PHHs could be infected. In contrast, only a subset of HepaRG cells stained positive for HBcAg at comparable or even higher mge. Infection predominantly occurred at the edges of islands of hepatocyte-like HepaRG cells. This indicates a limited accessibility of the HBV receptor, possibly as a result of its polar sorting. Multidrug resistance protein 2 (MRP2), a marker selectively transported to the apical (i.e., canalicular) cell membrane, revealed two polarization phenotypes of HepaRG cells. HBV infection within the islands of hepatocyte-like HepaRG cells preferentially occurred in cells that resemble PHH, exhibiting canalicular structures. However, disruption of cell-cell junctions allowed the additional infection of cells that do not display a PHH-like polarization. Conclusion: HBV enters hepatocytes via the basolateral membrane. This model, at least partially, explains the difference of PHH and HepaRG cells in infection efficacy, provides insights into natural HBV infection, and establishes a basis for optimization of the HepaRG infection system. (HEPATOLOGY 2012)

Human hepatitis B virus (HBV), a small, enveloped DNA virus, is the prototypic member of the family, hepadnaviridae, causing acute and persistant liver infections.1 Currently, 360 million people are chronically infected with HBV and are, consequently, prone to developing progressive liver diseases, such as cirrhosis or hepatocellular carcinoma. Hepadnaviruses exhibit a pronounced liver tropism and a narrow host range.2 HBV predominantly infects and replicates in hepatocytes, but virus-specific antigens and nucleic acids have also been found in a number of nonhepatic tissues, including kidney, pancreas, and peripheral blood mononuclear cells.3 However, whether replication in extrahepatic tissues contributes to virus propagation is unclear.4, 5 The preference for replication in the liver can only partially be explained by hepatocyte-specific transcription factors.6 Further liver specificity occurs at the stage of early infection, because the differentiation state of HepaRG cells and primary hepatocytes is a prerequisite for the expression of an HBVpreS1-specific receptor and subsequent infection (Meier et al., manuscript in preparation).

Two strategies are used for the investigation of HBV infection in vitro. One is the application of hepatoma cell lines (e.g., HuH7 or HepG2), which do not support HBV entry and hence cannot be infected, but allow viral replication after delivery of the viral genome by stable or transient transfection.7, 8 These cell lines allow the examination of late-replication steps, such as transcription, translation, assembly, and release of HBV particles. Alternatively, primary cultures of human9 or tupaia10 hepatocytes and the human hepatoma cell line, HepaRG,11 support HBV infection and replication and are employed for investigations addressing HBV entry (e.g., attachment, receptor interaction, viral uptake, covalently closed circular DNA [cccDNA] formation, and regulation of gene transcription). Important determinants for infectivity within the HBV envelope proteins were identified using mutational analyses. These include the N-terminal 75 amino acids of the preS1-domain of the HBV L-protein, its myristoylation, and the integrity of a region in the antigenic loop of the S-domain.12-15 Little is known about specific host factors, particularly HBV-specific receptor(s) that contribute to the tropism of the virus. We and others identified heparan sulfate proteoglycans as mandatory attachment factors for HBV.16, 17 However, because of their ubiquitous expression, this interaction does not explain the hepatotropism of HBV, but rather represents a first, nonspecific step of a multistep entry process. Potential HBV-receptor candidates have been described in the past, but none of them have been confirmed in a functional assay.2 Expression and accessibility of HBV-specific receptor(s) on the cell surface define, among other factors, the susceptibility of cells toward HBV. In contrast to most epithelial cells, which exhibit a polarity consisting of a single apical and basal pole that oppose each other, hepatocytes in the liver are polarized in a more complex manner, with distinct apical and basolateral domains facing the continuous network of bile canaliculi and the hepatic sinusoid.18 Tight junctions between hepatocytes prevent lateral diffusion of substrates, form the blood-bile barrier, and contribute to the maintenance of cell polarity.

Using HepaRG cells and primary human hepatocytes (PHHs), we performed a detailed analysis of the properties of in vitro HBV infection. We investigated whether cell polarization, in addition to cell differentiation, would play a role in the infection process. We took advantage of the fact that HepaRG cells, in contrast to other hepatoma-derived cell lines, constitutively and synchronously display both hepatocyte- and biliary-like epithelial phenotypes at confluence.19

We demonstrated that disrupting the epithelial barrier leads to an increased HBV infection, suggesting that the entry of HBV into hepatocytes occurs in a polarized manner and that hepatocyte polarization imposes a physical barrier that restricts the access of the virus to its receptor(s).