Among numerous established human hepatoma cell lines, none has been shown susceptible to hepatitis B virus (HBV) infection. We describe here a cell line, called HepaRG, which exhibits hepatocytelike morphology, expresses specific hepatocyte functions, and supports HBV infection as well as primary cultures of normal human hepatocytes. Differentiation and infectability are maintained only when these cells are cultured in the presence of corticoids and dimethyl sulfoxide. The specificity of this HBV infection model was ascertained by both the neutralization capacity of HBV-envelope protein-specific antibodies and the competition with an envelope-derived peptide. HepaRG cells therefore represent a tool for deciphering the mechanism of HBV entry. Moreover, their close resemblance to normal human hepatocytes makes them suitable for many applications including drug metabolism studies.H epatitis B, one of the major infectious diseases worldwide, is caused by a small enveloped DNA virus, the hepatitis B virus (HBV). HBV exhibits a very narrow host range and shows a strong tropism for liver parenchymal cells. It has therefore been assumed that susceptibility to HBV infection is restricted to differentiated cells. Accordingly, it was found that only human hepatocyte primary cultures were susceptible to HBV infection (1-4). However, the use of this model is hampered by the limited availability and the inherent variability of human liver material. Even though several human hepatoma-derived cell lines support HBV replication after HBV DNA transfection (5-9), none of them are susceptible to HBV infection.We describe here a hepatoma-derived cell line that expresses a representative panel of liver-specific genes and is susceptible to HBV infection. This goal was achieved by combining an original selection procedure applied early after the cell line establishment in culture and the use of appropriate culture conditions, allowing the commitment of these cells to an optimal differentiation status. MethodsIsolation of the Cell Line and Culture Conditions. Cells were isolated from a liver tumor of a female patient suffering from hepatocarcinoma and hepatitis C infection. All experimental procedures were conducted in conformity with French laws and regulations and were approved by the National Ethics Committee. The samples were minced into small pieces, washed with Hepes buffer (pH 7.7; 140 mM NaCl͞2.68 mM KCl͞0.2 mM Na 2 HPO 4 ͞10 mM Hepes), and digested with 0.025% collagenase D (Boehringer Mannheim) diluted in the same buffer supplemented with 0.075% CaCl 2 under gentle stirring at 37°C. The cell suspension was washed twice in Hepes buffer and resuspended in a William's E medium supplemented with 10% FCS, 100 units͞ml penicillin, 100 g͞ml streptomycin, 5 g͞ml insulin, and 5 ϫ 10 Ϫ7 M hydrocortisone hemisuccinate. Cell suspension was distributed in several dishes without any coating feeder layer. After several weeks, cell growth was sufficient to fulfill the culture dishes. Cells appeared well differentiated, with a hepatocyte-like ...

The lack of an appropriate in vitro infection system for the major human pathogen hepatitis B virus (HBV) has prevented a molecular understanding of the early infection events of HBV. We used the novel HBVinfectible cell line HepaRG and primary human hepatocytes to investigate the interference of infection by HBV envelope protein-derived peptides. We found that a peptide consisting of the authentically myristoylated N-terminal 47 amino acids of the pre-S1 domain of the large viral envelope protein (L protein) specifically prevented HBV infection, with a 50% inhibitory concentration (IC 50 ) of 8 nM. The replacement of myristic acid with other hydrophobic moieties resulted in changes in the inhibitory activity, most notably by a decrease in the IC 50 to picomolar concentrations for longer unbranched fatty acids. The obstruction of HepaRG cell susceptibility to HBV infection after short preincubation times with the peptides suggested that the peptides efficiently target and inactivate a receptor at the hepatocyte surface. Our data both shed light on the molecular mechanism of HBV entry into hepatocytes and provide a basis for the development of potent hepadnaviral entry inhibitors as a novel therapeutic concept for the treatment of hepatitis 〉.The human hepatitis B virus (HBV) causes acute and chronic liver infections in humans. Owing to the propensity of HBV to establish persistent infections, about 400 million people worldwide have an Ϸ100-fold higher risk of developing liver cirrhosis and hepatocellular carcinoma than uninfected people. As a consequence, about 1 million people die every year from HBV-related end-stage liver failure (27). Thus, regardless of the availability of a vaccine and the possibility to therapeutically interfere with genome replication in already infected cells, there is a vital need for the development of agents that protect healthy hepatocytes from infection (e.g., by interference with virus entry) and hence bear the potential to be curative (13).HBV belongs to the family Hepadnaviridae, which includes small enveloped partially double-stranded DNA viruses infecting primates, rodents, and birds (7). Hepadnaviruses possess remarkable species specificities and preferentially target parenchymal liver cells of their respective natural hosts. Experimental in vitro HBV infections have so far only been successful in highly differentiated primary hepatocytes of humans (PHH) (9) and, surprisingly, Tupaia belangeri (8) or in the recently described HepaRG cell line (11). In vivo studies have been restricted to chimpanzees or, as alternatives of unclear relevance, the animal models Pekin ducks (19) and woodchucks (30), using the corresponding duck HBV (DHBV) and woodchuck HBV, respectively. Since the delivery (e.g., by transfection) of hepadnaviral genomes into nonsusceptible cell lines of diverse origins results in the replication, assembly, and secretion of infectious particles (1, 5), it has been assumed that the described limitations are related to some early infection events (receptor recogniti...

During the life cycle of hepatitis B virus (HBV), the large envelope protein (L) plays a pivotal role. Indeed, this polypeptide is essential for viral assembly and probably for the infection process. By performing mutagenesis experiments, we have previously excluded a putative involvement of the pre-S2 domain of the L protein in viral infectivity. In the present study, we have evaluated the role of the pre-S1 region in HBV infection. For this purpose, 21 mutants of the L protein were created. The entire pre-S1 domain was covered by contiguous deletions of 5 amino acids. First, after transfection into HepG2 cells, the efficient expression of both glycosylated and unglycosylated L mutant proteins was verified. The secretion rate of envelope proteins was modified positively or negatively by deletions, indicating that the pre-S1 domain contains several regulating sequences able to influence the surface protein secretion. The ability of mutant proteins to support the production of virions was then studied. Only the four C-terminal deletions, covering the 17 amino acids suspected to interact with the cytoplasmic nucleocapsids, inhibited virion release. Finally, the presence of the modified pre-S1 domain at the external side of all secreted virions was confirmed, and their infectivity was assayed on normal human hepatocytes in primary culture. Only a short sequence including amino acids 78 to 87 tolerates internal deletions without affecting viral infectivity. These results confirm the involvement of the L protein in the infection step and demonstrate that the sequence between amino acids 3 and 77 is involved in this process.