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 ...

Patients affected by chronic inflammatory disorders display high amounts of soluble CD95L. This homotrimeric ligand arises from the cleavage by metalloproteases of its membrane-bound counterpart, a strong apoptotic inducer. In contrast, the naturally processed CD95L is viewed as an apoptotic antagonist competing with its membrane counterpart for binding to CD95. Recent reports pinpointed that activation of CD95 may attract myeloid and tumoral cells, which display resistance to the CD95-mediated apoptotic signal. However, all these studies were performed using chimeric CD95Ls (oligomerized forms), which behave as the membrane-bound ligand and not as the naturally processed CD95L. Herein, we examine the biological effects of the metalloprotease-cleaved CD95L on CD95-sensitive activated T-lymphocytes. We demonstrate that cleaved CD95L (cl-CD95L), found increased in sera of systemic lupus erythematosus (SLE) patients as compared to that of healthy individuals, promotes the formation of migrating pseudopods at the leading edge of which the death receptor CD95 is capped (confocal microscopy). Using different migration assays (wound healing/Boyden Chamber/endothelial transmigration), we uncover that cl-CD95L promotes cell migration through a c-yes/Ca2+/PI3K-driven signaling pathway, which relies on the formation of a CD95-containing complex designated the MISC for Motility-Inducing Signaling Complex. These findings revisit the role of the metalloprotease-cleaved CD95L and emphasize that the increase in cl-CD95L observed in patients affected by chronic inflammatory disorders may fuel the local or systemic tissue damage by promoting tissue-filtration of immune cells.

The hepatitis B virus (HBV) envelope contains equimolar amounts of three viral proteins: the major (S), middle, and large (L) polypeptides. Their roles in the adsorption and penetration of the virus have not yet been elucidated. We have used a highly efficient in vitro model that permits reproducible HBV infection to investigate whether N-myristylation, a posttranslational modification of the L protein, was essential for viral infectivity. A point mutation abolishing myristylation was introduced into the HBV genome. Mutant virions were produced by transfecting viral DNA into hepatoma cells and their infectivity was evaluated on primary human hepatocyte cultures. No difference between mutant and wild-type viral RNA production could be observed. Furthermore, intermediate DNA replicative forms were observed in transfected cells demonstrating replication competence of mutant viral genomes. In addition, complete virions were produced in the cell supernatant. However, we found that mutant viral particles contained viral DNA with a reduced mean size, probably corresponding to a larger single-stranded region in the relaxed circular DNA form. We have evidenced the presence of pre-S1, pre-S2, and S epitopes at the outer surface of these virions by using immunoprecipitation with specific monoclonal antibodies. This result confirmed that mutant viruses were normally assembled. By contrast, myristylation-defective mutants completely lost their infectivity for human hepatocytes in primary cultures as shown by the absence of HBs antigen production and viral intermediate replicative forms in hepatocytes. In conclusion, the myristylation of the L protein is not required for the production of Dane-like particles but it is absolutely necessary for HBV infectivity.