Despite important progress toward deciphering human hepatitis B virus (HBV) entry into host cells, many aspects of the early steps of the life cycle remained completely obscure. Following endocytosis, HBV must travel through the complex network of the endocytic pathway to reach the cell nucleus and initiate replication. In addition to guiding the viral particles to the replication site, the endosomal vesicles may play a crucial role in infection, providing the appropriate environment for virus uncoating and nucleocapsid release. In this work, we investigated the trafficking of HBV particles internalized in permissive cells. Expression of key Rab proteins, involved in specific pathways leading to different intracellular locations, was modulated in HepaRG cells, using a stable and inducible short hairpin RNA (shRNA) expression system. The trafficking properties of the newly developed cells were demonstrated by confocal microscopy and flow cytometry using specific markers. The results showed that HBV infection strongly depends on Rab5 and Rab7 expression, indicating that HBV transport from early to mature endosomes is required for a step in the viral life cycle. This may involve reduction of disulfide bond-linked envelope proteins, as alteration of the redox potential of the endocytic pathway resulted in inhibition of infection. Subcellular fractionation of HBV-infected cells showed that viral particles are further transported to lysosomes. Intriguingly, infection was not dependent on the lysosomal activity, suggesting that trafficking to this compartment is a "dead-end" route. Together, these data add to our understanding of the HBV-host cell interactions controlling the early stages of infection. C hronic hepatitis B virus (HBV) infections can lead to lifethreatening liver diseases, such as cirrhosis and hepatocellular carcinoma, the third cause of cancer deaths worldwide (1). Infectious viral particles consist of an icosahedral nucleocapsid made of the core protein and a lipid bilayer embedding the small (S), middle (M), and large (L) envelope proteins (2). The nucleocapsid surrounds the relaxed circular, partially double-stranded DNA genome, to which the viral polymerase is covalently attached (3). Apart from the crucial roles played in virus morphogenesis and infection, the envelope proteins bear the intriguing property to self-assemble into spherical and filamentous subviral particles (SVP), which are released from cells in vast excess over virions, accounting for more than 90% of the total particles in the serum of infected patients (4).Studies regarding the early events of the HBV life cycle have been particularly problematic, as human primary hepatocytes, the physiological host, are difficult to procure and maintain in tissue culture and are refractory to genetic manipulation. The development of alternative infectivity models for HBV, represented by proliferating liver-derived cell lines able to differentiate and support HBV infection in vitro, has considerably overcome many of these drawbacks (5-7). Moreo...

Investigation of the entry pathways of hepatitis B virus (HBV), a member of the family Hepadnaviridae, has been hampered by the lack of versatile in vitro infectivity models. Most concepts of hepadnaviral infection come from the more robust duck HBV system; however, whether the two viruses use the same mechanisms to invade target cells is still a matter of controversy. In this study, we investigate the role of an important plasma membrane component, caveolin-1 (Cav-1), in HBV infection. Caveolins are the main structural components of caveolae, plasma membrane microdomains enriched in cholesterol and sphingolipids, which are involved in the endocytosis of numerous ligands and complex signaling pathways within the cell. We used the HepaRG cell line permissive for HBV infection to stably express dominant-negative Cav-1 and dynamin-2, a GTPase involved in vesicle formation at the plasma membrane and other organelles. The endocytic properties of the newly established cell lines, designated HepaRG Cav-1 , HepaRG Cav-1⌬1-81 , HepaRG Dyn-2 , and HepaRG Dyn-2K44A , were validated using specific markers for different entry routes. The cells maintained their properties during cell culture, supported differentiation, and were permissive for HBV infection. The levels of both HBV transcripts and antigens were significantly decreased in cells expressing the mutant proteins, while viral replication was not directly affected. Chemical inhibitors that specifically inhibit clathrin-mediated endocytosis had no effect on HBV infection. We concluded that HBV requires a Cav-1-mediated entry pathway to initiate productive infection in HepaRG cells.

Liver infection with hepatitis B virus (HBV), a DNA virus of the Hepadnaviridae family, leads to severe disease, such as fibrosis, cirrhosis and hepatocellular carcinoma. The early steps of the viral life cycle are largely obscure and the host cell plasma membrane receptors are not known. HepaRG is the only proliferating cell line supporting HBV infection in vitro, following specific differentiation, allowing for investigation of new host host-cell factors involved in viral entry, within a more robust and reproducible environment. Viral infection generally begins with receptor recognition at the host cell surface, following highly specific cell-virus interactions. Most of these interactions are expected to take place at the plasma membrane of the HepaRG cells. In the present study, we used this cell line to explore changes between the plasma membrane of undifferentiated (−) and differentiated (+) cells and to identify differentially-regulated proteins or signaling networks that might potentially be involved in HBV entry. Our initial study identified a series of proteins that are differentially expressed in the plasma membrane of (−) and (+) cells and are good candidates for potential cell-virus interactions. To our knowledge, this is the first study using functional proteomics to study plasma membrane proteins from HepaRG cells, providing a platform for future experiments that will allow us to understand the cell-virus interaction and mechanism of HBV viral infection.

The surface relief gratings (SRGs) can be generated when azo-polymer films are exposed to laser beam interference as a result of mass migration. Despite considerable research effort over the past two decades this complex phenomenon remains incompletely understood. Here we show, for the first time, the athermal photofluidisation of azo-polysiloxane films exposed to 488 nm light, directly monitored by optical microscopy. A process of surface relief erasure occurring in parallel with its inscription was also observed during laser irradiation. We therefore propose a new mechanism of SRG formation, based on three different processes: (1) the polymer photo-fluidization in illuminated regions, (2) the mass displacement from illuminated to dark regions and (3) the inverse mass displacement, from dark to illuminated regions. The mechanical properties of the films during UV light irradiation were investigated by classical rheology and, for the first time, by using amplitude modulation-frequency modulation atomic force microscopy (AM-FM AFM).