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.

Hepatitis B virus (HBV) belongs to the Hepadnaviridae family of enveloped DNA viruses. It was previously shown that HBV can induce endoplasmic reticulum (ER) stress and activate the IRE1-XBP1 pathway of the unfolded protein response (UPR), through the expression of the viral regulatory protein X (HBx). However, it remained obscure whether or not this activation had any functional consequences on the target genes of the UPR pathway. Of these targets, the ER degradation-enhancing, mannosidase-like proteins (EDEMs) are thought to play an important role in relieving the ER stress during UPR, by recognizing terminally misfolded glycoproteins and delivering them to the ER-associated degradation (ERAD). In this study, we investigated the role of EDEMs in the HBV life-cycle. We found that synthesis of EDEMs (EDEM1 and its homologues, EDEM2 and EDEM3) is significantly up-regulated in cells with persistent or transient HBV replication. Co-expression of the wild-type HBV envelope proteins with EDEM1 resulted in their massive degradation, a process reversed by EDEM1 silencing. Surprisingly, the autophagy/lysosomes, rather than the proteasome were involved in disposal of the HBV envelope proteins. Importantly, inhibition of the endogenous EDEM1 expression in HBV replicating cells significantly increased secretion of both, enveloped virus and subviral particles. This is the first report showing that HBV activates the ERAD pathway, which, in turn, reduces the amount of envelope proteins, possibly as a mechanism to control the level of virus particles in infected cells and facilitate the establishment of chronic infections.

SummaryThe hepatitis C virus (HCV) is a major etiologic agent for severe liver diseases (e.g. cirrhosis, fibrosis and hepatocellular carcinoma). Approximately 140 million people have chronic HCV infections and about 500 000 die yearly from HCV‐related liver pathologies. To date, there is no licensed vaccine available to prevent HCV infection and production of a HCV vaccine remains a major challenge. Here, we report the successful production of the HCV E1E2 heterodimer, an important vaccine candidate, in an edible crop (lettuce, Lactuca sativa) using Agrobacterium‐mediated transient expression technology. The wild‐type dimer (E1E2) and a variant without an N‐glycosylation site in the E2 polypeptide (E1E2∆N6) were expressed, and appropriate N‐glycosylation pattern and functionality of the E1E2 dimers were demonstrated. The humoral immune response induced by the HCV proteins was investigated in mice following oral administration of lettuce antigens with or without previous intramuscular prime with the mammalian HEK293T cell‐expressed HCV dimer. Immunization by oral feeding only resulted in development of weak serum levels of anti‐HCV IgM for both antigens; however, the E1E2∆N6 proteins produced higher amounts of secretory IgA, suggesting improved immunogenic properties of the N‐glycosylation mutant. The mice group receiving the intramuscular injection followed by two oral boosts with the lettuce E1E2 dimer developed a systemic but also a mucosal immune response, as demonstrated by the presence of anti‐HCV secretory IgA in faeces extracts. In summary, our study demonstrates the feasibility of producing complex viral antigens in lettuce, using plant transient expression technology, with great potential for future low‐cost oral vaccine development.