The human hepatitis B virus (HBV) and the duck hepatitis B virus (DHBV) share several fundamental features. Both viruses have a partially double-stranded DNA genome that is replicated via a RNA intermediate and the coding open reading frames (ORFs) overlap extensively. In addition, the genomic and structural organization, as well as replication and biological characteristics, are very similar in both viruses. Most of the key features of hepadnaviral infection were first discovered in the DHBV model system and subsequently confirmed for HBV. There are, however, several differences between human HBV and DHBV. This review will focus on the molecular and cellular biology, evolution, and host adaptation of the avian hepatitis B viruses with particular emphasis on DHBV as a model system.

Little is known about cellular determinants essential for human hepatitis B virus infection. Using the duck hepatitis B virus as a model, we first established a sensitive binding assay for both virions and subviral particles and subsequently elucidated the characteristics of the early viral entry steps. The infection itinerary was found to initiate with the attachment of viral particles to a low number of binding sites on hepatocytes (about 10 4 per cell). Virus internalization was fully accomplished in less than 3 h but was then followed by a period of unprecedented length, about 14 h, until completion of nuclear import of the viral genome. Steps subsequent to virus entry depended on both intact microtubules and their dynamic turnover but not on actin cytoskeleton. Notably, cytoplasmic trafficking of viral particles and emergence of nuclear covalently closed circular DNA requires microtubules during entry only at and for specific time periods. Taken together, these data disclose for the first time a series of steps and their kinetics that are essential for the entry of hepatitis B viruses into hepatocytes and are different from those of any other virus reported so far.Infections by animal viruses begin with the attachment of the viruses to their respective host cells, followed by internalization, uncoating, and delivery of the viral genome to its cellular replication site. These early steps are poorly characterized for hepadnaviruses, pararetroviruses which almost exclusively infect hepatocytes, leading to both limited and persistent noncytotoxic infections (for a review, see reference 6). Studies addressing the early steps of the hepadnavirus life cycle were hampered mainly by the lack of permissive cell lines and low signal intensities. Despite these restrictions, a number of cellular proteins have been proposed to serve as receptors for human hepatitis B virus (HBV); however, none of these proteins renders infection-resistant cell lines or primary cells susceptible to HBV infection. Progress in this field has been greater in the surrogate model system of duck HBV (DHBV). A novel carboxypeptidase (CPD, also designated gp180) has been identified as a major pre-S binding protein and proposed to be the potential DHBV receptor (12,13,30,31,32). Consistent with this notion, CPD was shown to bind both viral particles and recombinant L protein with high affinity (33). More importantly, antibodies directed against CPD inhibit DHBV infection of primary duck hepatocytes (PDHs) (33), and heterologous expression of CPD leads to efficient internalization of viral particles in nonpermissive hepatoma cells but is not sufficient to confer susceptibility to DHBV infection (2,11,30). These studies suggest that additional or other factors are required for the establishment of a productive infection. Studies addressing the entry steps of the viral life cycle are further complicated by the fact that hepadnavirus infection leads to production of a huge excess of noninfectious, so-called subviral particles (SVPs) in addition to pro...

Viruses can spread by different mechanisms: via intracellular particles through cell junctions to neighboring cells or via secreted virions to adjacent or remote cells. The observation of clusters of hepadnavirus-infected cells both in vivo and in primary hepatocytes neither proves the first mechanism nor excludes the second. In order to test which mechanism, if not both, is used by hepatitis B viruses in order to spread, we used primary duck hepatocytes and duck hepatitis B virus (DHBV) as an infection model. If extracellular progeny virus alone determines spreading, neutralizing antisera or drugs blocking virus binding to hepatocytes should abolish secondary infection. In order to test this, we used DHBV envelope-specific neutralizing antisera, as well as suramin, a known inhibitor of infection. Both reagents strongly reduced hepatocellular attachment of viral particles and almost completely abolished primary infection, whereas an ongoing intracellular infection was not affected as long as no progeny virus was released. In contrast, incubation of infected primary hepatocytes with these reagents during release of progeny virus completely prevented secondary infection. Moreover, the combination of electron and immunofluorescence microscopy analyses revealed the residence of viral particles in cytoplasmic vesicles preferentially located near the basolateral membrane of infected hepatocytes. Taken together, these data strongly suggest that hepatitis B viruses mainly spread by secreted, extracellular progeny and point to polarized egress of viral particles into intercellular compartments, which restricts their diffusion and favors transmission of virus to adjacent cells.

The identity and functionality of biological membranes are determined by cooperative interaction between their lipid and protein constituents. Cholesterol is an important structural lipid that modulates fluidity of biological membranes favoring the formation of detergent-resistant microdomains. In the present study, we evaluated the functional role of cholesterol and lipid rafts for entry of hepatitis B viruses into hepatocytes. We show that the duck hepatitis B virus (DHBV) attaches predominantly to detergent-soluble domains on the plasma membrane. Cholesterol depletion from host membranes and thus disruption of rafts does not affect DHBV infection. In contrast, depletion of cholesterol from the envelope of both DHBV and human HBV strongly reduces virus infectivity. Cholesterol depletion increases the density of viral particles and leads to changes in the ultrastructural appearance of the virus envelope. However, the dual topology of the viral envelope protein L is not significantly impaired. Infectivity and density of viral particles are partially restored upon cholesterol replenishment. Binding and entry of cholesterol-deficient DHBV into hepatocytes are not significantly impaired, in contrast to their release from endosomes. We therefore conclude that viral but not host cholesterol is required for endosomal escape of DHBV.