Infectious entry of hepatitis B viruses (HBV) has nonconventional facets. Here we analyzed whether a cell-permeable peptide [translocation motif (TLM)] identified within the surface protein of human HBV is a general feature of all hepadnaviruses and plays a role in the viral life cycle. Surface proteins of all hepadnaviruses contain conserved functional TLMs. Genetic inactivation of the duck HBV TLMs does not interfere with viral morphogenesis; however, these mutants are noninfectious. TLM mutant viruses bind to cells and are taken up into the endosomal compartment, but they cannot escape from endosomes. Processing of surface protein by endosomal proteases induces their exposure on the virus surface. This unmasking of TLMs mediates translocation of viral particles across the endosomal membrane into the cytosol, a prerequisite for productive infection. The ability of unmasked TLMs to translocate processed HBV particles across cellular membranes was shown by confocal immunofluorescence microscopy and by infection of nonpermissive cell lines with HBV processed in vitro with endosomal lysate. Based on these data, we propose an infectious entry mechanism unique for hepadnaviruses that involves virus internalization by receptor-mediated endocytosis followed by processing of surface protein in endosomes. This processing activates the function of TLMs that are essential for viral particle translocation through the endosomal membrane into the cytosol and productive infection.cell permeability ͉ envelope protein ͉ virus entry I nfection with human hepatitis B virus (HBV) can cause acute or chronic inflammation of the liver (1, 2). HBV is the prototype member of the hepadnaviridae family, which encompasses members infecting woodchucks, ground squirrels, and avian viruses isolated from, e.g., pekin ducks, gray herons, and storks.Duck HBV (DHBV) is a well characterized model system of hepadnaviral infection (3). Cultures of primary duck hepatocytes (PDHs) can be readily established and efficiently infected (3, 4) and therefore provide a suitable tool for analyzing the early steps of hepadnaviral infection on the molecular level. As for HBV (5-7), it is known that DHBV infection is initiated by attachment of the virus particle to the hepatocyte surface via the pre-S domain of the viral surface protein L (8, 9). In DHBV there are two surface proteins embedded in the lipid envelope: The major S protein, a transmembrane protein that encompasses 167 aa, and the L protein, consisting of the S domain N-terminally extended by the 160-aa pre-S domain. Previous work suggested that DHBV enters the cell by receptor-mediated endocytosis (10-13). The mechanism that allows internalized viral particles to escape from the endocytic pathway remained elusive.Recently, a cell-permeable peptide [translocation motif (TLM)] was identified in the pre-S domain of HBV (14). The TLM is a 12-aa-encompassing domain that forms an amphipathic ␣-helix. It mediates an energy-and receptor-independent transfer of peptides, nucleic acids, and proteins when fus...

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