Identification of a structural motif crucial for infectivity of hepatitis B viruses Lars Stoeckl*†, Anneke Funk†‡, Ariane Kopitzki*, Boerries Brandenburg*, Stefanie Oess§, Hans Will‡,Hu¨ seyin Sirma‡, and Eberhard Hildt*¶ʈ** ¶Department of Internal Medicine II, University of Freiburg, Hugstetterstrasse 55, D-79106 Freiburg, Germany; ‡Department of General Virology, Heinrich Pette Institute, D-20251 Hamburg, Germany; §Institute of Biochemistry, Zentrum der Biologischen Chemie, D-60590 Frankfurt, Germany; *Department of Molecular Virology, Robert Koch Institute, D-13353 Berlin, Germany; and ʈInstitute of Virology, Humboldt University (Charite), D-13353 Berlin, Germany Edited by Jesse Summers, University of New Mexico, Albuquerque, NM, and approved March 3, 2006 (received for review November 16, 2005) Infectious entry of hepatitis B viruses (HBV) has nonconventional Because the membrane translocation function of the TLM is facets. Here we analyzed whether a cell-permeable peptide [trans- highly conserved among all hepadnaviridae tested we investi- location motif (TLM)] identified within the surface protein of gated whether the TLM function is of relevance for the viral life human HBV is a general feature of all hepadnaviruses and plays a cycle. role in the viral life cycle. Surface proteins of all hepadnaviruses contain conserved functional TLMs. Genetic inactivation of the Results duck HBV TLMs does not interfere with viral morphogenesis; The Pre-S Domain of Hepadnaviruses Harbors a Cell Permeability- however, these mutants are noninfectious. TLM mutant viruses Mediating Domain. Detailed analysis revealed that cell perme- bind to cells and are taken up into the endosomal compartment, ability mediated by TLMs does not depend on an unique amino but they cannot escape from endosomes. Processing of surface acid sequence but on the capacity to form an ␣-helix with an protein by endosomal proteases induces their exposure on the amphipathic structure. A homology search for potential TLMs virus surface. This unmasking of TLMs mediates translocation of in the pre-S domain of the surface proteins of various hepad- viral particles across the endosomal membrane into the cytosol, a naviridae predicted the existence of TLMs in the pre-S domain prerequisite for productive infection. The ability of unmasked TLMs of all hepadnaviridae (14). In the case of DHBV, the existence to translocate processed HBV particles across cellular membranes of two independent translocation motifs within the pre-S domain was shown by confocal immunofluorescence microscopy and by is predicted (Fig. 6A, which is published as supporting informa- infection of nonpermissive cell lines with HBV processed in vitro tion on the PNAS web site). with endosomal lysate. Based on these data, we propose an To analyze the potential of these predicted motifs to act as infectious entry mechanism unique for hepadnaviruses that in- cell-permeable peptides, recombinant fusion proteins with volves virus internalization by receptor-mediated endocytosis fol- eGFP were engineered. Immunoblot analysis was performed lowed by processing of surface protein in endosomes. This pro- with a GFP-specific antiserum and the cytosolic fraction derived cessing activates the function of TLMs that are essential for viral from HepG2 cells grown in medium containing these various particle translocation through the endosomal membrane into the purified TLM fusion proteins. The blot revealed comparable cytosol and productive infection. amounts of the TLM fusion proteins within the cytosolic frac- tions, whereas, in the case of cells grown in the presence of WT cell permeability ͉ envelope protein ͉ virus entry eGFP, no eGFP-specific protein was detectable (Fig. 6B). Mu- tation of TLMs prevented translocation of the fusion proteins nfection with human hepatitis B virus (HBV) can cause acute into the cytosol (Fig. 6C). These data indicate that the predicted Ior chronic inflammation of the liver (1, 2). HBV is the TLMs from the hepadnaviridae members analyzed display sim- prototype member of the hepadnaviridae family, which encom- ilar cell permeability when compared with the previously iden- passes members infecting woodchucks, ground squirrels, and tified HBV-TLM. avian viruses isolated from, e.g., pekin ducks, gray herons, and storks. Functionality of the TLMs Is Dispensable for DHBV Secretion. The Duck HBV (DHBV) is a well characterized model system of finding that the membrane translocation function of TLMs is hepadnaviral infection (3). Cultures of primary duck hepatocytes highly conserved throughout hepadnaviridae evolution is sug- (PDHs) can be readily established and efficiently infected (3, 4) gestive for a crucial role in the viral life cycle. To study this and therefore provide a suitable tool for analyzing the early steps observation, mutated 1.2 DHBV genomes were generated cod- ͞ of hepadnaviral infection on the molecular level. As for HBV ing for pre-S S proteins lacking a functional TLM1 (amino acids (5–7), it is known that DHBV infection is initiated by attachment 20–31) (DHBVD1), a functional TLM2 (amino acids 42–53) ͞ of the virus particle to the hepatocyte surface via the pre-S (DHBVD2), or both TLMs (DHBVD1 2) without impairing domain of the viral surface protein L (8, 9). In DHBV there are functionality of the polymerase. Transfection of LMH cells with two surface proteins embedded in the lipid envelope: The major these constructs, followed by cesium chloride centrifugation of S protein, a transmembrane protein that encompasses 167 aa, the culture supernatants and subsequent quantification of the and the L protein, consisting of the S domain N-terminally extended by the 160-aa pre-S domain. Previous work suggested Conflict of interest statement: No conflicts declared. that DHBV enters the cell by receptor-mediated endocytosis (10–13). The mechanism that allows internalized viral particles This paper was submitted directly (Track II) to the PNAS office. to escape from the endocytic pathway remained elusive. Abbreviations: HBV, hepatitis B virus; MGE, multiplicity of genome equivalents; TLM, translocation motif; PDH, primary duck hepatocyte; DHBV, duck HBV; cccDNA, covalently Recently, a cell-permeable peptide [translocation motif (TLM)] closed circular DNA; HBsAg, hepatitis B virus surface antigen; HBcAg, hepatitis B virus core was identified in the pre-S domain of HBV (14). The TLM is a antigen; HA, hemagglutinin. 12-aa-encompassing domain that forms an amphipathic ␣-helix. It †L.S. and A.F. contributed equally to this work. mediates an energy- and receptor-independent transfer of peptides, **To whom correspondence should be addressed. E-mail: eberhard.hildt@uniklinik- nucleic acids, and proteins when fused to them across membranes freiburg.de. without affecting their integrity (14–16). © 2006 by The National Academy of Sciences of the USA 6730–6734 ͉ PNAS ͉ April 25, 2006 ͉ vol. 103 ͉ no. 17 www.pnas.org͞cgi͞doi͞10.1073͞pnas.0509765103 Downloaded by guest on October 2, 2021 noblot analysis of cellular lysates from WT-, DHBVD2-, or DHBVD1͞2-infected cells (Fig. 1B). In contrast to WT-infected cells, cells infected with DHBD1͞2 or DHBVD2 showed no de novo synthesis of core protein. Furthermore, only in cells infected with WT DHBV, but not in cells infected with DH- BVD2 or DHBVD1͞2, replicative intermediates were found by Southern blot analysis (Fig. 1C). Moreover, covalently closed circular DNA (cccDNA) was detected only in PDHs infected with WT DHBV (Fig. 1D) when analyzed 5 days after infection by cccDNA-selective PCR. These data show that destruction of the TLMs abolishes DHBV infectivity. TLMs Are Dispensable for Attachment and Entry of Viral Particles to and into PDHs. To control whether the defect in infectivity of TLM mutant viruses is due to reduced binding to PDHs, attachment assays were performed. After inoculation cells were incubated for2hat4°C(viral binding occurs, and internalization is blocked). Then the amount of viral particles attached to the cell was determined by semiquantitative PCR and immunoblotting. Similar amounts of WT DHBV and the mutant viral particles were found to be attached to the cell surface (Fig. 8A Upper Left, Fig. 1. Destruction of the TLM abolishes infectivity of DHBV. (A) Immuno- which is published as supporting information on the PNAS web fluorescence microscopy of infected PDH using an L-specific antiserum. Cells site). were infected with 100 MGE WT DHBV, DHBVD2, or DHBVD1͞2 mutant. Cells were fixed 4 days after infection. Hoechst staining was used to visualize nuclei. A PCR-based analysis of the amount of viral particles that The photographs were taken at ϫ200 magnification. (B) Immunoblot analysis have entered the cells after 3 h revealed similar amounts of of lysates from PDHs infected with WT DHBV or the mutants by using a DHBV intracellular virus in cells inoculated with WT virus and the core-specific antiserum. Uninfected PDHs served as negative control. (C) PDHs TLM-deficient viruses (Fig. 8A Lower Left). These data collec- were infected with 100 MGE. Cells were harvested 7 days after infection and tively show that the loss of infectivity of TLM mutant viruses is analyzed for replicative intermediates by Southern blotting. (D) Analysis of due to neither an impaired attachment nor inhibition of virus cccDNA by PCR. The cccDNA was isolated 3 days after infection and amplified entry, but to a post entry block. by PCR by using cccDNA-selective primers. Uninfected PDHs served as negative control. TLM Integrity Is Essential for DHBV to Escape from the