Identification of a structural motif crucialfor infectivity of hepatitis B virusesLars Stoeckl*†, Anneke Funk†‡, Ariane Kopitzki*, Boerries Brandenburg*, Stefanie Oess§, Hans Will‡, Huseyin Sirma‡,and Eberhard Hildt*¶�**

¶Department of Internal Medicine II, University of Freiburg, Hugstetterstrasse 55, D-79106 Freiburg, Germany; ‡Department of General Virology, HeinrichPette Institute, D-20251 Hamburg, Germany; §Institute of Biochemistry, Zentrum der Biologischen Chemie, D-60590 Frankfurt, Germany; *Department ofMolecular Virology, Robert Koch Institute, D-13353 Berlin, Germany; and �Institute 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 nonconventionalfacets. Here we analyzed whether a cell-permeable peptide [trans-location motif (TLM)] identified within the surface protein ofhuman HBV is a general feature of all hepadnaviruses and plays arole in the viral life cycle. Surface proteins of all hepadnavirusescontain conserved functional TLMs. Genetic inactivation of theduck HBV TLMs does not interfere with viral morphogenesis;however, these mutants are noninfectious. TLM mutant virusesbind to cells and are taken up into the endosomal compartment,but they cannot escape from endosomes. Processing of surfaceprotein by endosomal proteases induces their exposure on thevirus surface. This unmasking of TLMs mediates translocation ofviral particles across the endosomal membrane into the cytosol, aprerequisite for productive infection. The ability of unmasked TLMsto translocate processed HBV particles across cellular membraneswas shown by confocal immunofluorescence microscopy and byinfection of nonpermissive cell lines with HBV processed in vitrowith endosomal lysate. Based on these data, we propose aninfectious entry mechanism unique for hepadnaviruses that in-volves virus internalization by receptor-mediated endocytosis fol-lowed by processing of surface protein in endosomes. This pro-cessing activates the function of TLMs that are essential for viralparticle translocation through the endosomal membrane into thecytosol and productive infection.

cell permeability � envelope protein � virus entry

Infection with human hepatitis B virus (HBV) can cause acuteor chronic inflammation of the liver (1, 2). HBV is the

prototype member of the hepadnaviridae family, which encom-passes members infecting woodchucks, ground squirrels, andavian viruses isolated from, e.g., pekin ducks, gray herons, andstorks.

Duck HBV (DHBV) is a well characterized model system ofhepadnaviral 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 stepsof hepadnaviral infection on the molecular level. As for HBV(5–7), it is known that DHBV infection is initiated by attachmentof the virus particle to the hepatocyte surface via the pre-Sdomain of the viral surface protein L (8, 9). In DHBV there aretwo surface proteins embedded in the lipid envelope: The majorS protein, a transmembrane protein that encompasses 167 aa,and the L protein, consisting of the S domain N-terminallyextended by the 160-aa pre-S domain. Previous work suggestedthat DHBV enters the cell by receptor-mediated endocytosis(10–13). The mechanism that allows internalized viral particlesto 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 a12-aa-encompassing domain that forms an amphipathic �-helix. Itmediates an energy- and receptor-independent transfer of peptides,nucleic acids, and proteins when fused to them across membraneswithout affecting their integrity (14–16).

Because the membrane translocation function of the TLM ishighly conserved among all hepadnaviridae tested we investi-gated whether the TLM function is of relevance for the viral lifecycle.

ResultsThe Pre-S Domain of Hepadnaviruses Harbors a Cell Permeability-Mediating Domain. Detailed analysis revealed that cell perme-ability mediated by TLMs does not depend on an unique aminoacid sequence but on the capacity to form an �-helix with anamphipathic structure. A homology search for potential TLMsin the pre-S domain of the surface proteins of various hepad-naviridae predicted the existence of TLMs in the pre-S domainof all hepadnaviridae (14). In the case of DHBV, the existenceof two independent translocation motifs within the pre-S domainis predicted (Fig. 6A, which is published as supporting informa-tion on the PNAS web site).

To analyze the potential of these predicted motifs to act ascell-permeable peptides, recombinant fusion proteins witheGFP were engineered. Immunoblot analysis was performedwith a GFP-specific antiserum and the cytosolic fraction derivedfrom HepG2 cells grown in medium containing these variouspurified TLM fusion proteins. The blot revealed comparableamounts of the TLM fusion proteins within the cytosolic frac-tions, whereas, in the case of cells grown in the presence of WTeGFP, no eGFP-specific protein was detectable (Fig. 6B). Mu-tation of TLMs prevented translocation of the fusion proteinsinto the cytosol (Fig. 6C). These data indicate that the predictedTLMs from the hepadnaviridae members analyzed display sim-ilar cell permeability when compared with the previously iden-tified HBV-TLM.

Functionality of the TLMs Is Dispensable for DHBV Secretion. Thefinding that the membrane translocation function of TLMs ishighly conserved throughout hepadnaviridae evolution is sug-gestive for a crucial role in the viral life cycle. To study thisobservation, mutated 1.2 DHBV genomes were generated cod-ing for pre-S�S proteins lacking a functional TLM1 (amino acids20–31) (DHBVD1), a functional TLM2 (amino acids 42–53)(DHBVD2), or both TLMs (DHBVD1�2) without impairingfunctionality of the polymerase. Transfection of LMH cells withthese constructs, followed by cesium chloride centrifugation ofthe culture supernatants and subsequent quantification of the

Conflict of interest statement: No conflicts declared.

This paper was submitted directly (Track II) to the PNAS office.

Abbreviations: HBV, hepatitis B virus; MGE, multiplicity of genome equivalents; TLM,translocation motif; PDH, primary duck hepatocyte; DHBV, duck HBV; cccDNA, covalentlyclosed circular DNA; HBsAg, hepatitis B virus surface antigen; HBcAg, hepatitis B virus coreantigen; HA, hemagglutinin.

†L.S. and A.F. contributed equally to this work.

**To whom correspondence should be addressed. E-mail: eberhard.hildt@uniklinik-freiburg.de.

© 2006 by The National Academy of Sciences of the USA

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viral particles by dot blot analysis, revealed reduced amounts ofsecreted viral particles in the supernatant of DHBVD1-transfected cells as compared with that of WT virus (Fig. 7A,which is published as supporting information on the PNAS website). In contrast, mutants DHBVD2 and DHBVD1�2 secretedsimilar amounts of viral particles as the WT virus. Therefore, theobserved phenotype of DHBVD1 cannot be due to the TLMdeficiency but must have another reason (see below). Compa-rable amounts of replicative intermediates in LMH cells trans-fected with WT DHBV, DHBVD2, or DHBVD1�2 and areduced amount in the case of DHBVD1 detected by Southernblotting (Fig. 7B) corroborates this conclusion. Because none ofthe TLM mutations introduced alter the viral polymerase pro-tein sequence, an effect of the D1 mutation on a regulatoryelement in pregenomic RNA encapsidation, in transcription oron the posttranscriptional level, are the most likely explanationsfor its reduced replication efficiency. Taken together, theseresults indicate that TLM functionality is dispensable for viralmorphogenesis.

Integrity of the TLM Is Crucial for DHBV Infectivity. To analyze therelevance of the TLMs for the infection process, PDHs wereinfected at a multiplicity of genome equivalents (MGE) per cellof 100 with WT DHBV and the mutants DHBVD2 or DH-BVD1�2. Because of the observed TLM-independent reductionin the replication efficiency of DHBVD1, it was excluded fromthese assays. The productivity of infection was analyzed 4 daysafter inoculation. Immunofluorescence staining of the PDHsusing surface protein-specific antiserum KpnI (17), which rec-ognizes the mutant proteins (data not shown), revealed de novosynthesis of surface proteins indicative for productive infectiononly in PDHs infected with WT virus. The TLM-deficientmutants DHBVD2 and DHBVD1�2 failed to infect PDHs (Fig.1A). This finding was confirmed by core protein-specific immu-

noblot analysis of cellular lysates from WT-, DHBVD2-, orDHBVD1�2-infected cells (Fig. 1B). In contrast to WT-infectedcells, cells infected with DHBD1�2 or DHBVD2 showed no denovo synthesis of core protein. Furthermore, only in cellsinfected with WT DHBV, but not in cells infected with DH-BVD2 or DHBVD1�2, replicative intermediates were found bySouthern blot analysis (Fig. 1C). Moreover, covalently closedcircular DNA (cccDNA) was detected only in PDHs infectedwith WT DHBV (Fig. 1D) when analyzed 5 days after infectionby cccDNA-selective PCR. These data show that destruction ofthe TLMs abolishes DHBV infectivity.

TLMs Are Dispensable for Attachment and Entry of Viral Particles toand into PDHs. To control whether the defect in infectivity of TLMmutant viruses is due to reduced binding to PDHs, attachmentassays were performed. After inoculation cells were incubatedfor 2 h at 4°C (viral binding occurs, and internalization isblocked). Then the amount of viral particles attached to the cellwas determined by semiquantitative PCR and immunoblotting.Similar amounts of WT DHBV and the mutant viral particleswere found to be attached to the cell surface (Fig. 8A Upper Left,which is published as supporting information on the PNAS website).

A PCR-based analysis of the amount of viral particles thathave entered the cells after 3 h revealed similar amounts ofintracellular virus in cells inoculated with WT virus and theTLM-deficient viruses (Fig. 8A Lower Left). These data collec-tively show that the loss of infectivity of TLM mutant viruses isdue to neither an impaired attachment nor inhibition of virusentry, but to a post entry block.

TLM Integrity Is Essential for DHBV to Escape from the Endosome.Recent work suggests that DHBV is internalized by receptor-mediated endocytosis (10–12, 18). Based on these data wehypothesized that TLM deficiency might result in retention ofthe virus within the endosomal compartment. To investigate thispossibility experimentally, we isolated endosomal and cytosolicfractions from infected PDHs 10 h after infection. The subcel-lular fractions were adjusted to identical protein concentrations,and the amount of DHBV-specific DNA in the endosomal andin the cytosolic compartments was quantified by TaqMan PCR.This study revealed similar amounts of DHBV DNA in theendosomal fraction derived from DHBVD1-, DHBVD2-, andDHBVD1�2-infected cells and a smaller amount in the case ofWT DHBV-infected cells. However, in the cytosolic fraction, asignificant number of viral genomes was detected only in the caseof WT DHBV-infected cells but not in the cytosol from PDHsinfected with the mutants (Fig. 2). These results indicate thatendosomal escape of internalized viral particles requires properfunction of the TLM.

Cleavage of Hepatitis B Virus Surface Antigen (HBsAg) by EndosomalProteases Results in Surface Exposure of the TLM. The data describedabove raise the question of why TLMs enable translocation ofviral particles across the endosomal membrane but not across theplasma membrane. It can be hypothesized that the TLMs aremasked in progeny viral particles, ensuring the hepadnaviralspecificity for hepatocytes. After receptor-mediated endocyto-sis, an unmasking of the TLMs may occur in the endosomalcompartment that exposes the TLMs on the surface of the virusparticle. Once unmasked, the TLMs allow efficient escape of theviral particles from the endosome.

To verify this hypothesis, accessibility of the TLMs wasanalyzed by immunoprecipitation of HBV particles with anHBV-TLM-specific antiserum and an HBsAg-specific serumused as a positive control. The precipitates were analyzed for thepresence of HBV particles by using a hepatitis B virus coreantigen (HBcAg)-specific antiserum. Western blot analysis re-

Fig. 1. Destruction of the TLM abolishes infectivity of DHBV. (A) Immuno-fluorescence microscopy of infected PDH using an L-specific antiserum. Cellswere infected with 100 MGE WT DHBV, DHBVD2, or DHBVD1�2 mutant. Cellswere fixed 4 days after infection. Hoechst staining was used to visualize nuclei.The photographs were taken at �200 magnification. (B) Immunoblot analysisof lysates from PDHs infected with WT DHBV or the mutants by using a DHBVcore-specific antiserum. Uninfected PDHs served as negative control. (C) PDHswere infected with 100 MGE. Cells were harvested 7 days after infection andanalyzed for replicative intermediates by Southern blotting. (D) Analysis ofcccDNA by PCR. The cccDNA was isolated 3 days after infection and amplifiedby PCR by using cccDNA-selective primers. Uninfected PDHs served as negativecontrol.

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vealed that native HBV particles could not be precipitated by theTLM-specific serum (Fig. 3, lane 2). However, if HBV particleswere incubated with endosomal lysate and then subjected toimmunoprecipitation by using the TLM-specific antiserum, asignificant amount of virus was precipitated (Fig. 3, lane 8). Thisresult demonstrates that an unmasking step must have occurredexposing the TLM on the viral particle surface and conferringaccessibility for the TLM-specific serum. To analyze whether theacidic environment of the endosome is sufficient to induceunmasking of the TLM, the viral particles were incubated for 30min at pH 5.0 (Fig. 3, lanes 4 and 6). The Western blot of theprecipitates shows that acidification alone is not sufficient tounmask the TLM. When a protease inhibitor mixture was addedto the endosomal lysate, no subsequent precipitation of viralparticles by the TLM-specific antiserum was detected (data notshown). Western blot analysis of viral particles that were incu-

bated with endosomal lysate in vitro confirmed proteolyticprocessing of viral particles in the endosome (Fig. 9A, which ispublished as supporting information on the PNAS web site). Weconcluded from these data that the TLM is not exposed on thesurface of mature viral particles. A proteolytic activity in theendosomal compartment is crucial for unmasking the TLM onthe viral particle surface.

Processing of HBV Particles by Endosomal Lysate Enables Infection ofNonpermissive Cells. The resulting question is whether surface-exposed TLMs indeed allow translocation of the processedparticle across membranes. HBV particles with unmasked TLMsshould be able to translocate across the plasma membrane andto establish infection in Huh7 cells. These cells normally cannotbe efficiently infected by HBV.

To test this hypothesis purified HBV particles were pretreated for60 min with endosomal lysate from HepG2 cells. These processedviral particles were then used to infect Huh7 cells with a MGE of102 to 103. As a control, cells were infected with a comparable MGEof unprocessed viral particles. Three days after infection de novosynthesis of HBsAg and HBcAg was analyzed by immunofluores-cence microscopy by using an HBsAg-specific antiserum (Fig. 4A,red fluorescence) or an HBcAg-specific antiserum (Fig. 4A, bluefluorescence). The immunofluorescence staining revealed no in-fected cells in cultures incubated with unprocessed virus whereas upto 40% of cells were stained after incubation with processed viralparticles. Similar results were obtained when DHBV particles wereprocessed by endosomal lysate from LMH cells and used forsuccessful infection of LMH cells (Fig. 4B). These cells are resistantto infection with authentic DHBV. Furthermore, infectivity wasabolished by addition of a protease inhibitor mixture to the endo-somal lysate (Fig. 4B). Processing of TLM-deficient mutants DH-BVD2 and DHBVD1�2 by endosomal lysate failed to establish an‘‘infection’’ in LMH cells (Fig. 4B). The results of these immuno-staining assays were confirmed and extended by analysis of the cellculture supernatants for viral progeny DNA at different times afterinoculation by PCR (Fig. 9B). These data demonstrate the abilityof unmasked TLMs to mediate translocation of processed viralparticles across membranes.

Previous experiments had shown that the TLM functions as acell-permeable and not a fusogenic peptide, which means thatpeptides or proteins fused to the TLM are translocated across themembrane into the cytoplasm. To analyze whether the TLMenables the translocation of processed viral particles across theplasma membrane into the cytoplasm, 293 cells were incubatedwith processed WT HBV, processed TLM-deficient HBV, andunprocessed virus. If a fusogenic step is the acting mechanism,then surface proteins should be enriched in the membrane.However, if cell permeability is enhanced because of membranetranslocation, then a prominent cytoplasmic staining should beobserved. The confocal f luorescence microscopy shows that cellsincubated with endosomally processed WT HBV particles ex-hibit strong cytoplasmic staining for viral envelope protein (Fig.9C). These data imply that translocation across the membraneindeed occurs and results in delivery of HBsAg to the cytosol.We conclude from these data that after receptor-mediatedendocytosis incoming viral particles are processed in the endo-some. This processing leads to exposure of the TLMs on theparticle surface, a crucial step for endosomal escape across themembrane into the cytosol (Fig. 5).

DiscussionPublished work suggested that hepadnaviral infection relies onan endocytic process (10–12, 18), and the presence of envelopedviral particles in purified endosomal fractions at an early stageof infection was very recently visualized by electron microscopy(unpublished data). However, the mechanisms allowing escapeof the virus and the viral nucleocapsid from the endocytotic

Fig. 2. TLM-deficient viral particles are trapped in the endosome. PDH wereinoculated with WT or mutant DHBV (200 MGE). After 10 h of incubation withWT DHBV, DHBVD1�2, DHBVD2, and DHBVD1, cells were harvested andsubfractionated. Cytosolic and endosomal fractions were adjusted to identicalprotein concentrations, and their purity was controlled by immunoblotting byusing grb2 (cytoplasm)- and clathrin HC (endosomes)-specific antisera. Fordetection of viral DNA in the cytosolic (c) and endosomal (e) fractions, TaqManPCR was performed. The y axis indicates the number of viral genomes per 25�l of resuspended subcellular fraction.

Fig. 3. Cleavage of HBsAg by endosomal proteases results in surface expo-sure of the TLM. Purified HBV particles were subjected to immunoprecipita-tion with either an HBV-TLM-specific antiserum (lanes 2, 4, 6, and 8), or anHBsAg-specific serum as a positive control (lanes 1, 3, 5, and 7). The precipi-tated material was immunoblotted by using an HBcAg-specific antiserum. Inlanes 1 and 2 precipitates of untreated HBV particles were loaded using anHBsAg-specific serum or a TLM-specific serum. Purified HBV particles wereincubated for 30 min at pH 5.0 and immunoprecipitated by using an HBV-TLM-specific antiserum (lane 4) and an HBsAg-specific serum (lane 3). PurifiedHBV particles were incubated for 30 min at pH 5.0, then by addition of 10� PBSthe pH was shifted to �7. Afterward, immunoprecipitation was performed byusing an HBV-TLM-specific antiserum (lane 6) and an HBsAg-specific serum(lane 5). Purified HBV particles were incubated for 30 min at pH 5.0 inendosomal lysate from HepG2 cells and immunoprecipitated by using anHBV-TLM-specific antiserum (lane 8) and an HBsAg-specific serum (lane 7).Recombinant HBcAg (lane 9) served as positive control.

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pathway are unknown. Here we show that integrity of the TLMis a crucial prerequisite for infectivity of hepadnaviruses. Func-tional impairment of the TLM results in a block of the infectiousprocess at a post-entry step. TLM-deficient viral particles areunable to escape from the endosome into the cytosol whereasWT particles can partially and time-dependently escape. Pro-teolytic processing of the viral particle in the endosome inducesthe exposure of the TLMs on the surface of the viral particle,enabling translocation across the membrane into the cytoplasm.The unmasking or generation of entry-mediating sequencesduring a viral infection process is not unprecedented: Influenzavirus homotrimers are assembled as hemagglutinin (HA) 0precursors that display no fusogenic activity. Endolytic cleavageby a furin-like protease results in formation of HA1 and HA2.This cleavage step primes HA for fusion by liberating sequencesin HA for fusion-related conformational changes (19, 20). In the

case of poliovirus, the interaction with the receptor results inexternalization of the N-myristoylated VP4 peptide and expo-sure of amphipathic sequences of VP1 that can insert intomembranes mediating entry in the cytoplasm (21, 22).

In contrast to other enveloped viruses, the TLM-mediatedescape from the endosome is not a fusogenic process. The differ-ence from fusogenic mechanisms is that the TLM does not rest inthe membrane at the end of the translocation process (14–16).Therefore, it is likely that the cleaved surface protein remainsassociated with the nucleocapsid during escape from the endocy-totic pathway. The surface protein processing in the endosome andthe reducing conditions in the cytoplasm, which destroy disulfidebridges in the S-domain, affect the HBsAg–nucleocapsid interac-tion and allow removal of the envelope from the nucleocapsid.

Endosomal processing of HBV particles and subsequent in-fection of nonpermissive hepatoma cell lines demonstrate thepotential of surface-exposed TLMs to translocate particlesacross membranes. Moreover, this experimental procedure canbe used to investigate post-entry steps in HBV infection inimmortalized hepatoma cell lines.

A previous report describing that HepG2 cells can be infectedwith very low efficiency by HBV particles after preincubation withV8 proteases (23) has to be reconsidered. It is conceivable that thespecificity of V8 protease used in the study is not suitable to induceproper processing and unmasking of the TLMs. Unspecific orcontaminating protease activity might have processed a smallfraction of viral particles and generated unmasked TLMs.

Our data identify TLMs as conserved motifs of HBVs that areessential for infectivity and suggest an entry mechanism for theseand possibly other enveloped viruses, as summarized in thefollowing model (Fig. 5): after receptor-mediated endocytosis ofthe viral particle and endocytic entry, proteolytic processing ofsurface proteins occurs, which results in unmasking of TLMs.

Fig. 4. Processing of HBV particles by endosomal lysate enables infection ofnonpermissive cells. (A) Confocal microscopy of Huh7 cells infected withunprocessed HBV particles (Left) or with particles incubated with endosomallysate from HepG2 cells for 30 min before infection (Right). The MGE in bothcases was 103. For analysis of the infectivity, HBsAg- and HBcAg-specificantisera were used. Their antigen-specific binding was detected by secondaryantibodies visualized by red and blue fluorescence, respectively. Actin fila-ments were stained by using FITC-conjugated phalloidine. Photographs weretaken at �200 and �630 magnification. (B) Confocal microscopy of LMH cellsinfected with unprocessed DHBV particles or with processed WT DHBV, DH-BVD1�2, and DHBVD2 particles that had been preincubated with endosomallysates from LMH cells for 60 min. As a control, processing of WT DHBV wasperformed in the presence of a protease inhibitor mixture (Roche). The MGEin all cases was 103. For analysis of the infectivity, a surface-specific serumvisualized by the blue fluorescence was used. Photographs were taken at�630 magnification.

Fig. 5. Model of the endosomal processing of hepadnaviral particles. Hep-adnaviruses are internalized by receptor-mediated endocytosis. In the endo-somal compartment proteolytic cleavage of the surface protein occurs, result-ing in a conformational change that exposes the TLMs (shown as red circles)on the surface of the viral particle. The high density of TLMs exposed on thesurface of the particle allows endosomal escape into the cytosol to initiateinfection.

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The endosomal processing generates a modified viral particlethat is decorated on its surface with TLMs. The high density ofTLMs exposed on the particle surface allows exit from theendosome and subsequent establishment of infection.

Materials and MethodsGeneration of TLM-Deficient Genomes. Generation of mutantDHBV genomes lacking a functional TLM was performed based onthe subcloned DHBV genome pDHBV16�1.1 (24) by site-directedmutagenesis using the QuikChange site-directed mutagenesis kitfrom Stratagene. The sequence nucleotides 858–893 encoding theTLM1 was mutated from CTGTTAAACCAACTTGCCG-GAAGGATGATCCCAAAA to CTGTCAAACCAACGTGC-CGGAAGGACGACCCCAAAA, resulting in conversion of thecorresponding amino acid sequence from LLNQLAGRMIPK toLSNQRAGRTTPK. The sequence nucleotides 924–959 encodingTLM2 was mutated from ACACTAGATCACGTGTTAGAC-CATGTGCAAACAATG to ACACCAGATCACGAGTCA-GACCATGCGCAAACAATG, resulting in conversion of TLDH-VLDHVQTM to TPDHESDHAQTM.

Generation of mutant HBV genomes lacking a functionalTLM was performed based on the subcloned HBV genomepSPT1.2HBV as described above. The TLM-encoding sequencewas mutated from CCCATATCGTCAATCTCCTCGAGGAT-TGGGGACCCT to CCCACATCGTCAACCTTCTCGAC-GATTGGGGACCCT, resulting in conversion of PISSISSRT-GDP to PTSSTFSTTGDP. Mutated nucleotides are highlightedin bold.

Protein Analysis. SDS�PAGE was performed according to Lae-mmli (25). Before loading, samples were adjusted to identicalprotein concentrations. Gels were loaded with 20 �g of totalprotein per lane.

For immunoblot analysis, a DHBV-pre-S-specific serum, aDHBV-core-specific serum, and a HBV-core-specific serum gen-erated by immunization of rabbits with the respective purifiedprotein were instrumental. Hexokinase (cytoplasm)-specific, his-tone H1 (nucleus)-specific, cathepsin B (lysosome)-specific, grb2(cytoplasm)-specific, clathrin HC (endosome)-specific, andTNF-RI (microsomes)-specific sera (Santa Cruz Biotechnology)were used as controls for equal loading and purity control ofsubcellular fractions. For detection of eGFP, a polyclonal rabbitserum (Invitrogen) was used. Immunoprecipitations were per-formed as described recently (26) by using a rabbit-derived TLM-specific serum or a goat-derived HBsAg-specific serum (DAKO).The HBV-TLM-specific antiserum was generated by immunizationof rabbit with crosslinked peptide.

Indirect immunofluorescence labeling was performed as de-scribed in refs. 17 and 26. cccDNA isolation and detection,

isolation of replicative intermediates, and Southern blot analysiswere performed as described in refs. 13 and 17.

Cell Culture, Transfection, and Subcellular Fractionation. Fetal PDHswere prepared and cultivated as described (27). PDHs were seededinto 12-well plates at a density of �5 � 105 liver cells per well.

Huh7, HepG2, 293, and LMH cells were cultured in DMEMsupplemented with 10% FCS. For production of DHBV particles,20 6-cm dishes with 0.8 � 106 LMH cells were transfected with 2.5�g of pDHBV16.1.1 or the respective mutants using N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium methylsulfate(Roche). Cell culture supernatants were harvested daily betweendays 3 and 7 after transfection, clarified by centrifugation, andstored at 4°C. Aliquots were polyethylene glycol-precipitated aspreviously described and resuspended in culture medium. Subcel-lular fractionation was performed as described (14, 16).

TaqMan PCR. To quantify virus production after transfection exper-iments, transfected DNA was first eliminated by DNase I treatment.DNase was then destroyed by heating to 94°C, and viral DNA waspurified by using the High Pure viral nucleic acid kit (Roche). Forquantification of viral particles in subcellular compartments, pro-teins were removed by purification of the viral nucleic acid by usingthe same kit. To quantify virus-specific DNA, primers ntDHBV1311–1331 and ntDHBV 1398–1377 were instrumental. The probecorresponded to ntDHBV 1343–1376 and was synthesized by IBA(Goettingen, Germany). The assay was calibrated in a rangecorresponding to 102 to 109 DHBV genomes.

In Vitro Processing of Viral Particles. HBV- or DHBV-positive serawere subjected to gel filtration on a Superose 6 column (AmershamPharmacia) on an Aekta purifier system (Amersham Pharmacia).The HBV- or DHBV-positive fractions (nearly identical with theexclusion volume) were pooled, and the amount of viral genomeswas quantified by TaqMan PCR. A total of 105 genome equivalentswere incubated with endosomal lysate isolated from 2 � 105 HepG2or LMH cells for 30–60 min. Endosomes were prepared as de-scribed (14, 28). Briefly, endosomal lysate was obtained by soni-cation of the endosomal fraction and resuspended in 20 mM Hepes(pH 5.8)�200 mM NaCl. Protease activity was inhibited with acommercial protease inhibitor mixture (Roche) (see SupportingMaterials and Methods, which is published as supporting informa-tion on the PNAS web site).

We thank Dr. P. H. Hofschneider for many helpful and stimulatingdiscussions and for support. We thank Sarah Kinkley for critical readingof the manuscript. The Heinrich Pette Institute is supported by the Freieund Hansestadt Hamburg and the Bundesministerium fur Gesundheitund Soziale Sicherung. This work was supported by grants from theDeutsche Forschungsgemeinschaft and by the German CompetenceNetwork for Viral Hepatitis funded by German Ministry of Educationand Research Grant TP13.1.

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