JOURNAL OF VIROLOGY, Jan. 2010, p. 387–396 Vol. 84, No. 10022-538X/10/$12.00 doi:10.1128/JVI.01921-09Copyright © 2010, American Society for Microbiology. All Rights Reserved.

Production and Function of the Cytoplasmic Deproteinized RelaxedCircular DNA of Hepadnaviruses�

Haitao Guo,1* Richeng Mao,1 Timothy M. Block,1,2 and Ju-Tao Guo1*Drexel Institute for Biotechnology and Virology Research, Department of Microbiology and Immunology,

Drexel University College of Medicine,1 and Institute for Hepatitis and Virus Research,Hepatitis B Foundation,2 3805 Old Easton Road, Doylestown, Pennsylvania 18902

Received 10 September 2009/Accepted 20 October 2009

Removal of genome-bound viral DNA polymerase ought to be an essential step in the formation of hepad-navirus covalently closed circular DNA (cccDNA). We previously demonstrated that deproteinized (DP)relaxed circular DNA (rcDNA) of hepatitis B virus (HBV) existed in both the cytoplasm and nuclei of infectedcells and the vast majority of cytoplasmic DP rcDNA was associated with DNase I-permeable nucleocapsids.In our efforts to investigate the role of the cytoplasmic DP rcDNA in cccDNA formation, we demonstrated thatrcDNA deproteinization could occur in an endogenous DNA polymerase reaction with either virion-derived orintracellular nucleocapsids. As observed in the cytoplasm of virally infected cells, in vitro deproteinizationrequires the maturation of plus-strand DNA and results in changes in nucleocapsid structure that render theDP rcDNA susceptible to DNase I digestion. Remarkably, we found that the cytoplasmic DP rcDNA-containingnucleocapsids could be selectively immunoprecipitated with an antibody against the carboxyl-terminal peptideof HBV core protein and are associated with cellular nuclear transport receptors karyopherin-� and -�.Moreover, transfection of small interfering RNA targeting karyopherin-�1 mRNA or expression of a dominant-negative karyopherin-�1 in a stable cell line supporting HBV replication resulted in the accumulation of DPrcDNA in cytoplasm and reduction of nuclear DP rcDNA and cccDNA. Our results thus favor a hypothesis thatcompletion of plus-strand DNA synthesis triggers the genomic DNA deproteinization and structural changesof nucleocapsids, which leads to the exposure of nuclear localization signals in the C terminus of core proteinand mediates the nuclear transportation of DP rcDNA via interaction with karyopherin-� and -�.

Hepatitis B virus (HBV) is the prototype member of theHepadnaviridae family and contains a relaxed circular (rc) par-tially double-stranded DNA (3.2 kb in length) genome with itsDNA polymerase protein covalently attached to the 5� termi-nus of minus-strand DNA (10, 26, 38). One of the most in-triguing biological features of hepadnaviruses is that the viralgenomic DNA is replicated via protein-primed reverse tran-scription of an RNA intermediate called pregenomic RNA(pgRNA) in the cytoplasmic nucleocapsids (37). However, un-like classical retroviruses, the integration of hepadnavirusgenomic DNA into host cellular chromosomes is not an oblig-atory step in its life cycle. Instead, a nuclear episomal co-valently closed circular DNA (cccDNA) is formed from thercDNA genome in nucleocapsids, either from incoming virionsduring initial infection or from the pool of progeny nucleocap-sids formed in the cytoplasm during replication (40, 42). Thosetwo pathways culminate in the formation of a regulated steady-state population of 10 to 50 cccDNA molecules per infectedcell (3, 29, 34). The cccDNA exists as a minichromosome in thenucleus and serves as the template for the transcription of viralRNAs (47). The stability of this key replication intermediate isstill in debate, but a continued productive hepadnavirus infec-

tion clearly requires a persistent population of cccDNA as thesource of viral RNAs for viral replication and production ofvirions (27, 40, 42, 44). Thus far, therapeutic elimination ofcccDNA with highly active viral DNA polymerase inhibitorshas not been achieved in chronically HBV-infected patientsand remains a major challenge for a cure of chronic hepatitisB (18, 20, 23, 45).

Concerning the molecular mechanism of cccDNA formationfrom its precursor, the cytoplasmic nucleocapsid-associatedrcDNA, one of the most obvious biochemical reactions thatought to occur is the removal of genome-bound viral DNApolymerase. In principle, the resulting protein-free or depro-teinized (DP) rcDNA could be an essential intermediate ofcccDNA formation. Recently, we and others rigorously dem-onstrated that such predicted DP rcDNA species indeed existin the hepadnavirus-infected cells (9, 12). Detailed analysis ofthe structural features revealed that DP rcDNA containedexclusively complete plus-strand DNA, suggesting that the re-moval of covalently genome-bound polymerase may requirethe completion of plus-strand DNA synthesis (9, 12).

In an effort to determine where rcDNA deproteinizationmay occur and the role of DP rcDNA in cccDNA formation,we found previously that (i) the DP rcDNA existed in both thecytoplasm and the nucleus; (ii) while the majority of the cyto-plasmic DP rcDNA presented in DNase I-permeable nucleo-capsids, a small portion (�10%) of cytoplasmic DP rcDNAwas located in DNase I-resistant, presumably intact nucleocap-sids; (iii) the nuclear DP rcDNA was DNase I sensitive and didnot associate with nucleocapsids. Moreover, we showed thatthe DP rcDNA appeared earlier than cccDNA during hepad-

* Corresponding author. Mailing address: Drexel Institute for Bio-technology and Virology Research, Department of Microbiology andImmunology, Drexel University College of Medicine, 3805 Old EastonRoad, Doylestown, PA 18902. Phone for J.-T. Guo: (215) 489-4929.Fax: (215) 489-4920. E-mail: Ju-Tao.Guo@drexelmed.edu. Phone forH. Guo: (215) 489-4928. Fax: (215) 489-4920. E-mail: Haitao.Guo@drexelmed.edu.

� Published ahead of print on 28 October 2009.

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navirus DNA replication and that transfection of purified duckHBV (DHBV) DP rcDNA into chicken hepatoma cells initi-ated cccDNA formation and viral DNA replication (12).

Based on the experimental evidence summarized above, weproposed that the removal of genome-bound polymerase pro-tein initiates inside the nucleocapsid and may even triggernucleocapsid disassembly, which, in turn, leads to the exposureof a nuclear localization signal (NLS) at the carboxyl terminusof capsid protein to mediate the import of the DP rcDNA intothe nucleus through the nuclear pore complex (31, 46). Sub-sequently, the DP rcDNA is converted into cccDNA by cellularDNA repair machinery (15).

In order to test this hypothesis, we focused our research effortson elucidating the molecular mechanism of the production, un-coating, and nuclear transportation of cytoplasmic DP rcDNA.Our results for the first time demonstrate that hepadnavirus nu-cleocapsid contains sufficient information and factors to allow fordeproteinization of the associated viral genome and provide evi-dence suggesting that the deproteinization reaction requires ac-tivities of both a viral DNA polymerase and a putative serineprotease. Consistent with the notion that the cytoplasmic DPrcDNA is the functional precursor of cccDNA formation (12), weobtained evidence showing that rcDNA deproteinization wastightly linked with nucleocapsid disassembly and exposure of anNLS located at the carboxyl-terminal portion of the core protein.Furthermore, we showed that the transfection of small interferingRNA (siRNA) targeting karyopherin-�1 mRNA or expression ofa dominant-negative karyopherin-�1 in a stable cell line support-ing HBV replication resulted in the accumulation of cytoplasmicDP rcDNA and reduction of nuclear DP rcDNA and cccDNA.

Our findings presented herein provide insight on the molec-ular mechanism of cccDNA formation and clues on the devel-opment of novel intervention strategies to control chronicHBV infection.

MATERIALS AND METHODS

Preparation of DHBV virions and intracellular DHBV nucleocapsids. DHBVvirions were prepared from sera of congenitally infected ducks (kindly providedby William S. Mason, Fox Chase Cancer Center, Philadelphia, PA) by sucrosegradient centrifugation (24). Briefly, 1 ml of duck sera was layered onto a 5-ml10-to-20% (wt/vol) sucrose gradient in 0.15 M NaCl–0.02 M Tris-HCl (pH 7.4)and centrifuged for 3 h at 45,000 rpm in SW55 rotor at 4°C. The supernatant fluidwas removed, and the pellet was resuspended in TNE buffer (0.15 M NaCl, 0.01M Tris-HCl [pH 7.4], and 0.1 mM EDTA).

To prepare intracellular immature DHBV nucleocapsids, Dstet5 cells werecultured in medium without tetracycline, but in the presence of 1 mM foscarnet(PFA), for 3 days with daily medium change and followed by removal of PFA for4 h to resume viral DNA synthesis (13). The cells were then lysed with chilledlysis buffer containing 10 mM Tris-HCl (pH 8.0), 1 mM EDTA, 0.1% NonidetP-40, and 8% sucrose on ice for 10 min, the lysate was centrifuged at 10,000 �g for 5 min to remove the nuclei and cell debris. The clarified supernatant wasoverlaid onto a 10 to 55% (wt/wt) sucrose gradient and centrifuged at 24,000 rpmfor 16 h at 4°C using a Beckman SW28 rotor. Nineteen 2-ml fractions werecollected from the bottom of the cushion. Ten microliters of each fraction wasdot-blotted onto the nitrocellulose membrane, and DHBV core protein wasdetected by sequential incubation with a rabbit antibody against DHBV coreprotein and horseradish peroxidase-labeled antibody against rabbit immunoglob-ulin G (IgG). The bound antibody is revealed by enhanced chemiluminescence(12). DHBV core protein-positive fractions were pooled together, and the su-crose concentration was adjusted to 10% by addition of TNE buffer. The dilutedsample was overlaid on a 20% sucrose cushion and centrifuged at 45,000 rpm for3 h at 4°C using the Beckman SW55 rotor. The pellet was resuspended in TNEbuffer.

Endogenous DNA polymerase reaction. A typical endogenous DNA polymerasereaction (EPR) mixture was assembled with 40 �l of DHBV virion preparation,50 �l of 2� EPR buffer which consisted of 0.3 M NaCl, 0.1 M Tris-HCl (pH 8.0),20 mM MgCl2, 2 mM dithiothreitol, 0.2% (vol/vol) Nonidet P-40, and 0.2 mM ofdeoxynucleoside triphosphate (dNTP). DNA polymerase and/or protease inhib-itors (Pierce and Calbiochem) were added as indicated below, and water wasprovided to bring the reaction volume to 100 �l. After incubation at 37°C for theindicated period of time, the reaction volume was subjected to the extraction ofviral DNA with or without prior DNase I digestion. Occasionally, DHBV DNApolymerase activity is measured by a [�-32P]dCTP incorporation assay. Briefly,0.2 mM of dCTP in the 2� EPR buffer was replaced with 10 �M [�-32P]dCTPand followed by incubation of the endogenous DNA polymerase reaction volumeat 37°C for 1 h. The acid insoluble 32P was counted with a liquid scintillationcounter (PerkinElmer).

Analysis of viral DNA. Total viral DNA from the EPR sample was extractedby adding equal volumes of DNA extraction buffer that contained 20 mM EDTA,20 mM Tris-HCl (pH 8.0), 0.2% sodium dodecyl sulfate (SDS), and 1 mg/mlpronase, followed by incubation for 1 h at 37°C. The digestion mixture wasextracted twice with phenol, and DNA was precipitated with ethanol and dis-solved in TE buffer (10 mM Tris-HCl [pH 8.0], 1 mM EDTA). Protein-free orDP DHBV DNA from the EPR samples were prepared by direct phenol extrac-tion without prior pronase digestion (Hirt procedure) (12). Briefly, EPR mixturewas brought up to 1.5 ml by addition of TE buffer (10 mM Tris-HCl [pH 7.5], 10mM EDTA) and mixed with 100 �l of 10% SDS. After 30 min incubation atroom temperature, 5 M NaCl was added to bring the final concentration of NaClto 1 M and continued to be incubated at 4°C overnight. The sample was thenclarified by centrifugation at 12,000 � g for 30 min at 4°C and extracted twicewith phenol and once with phenol-chloroform. DNA was precipitated with 2volumes of ethanol overnight at room temperature and dissolved in TE buffer.One-half of the DNA sample from each EPR was resolved by electrophoresisinto a 1.5% agarose gel. The gel was then subjected to denaturation in a solutioncontaining 0.5 M NaOH and 1.5 M NaCl, followed by neutralization in a buffercontaining 1 M Tris-HCl (pH 7.4) and 1.5 M NaCl. DNA was then blotted ontoa Hybond-XL membrane (GE Healthcare) in 20� SSC buffer (1� SSC is 0.15 MNaCl plus 0.015 M sodium citrate). For the detection of HBV DNA, membraneswere probed with an [�-32P]UTP (800 Ci/mmol; Perkin Elmer)-labeled minus-strand specific full-length HBV riboprobe. Hybridization was carried out in 5 mlEkono hybridization buffer (Genotech) with 1-h prehybridization at 65°C andovernight hybridization at 65°C, followed by a 1-h wash with 0.1� SSC and 0.1%SDS at 65°C. The membrane was exposed to a phosphorimager screen, andhybridization signals were quantified with QuantityOne software (Bio-Rad).

Immunoprecipitation assay. The cytoplasmic fractions of HepDES19 cellsthat were cultured in the absence of tetracycline for 12 days were prepared witha Qproteome cell compartment kit (Qiagen) by following the manufacturer’sdirections (12). For the immunoprecipitation assay, 1 ml of cytoplasmic lysateprepared from approximately 5 � 106 cells was mixed with 35 �l of protein A/Gplus beads (Santa Cruz) that were preabsorbed with antibodies against karyo-pherin-�1 (Zymed), karyopherin-�2 (Santa Cruz), karyopherin-�1 (Abcam), orHBsAg (Dako), respectively. The mixtures were incubated at 4°C overnight.Beads were washed four times with TNE buffer. Core and DP DNA wereextracted with or without prior digestion of DNase I, respectively. Viral DNAwas analyzed by Southern blot hybridization.

Plasmid DNA and siRNA transfection. HepDES19 cells cultured in a collagen-coated 35-mm dish were transfected by Lipofectamine 2000 (Invitrogen) with a4 �g control vector plasmid or a plasmid expressing dominant negative karyo-pherin-�1, dominant negative karyopherin-�1, or 90 nM of control siRNA(Santa Cruz), or Smartpool siRNA targeting karyopherin-�1 (Dharmacon). Thefull-length karyopherin-�1 was amplified from the cDNA synthesized fromHepG2 cell total RNA and cloned into pcDNA3.1 (Invitrogen). The C-terminalfragment from aa 256 to aa 876 of karyopherin-�1, which can bind to karyo-pherin � but fails to bind Ran GTPase (17), was PCR amplified from thekaryopherin-�1 plasmid and cloned into pcDNA3.1-TOPO-V5 (Invitrogen) toexpress the dominant negative karyopherin-�1 with a C-terminal V5 tag. All thecDNA clones were confirmed by DNA sequencing. N-FLAG-tagged dominantnegative karyopherin-�1 was kindly provided by Christopher Basler (MountSinai School of Medicine, New York, NY) (32). The transfected cells werecultured in tetracycline-free medium for 5 days, followed by a second round oftransfection and continued culture with tetracycline-free media for another 5days. Total core DNA, DP DNA, and cccDNA were extracted and analyzed asdescribed previously (12).

Western blot assay. The transfected cells were lysed in 300 �l of 1 � Laminibuffer, a total of 30 �l of the cell lysate was resolved on an SDS-12% polyacryl-amide gel and transferred onto Immobilon-FL polyvinylidene difluoride mem-

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brane (Millipore). The membrane was blocked with Western Breeze blockingbuffer (Invitrogen) and probed with specific antibody against karyopherin-�1(SC-1919; Santa Cruz), V5 epitope (Invitrogen), FLAG peptide (Sigma), andHBc170 (produced in Genscript Facility, NJ), and bound antibody was revealedby IRDye secondary antibodies and visualized by the Li-COR Odyssey system.

RESULTS

DHBV virion-associated rcDNA deproteinization and nu-cleocapsid disassembly occur following endogenous DNA poly-merase reaction in vitro. As stated above, hepadnavirus con-tains a partially double-stranded rcDNA genome that the viralDNA polymerase covalently attaches to the 5� terminus of itsminus strand, and the plus strand is incompletely synthesizedand heterogeneous in length (38). It has been demonstratedseveral decades ago that incubation of purified virion particlesin solution containing nonionic detergent and dithiothreitol(DTT) to disrupt viral envelope and dNTP as substrates andthat viral DNA polymerase can elongate the incomplete plusstrand DNA chain in vitro. This is the so-called endogenousDNA polymerase reaction (33). Based on our observation thatDP rcDNA contains full-length plus-strand DNA, we hypoth-esized that the completion of the plus-strand DNA synthesismight serve as a trigger for rcDNA deproteinization and/ornucleocapsid disassembly (12).

To test this hypothesis, endogenous DNA polymerase reac-tions were performed with purified DHBV virions. The reac-tion mixtures were either without incubation (Fig. 1, lanes 2 to5) or incubated at 37°C for 16 h (Fig. 1, lanes 6 to 9). Total viralDNA was extracted by protease digestion and phenol extrac-tion. DP viral DNA was extracted via the Hirt procedure (14).The Hirt procedure involves lysis of cells and/or viral particles

with SDS and high concentrations of salts to disassociate phys-ically associated proteins, such as capsid proteins, from DNA,and was followed by direct phenol extraction of DNA from thelysates without protease digestion (14). Therefore, DNA co-valently linked to protein is partitioned into phenol or inter-phase, and only the DNA molecules that are not covalentlyattached to protein are selectively extracted. Hence, the viralDNA species in the Hirt preparations should be free of co-valently genome-bound viral DNA polymerase. To monitor thedisassembly of nucleocapsids, the indicated samples were sub-jected to DNase I digestion for 30 min at 37°C prior to totaland DP DNA extraction.

Consistent with previous observations, there is no detectableDP DNA in nonincubated virions, suggesting that all DHBVrcDNA in virion particles is covalently attached to DNA poly-merase (11, 26). Interestingly, after a prolonged endogenouspolymerase reaction, a small amount of DP rcDNA could bedetected. As observed in DHBV-infected duck livers andDstet5 cells, the DP rcDNA species is exclusively DNase Isensitive (12). These results hence indicate that the rcDNAdeproteinization and capsid disassembly occur following the invitro endogenous DNA polymerase reaction, albeit at a verylow efficiency.

DHBV rcDNA deproteinization requires viral DNA poly-merase activity. To further characterize the requirements forthis in vitro genomic DNA deproteinization reaction, the en-dogenous polymerase assay was performed under the condi-tion that dNTPs were omitted or DNA polymerase inhibitorddCTP or PFA were added into the reaction and incubated atvarious periods of time. DP DNA were then extracted by theHirt procedure. Total virion DNA and DP virion DNA wereextracted and served as positive and negative controls. Theresults revealed the following observations. First, as expected,untreated DHBV virion particles do not contain any detectablelevel of DP DHBV DNA (Fig. 2, lane 3). Second, DP DHBVDNA is detectable at 1 h and accumulated to a higher level

FIG. 2. DHBV virion-associated rcDNA deproteinization requiresDNA polymerase activity. Approximately 108 DHBV virion particlesprepared from DHBV-positive duck serum were contained in each100-�l EPR mixture as described in the legend of Fig. 1. The reactionmixtures were either without incubation (lanes 2 and 3) or incubated(lanes 4 to 10) at 37°C for the indicated periods of time. For thesamples analyzed in lanes 8 to 10, dNTP was omitted (lane 8), or dCTPwas replaced by ddCTP (lane 9), or 1 mM PFA was added into theEPR mixture (lane 10). Core and DP DNA were extracted from thereaction mixtures and analyzed by Southern blot hybridization assay.One hundred picograms of unit-length DHBV DNA served as themolecular weight marker (lane 1). The position of rcDNA (RC) isindicated.

FIG. 1. Removal of genome-bounded polymerase and capsid dis-assembly occur following endogenous polymerase reaction in vitro.Approximately 108 DHBV virion particles prepared from DHBV-pos-itive duck serum were contained in each 100-�l EPR mixture including150 mM NaCl, 50 mM Tris-HCl (pH 8.0), 10 mM MgCl2, 1 mM DTT,0.1% NP-40, and 0.1 mM dNTP. The reaction mixtures were eitherwithout incubation (lanes 2 to 5) or incubated at 37°C for 16 h (lanes6 to 9), followed by total DNA and DP DNA extraction without (lanes2, 3, 6, and 7) or with (lanes 4, 5, 8 and 9) prior DNase I digestion,respectively. The viral DNA were resolved in agarose gel and detectedby Southern blot hybridization with full-length riboprobe recognizingminus-strand DNA. One hundred picograms of 3.0-kb unit-lengthDHBV DNA served as the quantification standard and molecularweight marker (lane 1). The position of rcDNA (RC) is indicated.

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after 16 h of incubation (Fig. 2, lanes 4 to 7). Third, bothomission of dNTPs and addition of DNA polymerase inhibi-tors reduced the levels of DP DHBV DNA (Fig. 2, lanes 8 to10), indicating that the deproteinization reaction requiresDNA polymerase activity.

DHBV DNA deproteinization and nucleocapsid disassemblyrequire the completion of plus-strand DNA synthesis. Onesimple explanation for the requirement of viral DNA polymer-ase activity in the deproteinization reaction is that the comple-tion or extension of plus-strand DNA synthesis beyond a cer-tain length is an essential signal to trigger the biochemicalreaction. However, because DHBV virion-associated rcDNAcontains an almost full-length plus-strand DNA (21) and theobserved deproteinization reaction is at such a low efficiency, itis difficult to rule out the possibility that the observed depro-teinization reaction might occur randomly in capsids after a longperiod of incubation. To firmly establish the role of plus-strandDNA synthesis in triggering DHBV genome deproteinization, wetook advantage of our previous observation that the heteroge-neous lengths of DHBV DNA, from less-than-full-length minus-strand DNA to full-length double-stranded DNA, could be syn-thesized in purified intracellular pgRNA-containing DHBVnucleocapsids via an in vitro endogenous DNA polymerase reac-tion (13).

Accordingly, DHBV intracellular nucleocapsids were puri-fied from Dstet5 cells that were cultured in the absence oftetracycline and presence of PFA for 3 days to allow the ac-cumulation of pgRNA-containing capsids and were then cul-tured in the absence of PFA for an additional 4 h to resume

minus-strand DNA synthesis (13). Such nucleocapsids con-tained only pgRNA and heterogeneous lengths of minus-strand DHBV DNA, based upon their characteristic mobilityin the agarose gel (Fig. 3A, lane 2). However, incubation of thenucleocapsids in the endogenous DNA polymerase reactionresulted in the sequential appearance of more full-length mi-nus-strand DNA (Fig. 3A, upper panel, lanes 3 to 5), partiallydouble-stranded DNA (the smear between single stranded [ss]and double-stranded linear [dsl] DNA bands [Fig. 3A, upperpanel, lanes 4 to 6] and full-length rc- and dslDNA [Fig. 3A,upper panel, lane 6]). Compared with that observed in DHBV-replicating cells where rcDNA is the predominant form (ap-proximately 90%) of mature double-stranded viral DNA (36),the in vitro endogenous DNA polymerase reaction with puri-fied intracellular nucleocapsids produced more dslDNA, butless rcDNA (Fig. 3A and B), suggesting a less efficient primertranslocation during the initiation of plus-strand DNA synthe-sis under the in vitro conditions (36). Interestingly, as shown inthe lower panel of Fig. 3A, the DP DNA appeared only after6 h of incubation and was presented exclusively as full-lengthdslDNA. While the result clearly indicates that completion orat least near completion of the plus-strand DNA synthesis is anessential signal to trigger deproteinization, it is not yet clearwhy DP rcDNA could not be produced in this in vitro reaction,although mature rcDNA was made.

To test the possibility that rcDNA deproteinization mayrequire a longer time of incubation and determine whetherdslDNA deproteinization could also result in nucleocapsid dis-assembly, the purified intracellular DHBV nucleocapsids, as

FIG. 3. Mature double-stranded DNA synthesis and deproteinization occur in purified intracellular DHBV nucleocapsids following endoge-nous DNA polymerase reaction. (A) EPRs were performed with immature intracellular nucleocapsids prepared from Dstet5 cells (see Materialsand Methods for details) in a 100-�l EPR mixture as described in the legend of Fig. 1. The reaction mixtures were either without incubation (lane2) or incubated at 37°C for the indicated periods of time (lanes 3 to 6). Core DNA and DP DNA were analyzed by Southern blot hybridization.The amount of core DNA (upper panel) and DP DNA (lower panel) loaded onto each lane was derived from 4 � 106 and 4 � 107 cells. (B) EPRswere performed with immature intracellular DHBV nucleocapsids, and the reaction mixtures were either without incubation (lanes 2 and 6) orincubated at 37°C for the indicated periods of time (lanes 3 to 5 and 7 to 11). Core DNA and DP DNA were extracted without (lanes 2 to 9) orwith (lanes 10 and 11) prior DNase I digestion and detected by Southern blot hybridization with a DHBV minus-strand specific �-32P-riboprobe.Fifty picograms of 3.0-kb unit-length DHBV DNA was loaded as the hybridization size marker (lane 1). The positions of rcDNA (RC), dslDNA(DSL), single-stranded DNA (SS), and partial single-strand DNA are indicated.

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described above, were incubated under standard EPR condi-tions for 6, 12, and 16 h, respectively. Total and DP viral DNAwere extracted and analyzed by Southern blot hybridization. Inagreement with the results presented in Fig. 3A, after 6 h ofincubation, a large amount of full-length minus-strand DNA,heterogeneous lengths of double-stranded DNA and matureforms of dslDNA and rcDNA were synthesized. Further ex-tension of the incubation period did not produce a significantadditional amount of matured forms of double-stranded viralDNA (Fig. 3B, compare lanes 3 to 5). The DP dslDNA couldbe detected after 6 h, and the amount slightly increased withadditional incubation time (Fig. 3B, compare lanes 7 to 9).Although the mature rcDNA were made under these experi-mental conditions, the DP rcDNA was not detected even after16 h of incubation. Furthermore, while the vast majority ofviral DNA synthesized under the EPR conditions were resis-tant to DNase I digestion (Fig. 3B, lane 10), suggesting theirexistence in intact nucleocapsids, the DP dslDNA is completelysusceptible to DNase I treatment (Fig. 3B, lane 11). Hence, asobserved for rcDNA, the dslDNA deproteinization was alsoassociated with nucleocapsid disassembly.

Taken together, our results presented in the above sectionsclearly demonstrate that DHBV nucleocapsids contain suffi-cient information and factors to allow for deproteinization ofthe associated viral genomes. Although the sequence of theevents remains to be determined, the DNA deproteinizationand nucleocapsid disassembly are tightly linked events. More-over, while our results strongly support the notion that thematuration of plus-strand DNA synthesis is an essential signalto trigger the DNA deproteinization and nucleocapsid disas-sembly, an additional signal(s) may be required for the depro-teinization reaction to occur. This conclusion is based upon thefollowing two experimental evidence: (i) less than 1% of ma-ture rcDNA and/or dslDNA underwent deproteinization un-der the in vitro EPR conditions (Fig. 1 to 3), and (ii) only thedeproteinization of mature dslDNA, but not rcDNA, occurredunder the EPR conditions with purified intracellular nucleo-capsids.

DHBV DNA deproteinization appears to require a serineprotease activity. As extensively discussed in our previous re-port (12) and the report by Gao and Hu (9), removal of thegenome-bound DNA polymerase could potentially occur viathe following three biochemical reactions: (i) endonucleolyticcleavage of DNA sequence near the 5� terminus of the minus-strand DNA; (ii) hydrolysis of the phosphodiester bond be-tween tyrosine residue in the terminal protein (TP) domain ofpolymerase and the 5� end of the minus-strand DNA; (iii)proteolytic cleavage of DNA polymerase. The three reactionswill yield DP DNA bearing distinct structural features at the 5�end of the minus-strand DNA. For instance, the hydrolysis ofphosphodiester bond and proteolytic cleavage of polymerasewill yield DP rcDNA with its 5� end at its authentic position,and an endonuclease reaction will trim the 5� end of the minus-strand DNA. We had previously demonstrated by a primerextension assay that the minus strand of DP rcDNA containsan authentic 5� end (12). Thus, the possibility of endonucleasereaction for the removal of genome-bound DNA polymerasecan be ruled out. To further distinguish the two other possi-bilities, we took the advantage of our in vitro deproteinization

assay and determined whether the deproteinization reactioncould be inhibited by protease inhibitors.

To this end, the in vitro deproteinization assay was per-formed in the presence of the Halt protease inhibitor cocktail(PIC; without EDTA). DHBV DNA polymerase inhibitorddCTP was used as a positive control. As shown in Fig. 4A,ddCTP efficiently inhibited DHBV DNA deproteinization, andsimilarly, the PIC also prevented viral DNA deproteinization.The result appeared to suggest that the deproteinization wasmediated by proteolytic reaction. However, one argument isthat the PIC may actually inhibit the polymerase activity. Toaddress this issue, effects of the PIC on DHBV DNA polymer-ase activity was measured by [�-32P]dCTP incorporation. Asshown in Fig. 4B, while the polymerase inhibitors ddGTP andPFA potently inhibited [�-32P]dCTP incorporation, the poly-merase activity was only modestly reduced in the presenceof PIC.

To determine the active components in the Halt PIC, eachindividual component was tested, and we demonstrated thatthe in vitro DHBV rcDNA deproteinization reaction can bespecifically inhibited by a serine protease inhibitor AEBSF, butnot the cysteine (E-64) or calpain (MDL28170) protease in-hibitor (Fig. 4C). Moreover, AEBSF did not apparently inhibitDHBV DNA polymerase activity, as revealed by [�-32P]dCTPincorporation assay (Fig. 4D). Our results hence suggest thatthe removal of genome-bound DNA polymerase might be cat-alyzed by a cellular serine protease. However, due to the celltoxicity of the compound, its effect on deproteinization andcccDNA formation of DHBV in cultured cells could not bedetermined. Moreover, it should be pointed out that removalof the polymerase by such a proteolytic reaction will leave ashort peptide or at least one amino acid residue (tyrosine) thatstill attaches onto the 5� terminus of the minus-strand DNA.Therefore, to create a 5� end of minus-strand DNA that is ableto be ligated, the DNA end must be further processed by eitheran endonucleolytic cleavage of DNA sequence near the 5�terminus of the minus-strand DNA or a reaction that cleavethe phosphodiester bond between the tyrosine residue and firstnucleotide of minus strand DNA. The later reaction couldpotentially been catalyzed by a cellular DNA repair enzymecalled tyrosyl-DNA phosphodiesterase 1 (TDP1), which is ableto remove topoisomerase (Topo) II from Topo II inhibitorsinduced DNA strand break by cleavage of the tyrosine and5�-phosphotyrosyl linkage between Topo II and chromosomalDNA (2, 30). Involvement of TDP1 in DP-DNA productionand cccDNA synthesis is currently under investigation in ourlaboratory.

The carboxyl terminus of the core protein is exposed onHBV DP rcDNA-associated nucleocapsids. The results ob-tained from our previous cell fractionation studies (12) and thein vitro deproteinization assays presented above clearly dem-onstrate that the deproteinization of the mature forms ofhepadnavirus DNA were initiated inside the nucleocapsids inthe cytoplasm and associated with nucleocapsid disassembly.However, considering the fact that although the cytoplasmicDP rcDNA was largely DNase I sensitive, the DNA species wasstill associated with the core protein and cosedimented withnucleocapsids by sucrose gradient centrifugation, it was rea-sonable to believe that the deproteinization reaction most

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likely resulted in a drastic structural change in the nucleocap-sids, but not a complete capsid disassembly (12).

Biologically, to serve as a functional precursor for cccDNAformation, the cytoplasmic DP rcDNA must be transportedinto the nucleus. The deproteinization-associated nucleocapsidstructural change may thus be essential for delivery of the DPrcDNA into the nuclei for cccDNA formation. For instance, itwas demonstrated previously that the HBV core protein(HBcAg) contained a classical bipartite NLS at its C-terminalportion (7, 22, 43). This portion was not required for emptycapsid assembly but was essential for pgRNA assembly intonucleocapsid and viral DNA replication (22). However, cryo-electron microscopy study of nucleocapsids assembled withfull-length HBcAg in vitro revealed that the C-terminal por-tion of HBcAg was localized inside of the capsid shell (46).Hence, we hypothesized that the C-terminal tail of HBcAgought to flip out upon deproteinization of the matured capsid

DNA, which will lead to a selective recognition and nuclearpore complex binding of DP rcDNA-containing nucleocapsidsby cellular nuclear transport receptor karyopherins (4).

To determine the localization of the C-terminal portion ofthe HBV core protein in the various forms of nucleocapsids,we raised a rabbit polyclonal antibody against a peptide de-rived from the 14 C-terminal amino acid residues, which wasdesignated HBc170 (Fig. 5A). Western blot analysis demon-strated that the antibody specifically recognizes the HBV coreprotein in HepDES19 cell lysates (Fig. 5B). As input materialfor an immunoprecipitation assay, nucleocapsids in the cyto-plasmic lysate of HepDES19 cells contain heterogenouslengths of HBV DNA replicative intermediates, and the cyto-plasmic DP HBV DNA exists only as matured rcDNA. Con-sistent with our previous report (12), approximately 10% cy-toplasmic DP rcDNA is DNase I resistant (Fig. 5C, lanes 1 to4), suggesting that a deproteinization reaction might occur

FIG. 4. A serine protease inhibitor inhibits DHBV DNA deproteinization and nucleocapsid disassembly in vitro (A) EPRs were performed withDHBV virions in a 100-�l EPR mixture as described in the legend of Fig. 1. The reaction mixtures were either without incubation (lane 2) orincubated at 37°C for 16 h (lanes 3 to 5). For the samples analyzed in lanes 4 and 5, dCTP was replaced by ddCTP (lane 4) or 1� Halt PIC (Pierce)was added in the EPR mixture (lane 5). Core DNA and DP DNA were analyzed by Southern blot hybridization. (B) EPRs were performed with2 � 107 DHBV virions in a 10-�l EPR mixture including 150 mM NaCl, 50 mM Tris-HCl (pH 8.0), 10 mM MgCl2, 1 mM DTT, 0.1% NP-40, 0.1mM of dATP, dGTP, and dTTP, and 5 �M [�-32P]dCTP (800 Ci/mmole; Perkin Elmer). When indicated, dGTP was replaced by ddGTP or 1 mMPFA, or 1� EDTA-free Halt PIC was added. The reaction mixture was incubated at 37°C for 1 h; the mixture was blotted onto the 3MM Whatmanfilter, rinsed by 10% acetic acid for 15 min three times and briefly washed by 95% ethanol three times; the filter was then air dried; and theincorporated [32P]dCTP was counted with a liquid scintillation counter (Perkin Elmer). (C) EPRs were performed in the absence or presence of1� Halt PIC (lane 6) or its three individual active components—1 mM of wide-spectrum serine protease inhibitor AEBSF (lane 7), 15 �M ofcysteine protease inhibitor E-64 (lane 8), and 10 �M of calpain protease inhibitor MDL28170 (lane 9)—at 37°C for 16 h. Core DNA (lanes 2, 4,and 5) and DP DNA (lanes 3 and 6 to 9) were extracted and detected by Southern blot hybridization. One hundred picograms of 3.0-kb unit-lengthDHBV DNA served as the quantification standard and molecular weight marker (lane 1). The position of rcDNA (RC) is indicated. (D) EPRswere performed as described in legend for panel B of this figure in the absence or presence of 1 mM AEBSF. The incorporated [32P]dCTP wascounted with a liquid scintillation counter as described above.

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prior to nucleocapsid partial disassembly. Interestingly, theimmunoprecipitation assay demonstrated that while a rabbitpolyclonal antibody against the native HBV core protein(Dako) efficiently precipitated nucleocapsids containing allforms of HBV DNA intermediates and DP rcDNA (Fig. 5C,lanes 5 to 8), the antibody HBc170 precipitated only nucleo-capsids containing mature rcDNA and DP rcDNA, but not theimmature DNA replicative intermediates (Fig. 5C, lanes 9 to12). Because HepDES19 cells are deficient in producing viralenvelope proteins (12), as expected, antibody against HBsAgfailed to precipitate any form of HBV DNA (Fig. 5C, lanes 13and 14). Taken together, these results are in agreement withthe notion that the C-terminal portion of HBcAg is exposedupon the completion of plus-strand DNA synthesis and/or viralDNA deproteinization-induced capsid partial disassembly.

Cytoplasmic HBV DP rcDNA associates with karyopherin-�and k�. To investigate whether the C-terminal exposure ofHBcAg on DP rcDNA-containing nucleocapsid resulted in theaccessibility of the capsids to cellular nuclear transport recep-tors, an immunoprecipitation assay was performed with anti-bodies against karyopherin-�1 and HBsAg in the lysates ofHepDES19 cells. The results shown in Fig. 6A revealed thefollowing. (i) Comparing lanes 2 and 3 shows that prior DNaseI treatment of the input lysate reduces only the level of maturercDNA, but not that of immature DNA replicative intermedi-ates. Moreover, consistent with our previous observation, themajority of DP rcDNA in the cytoplasm is DNase I sensitive(compare lanes 4 and 5) (12). These results indicate that asignificant amount of intracellular mature rcDNA is actuallyDP rcDNA and exists in DNase I-permeable nucleocapsids.(ii) Only mature rcDNA can be immunoprecipitated andpresent in both total core DNA and DP DNA preparations

(lanes 6 and 7). The difference in amount of rcDNA in the totalcytoplasmic core DNA and Hirt DNA preparations could bedue to a higher recovery rate of core DNA extraction proce-dure. Finally, the immunoprecipitated rcDNA in both the totalcore DNA and DP DNA preparations are DNase I sensitive(lanes 8 and 9). As a negative control, antibody against HBsAgfailed to precipitate any form of HBV DNA. In addition,similar immunoprecipitation assays were performed with anti-bodies against two types of karyopherin-� and demonstratedthat like karyopherin-�1, both karyopherin-�1 and karyo-pherin-�2 selectively associated with only the cytoplasmic ma-ture rcDNA and/or DP rcDNA, but not nucleocapsids contain-ing immature viral DNA (Fig. 6B).

In summary, the results presented in the above two sectionsdemonstrate that the deproteinization of viral genomic DNAresulted in drastic structural changes in nucleocapsids, whichlead to the exposure of the C-terminal portion of HBcAg andinteraction with cellular nuclear transport receptors, such askaryopherin-� and -�.

Role of cytoplasmic DP rcDNA in cccDNA formation. Toinvestigate the functional role of the cytoplasmic DP rcDNA incccDNA formation, the interaction of cytoplasmic DP rcDNA-containing nucleocapsid with karyopherin-� and DP rcDNAnuclear transportation in HepDES19 cells were disrupted ei-ther by expression of a dominant-negative form of karyo-pherin-�1 or by reducing karyopherin-�1 expression by siRNAtransfection. The accumulation of DP rcDNA in the cytoplasmand nuclei and cccDNA formation were determined by aSouthern blot hybridization assay. As a confirmation, we havedemonstrated that the truncated karyopherin-�1 was properlyexpressed in the transfected cells and expression of the proteinwas reduced by approximately 50% in siRNA-transfected cells

FIG. 5. The carboxyl terminus of HBcAg is exposed on the surface of DP rcDNA-containing nucleocapsid. (A) A rabbit polyclonal antibody(HBc170) was raised against the synthetic peptide corresponding to the carboxy-terminal 14 amino acid residues of the HBV core protein. Theunderlined 12-amino-acid peptide within the C-terminal 14 amino acid residues is one of the bipartite NLSs of HBcAg. (B) Proteins in cell lysatesof HepDES19 cells cultured in the presence or absence of tetracycline for 8 days were resolved by SDS-polyacrylamide gel electrophoresis,transferred onto the membrane, probed with the antibody HBc170, and visualized by Li-COR. A cross-reactive cellular protein band is indicatedwith an asterisk, and �-actin served as the loading controls. (C) Cytoplasmic lysate of HepDES19 cells were subjected to immunoprecipitation withantibodies against the HBV core protein (Dako), C-terminal 14-amino-acid peptide (HBc170), and HBsAg. Core DNA and DP DNA wereextracted with or without prior DNase I digestion from the original lysate (input) and immunocomplexes on beads (IP) and analyzed by Southernblot hybridization. Lanes 1 and 2 were loaded with 1/10 of core DNA from the cytoplasmic fraction of one 60-mm dish; lanes 3 to 14 were loadedwith half of the indicated DNA samples from the cytoplasmic fraction of one 60-mm dish. The positions of rcDNA (RC) and single-stranded DNA(SS) are indicated. MW, molecular size.

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by Western blot analyses (Fig. 7E). As expected, neither ex-pression of truncated karyopherin-�1 nor reducing its expres-sion by siRNA transfection affected HBV DNA replication(Fig. 7A), core protein synthesis (Fig. 7E, lower panel), or totalcellular DP rcDNA production (Fig. 7B). However, both inhi-bition of karyopherin-�1 and reducing its expression led to theaccumulation of cytoplasmic DP rcDNA (Fig. 7C, lanes 2 and6) and reduction in the amounts of nuclear DP rcDNA andcccDNA (Fig. 7E, lanes 2 and 6). However, we noticed thatunlike dominant negative karyopherin-�, expression of domi-nant negative forms of karyopherin-�1 did not affect DP

rcDNA nuclear transportation or cccDNA formation. Consid-ering that the karyopherin-� family contains six isoform mem-bers (32) and both karyopherin-�1 and -�2 efficiently boundDP rcDNA (Fig. 6B), it is possible that inhibition of functionof a single species of karyopherin-� can be compensated by

FIG. 6. Cytoplasmic DP rcDNA were associated with karyo-pherins. One milliliter of cytoplasmic lysate prepared from 5 � 105

HepDES19 cells that were cultured in the absence of tetracycline for12 days was mixed with 35 �l of protein A/G plus beads (Santa Cruz)preabsorbed with antibodies against karyopherin-�1 (k�1) (Abcam) orHBsAg (Dako) (panel A) and against karyopherin-�1 (k�1; clone114-E12; Zymed) or karyopherin-�2 (k�2; sc-55537; Santa Cruz)(panel B). The mixtures were incubated at 4°C overnight. Beads werewashed four times with TNE buffer, and core DNA and DP DNA wereextracted from the beads with or without prior DNase I digestion asindicated. Viral DNA were analyzed by Southern blot hybridization.Lane 1 was loaded with 50 pg of 3.2-kb HBV DNA. In panel A, lanes2 and 3 were loaded with 1/20 of core DNA from the cytoplasmicfraction of one 60-mm dish, and lanes 4 and 5 were loaded with half ofDP DNA from the cytoplasmic fraction of one 60-mm dish. In panel B,lane 2 was loaded with 1/30 of core DNA from the cytoplasmic fractionof one 60-mm dish, and lane 3 was loaded with 1/10 of DP DNA fromthe cytoplasmic fraction of one 60-mm dish. Half the volume of im-munoprecipitated core DNA and DP DNA recovered from one 60-mmdish was loaded onto the gel (panel A, lanes 6 to 11; panel B, lanes 4to 11) The positions of rcDNA (RC) and single-stranded DNA (SS)are indicated.

FIG. 7. Nuclear transportation of HBV DP rcDNA and cccDNAformation was inhibited by expression of dominant negative karyo-pherin-�1 or knockdown of endogenous karyopherin-�1 expression.HepDES19 cells in a collagen-coated 35-mm dish were transfected byLipofectamine 2000 (Invitrogen) with 4 �g plasmid of control vector(lanes 1 and 4), dominant negative karyopherin-�1 (D.N-k�1; lane 2),dominant negative karyopherin-�1 (D.N-k�1; lane 3), 90 nM of con-trol siRNA (lane 5; Santa Cruz), or Smartpool siRNA for karyo-pherin-�1 (lane 6; Dharmacon). The transfected cells were cultured intetracycline-free medium for 5 days, followed by a second round oftransfection and continued culture under tetracycline-free condi-tions for another 5 days. The intracellular core DNA (A), whole-cellHirt DNA (B), cytoplasmic DP DNA (C), and nuclear DP DNA(D) were extracted as previously described and subjected to South-ern blot and DNA hybridization. The positions of rcDNA (RC),single-stranded DNA (SS), and cccDNA are indicated. Expressionof wild-type or recombinant proteins and HBcAg was assessed by aWestern blot assay (E).

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other subtypes of karyopherin-�. Nevertheless, our resultsdemonstrated that karyopherins are essential for DP rcDNAnuclear import and cccDNA formation. This observation isconsistent with the model that the cytoplasmic DP rcDNA is aprecursor of nuclear DP rcDNA and cccDNA.

DISCUSSION

cccDNA is the first viral product made from the incomingnucleocapsid-associated rcDNA during initial infection ofhepadnaviruses (39). In addition, cccDNA can also be pro-duced from the rcDNA in the progeny nucleocapsids in thecytoplasm of infected cells via an intracellular amplificationpathway (40, 42). Considering the molecular pathway ofcccDNA formation, the essential events should includegenomic rcDNA uncoating (nucleocapsid disassembly), nu-clear transportation, and conversion of rcDNA into cccDNA(19, 28). Biochemically, removal of genome-bound viral DNApolymerase is among the most obvious reactions required forthe conversion of rcDNA into cccDNA (9, 12). Detailed un-derstanding of the underlying molecular mechanisms of thesemolecular events should not only advance our knowledge inHBV biology, but also provide a basis for the development oftherapeutic strategies to inhibit cccDNA formation, which isessential to cure chronic HBV infection (16).

Based on our previous discovery and characterization of DPrcDNA, a product derived from the removal of genome-linkedviral DNA polymerase, we proposed that cytoplasmic DPrcDNA is the functional precursor of cccDNA. In the studiespresented in this report, we further elucidated the molecularmechanism of cytoplasmic DP rcDNA production, uncoating,and nuclear transportation and investigated its role in cccDNAformation. Our results demonstrated that the deproteinizationof DHBV rcDNA could occur in a prolonged endogenousDNA polymerase reaction with either virion-derived or intra-cellular nucleocapsids (Fig. 1 and 3). We also showed that thein vitro deproteinization reaction could be inhibited by viralreverse transcriptase inhibitors (Fig. 2) and a broad-spectrumserine protease inhibitor (Fig. 4). Moreover, as observed invirally infected cells, hepadnavirus genome deproteinizationwas tightly linked with nucleocapsid disassembly which leads tothe exposure of the C-terminal polypeptide of core protein andNLS on DP rcDNA-associated nucleocapsids. This was dem-onstrated by the following two independent experiments. First,the cytoplasmic HBV DP rcDNA-containing nucleocapsidscould be selectively immunoprecipitated with an antibodyagainst carboxyl-terminal peptide of capsid protein (Fig. 5).Second, DP rcDNA could be selectively immunoprecipitatedwith antibodies against cellular nuclear transport receptorskaryopherin-� and -� (Fig. 6). Furthermore, consistent withthe proposed role that the cytoplasmic DP rcDNA is a func-tional precursor of cccDNA formation, we provided evidenceshowing that inhibition of karyopherin-mediated nuclear trans-portation by either transfection of siRNA targeting karyo-pherin-�1 mRNA or expression of a dominant negative karyo-pherin-�1 in a stable cell line supporting HBV replicationresulted in the accumulation of cytoplasmic DP rcDNA andreduction of nuclear DP rcDNA and cccDNA (Fig. 7).

One of the most remarkable phenomena in cccDNA biosyn-thesis is that cccDNA can be formed from the rcDNA in the

progeny nucleocapsids in the cytoplasm of infected cells. Thisphenomenon suggests that the disassembly of progeny rcDNA-containing nucleocapsids occurs in the host cells without anextracellular virion phase (40, 42). This is in marked contrast tomany other animal viruses whose genome uncoating strictlyrelies on extracellular virion phase, due to the fact that thenucleocapsids of these viruses either become matured onlyduring budding and secretion process (8, 25) or require inter-action with viral receptors or an acid bath in endosomes duringentry of host cells to trigger disassembly (1, 6, 35). Hence, theability of intracellular progeny nucleocapsid to uncoat in hostcells suggests that the disassembly of hepadnaviral capsids maybe triggered directly either by maturation of DNA synthesisfrom inside of capsids or by attacking cellular factors thatrecognize structural features presented only on the surface ofmature capsids. Our results obtained from the in vitrodeproteinization assay strongly support the notion that viri-on-associated or intracellular nucleocapsids contain all theinformation and factors required for the viral genomic DNAdeproteinization and uncoating. Moreover, we providedcompelling evidence suggesting that the completion of plus-strand DNA synthesis is an essential, but not sufficient,signal to trigger genome deproteinization and capsid disas-sembly.

Considering the biochemical mechanism of deproteiniza-tion, our results indicated that a serine protease activity wasrequired for the deproteinization reaction. A simple explana-tion for this observation is that a host cellular serine proteasemay be packaged in nucleocapsid and cleave the polymeraseupon the completion of plus-strand DNA synthesis. Alterna-tively, the completion of plus-strand DNA synthesis may trig-ger nucleocapsid partial disassembly that makes rcDNA acces-sible to a copurified cellular serine protease. Nevertheless, theproteolytic removal of DNA polymerase is consistent with ourprevious observation that DP rcDNA purified from DHBVreplicating cells has an authentic 5� end (12).

The results presented herein (Fig. 6 and 7) and in our pre-vious report demonstrated that approximately 15% of the totalintracellular mature rcDNA were DP rcDNA (9, 12). How-ever, the deproteinization in our in vitro endogenous DNApolymerase reaction occurred at a very low efficiency. We es-timated that after 16 h of incubation, only �1% of mature rc-or dslDNA were deproteinized. While this is most likely due tothe in vitro reaction that does not recapitulate the intracellularenvironment, it is also entirely possible that efficient depro-teinization requires recruitment of additional cellular factorswhich did not exist in virion particle or were lost during puri-fication of intracellular nucleocapsids.

Macromolecular traffic between the cytoplasm and nucleo-plasm occurs through nuclear pore complexes (5). As for manyother viruses, the genomic DNA nuclear import is an obliga-tory step in the life cycle of hepadnaviruses (15, 41). Consistentwith a previous report (31), our results obtained from thisstudy help to reveal a very important feature of hepadnavirusnucleocapsids—that the carboxyl-terminal portion of core pro-tein is exposed only in mature double-stranded DNA-contain-ing nucleocapsids. In agreement with this observation, weshowed in this report that only DP rcDNA-containing nucleo-capsids are selectively associated with nuclear import receptor

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karyopherins and karyopherin-�1 is essential for DP rcDNAnuclear transportation and cccDNA formation.

Our work reported herein further elucidated the molecularmechanism of cytoplasmic DP rcDNA production and pro-vided additional evidence supporting the hypothesis that cyto-plasmic DP rcDNA is the functional precursor of cccDNAformation. The molecular pathway of hepadnavirus cccDNAformation proposed in this report will guide our future inves-tigation toward understanding the mechanism of cccDNA for-mation and development of antiviral strategies to cure chronicHBV infections.

ACKNOWLEDGMENTS

We thank Jinhong Chang and Pamela Norton for critical reading ofthe manuscript and helpful suggestions.

This work was supported by grants from the Commonwealth ofPennsylvania through the Hepatitis B Foundation. H.G. and J.-T.G.are Bruce Witte fellow and scholar of Hepatitis B Foundation, respec-tively.

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