Antisense Oligodeoxynucleotides Targeted against Molecular Chaperonin Hsp60 Block Human Hepatitis B Virus Replication*

The major role of hepatitis B virus polymerase (HBV pol) is polymerization of nucleotides, but it also participates in protein priming and the packaging of its own genome into capsids. Therefore, HBV pol may require many assistance factors for its roles. Previous reports have shown that Hsp60, a molecular chaperone, activates HBV pol both in vitro and ex vivo, such as inside insect cells. Moreover, HBV pol binds to Hsp60 in the HepG2 host cell line. In this report, we show that Hsp60 plays a role in the in vivo replication of HBV. Antisense oligodeoxynucleotides (A-ODNs) specifically directed against Hsp60 induced its down-regulation, severely reducing the level of replication-competent HBV without influencing cell proliferation and capsid assembly under these conditions. Furthermore, we found that Hsp60 did not encapsidate into nucleocapsids. Our results indicate that Hsp60 is important for HBV replication in vivo, presumably through activation of HBV pol before encapsidation of HBV pol into HBV core particle. In addition, A-ODNs specific for Hsp60 also inhibit replication of a mutant HBV strain that is resistant to the nucleoside analogue 3TC, which is the main drug used for HBV treatment, and we suggest that A-ODNs directed against Hsp60 are possible reagents as anti-HBV drugs. Conclusively, this report shows that the host factor, Hsp60, is essential for in vivo HBV replication and that mechanism of Hsp60 is probably through an activation of HBV pol by Hsp60.

The major role of hepatitis B virus polymerase (HBV pol) is polymerization of nucleotides, but it also participates in protein priming and the packaging of its own genome into capsids. Therefore, HBV pol may require many assistance factors for its roles. Previous reports have shown that Hsp60, a molecular chaperone, activates HBV pol both in vitro and ex vivo, such as inside insect cells. Moreover, HBV pol binds to Hsp60 in the HepG2 host cell line. In this report, we show that Hsp60 plays a role in the in vivo replication of HBV. Antisense oligodeoxynucleotides (A-ODNs) specifically directed against Hsp60 induced its down-regulation, severely reducing the level of replication-competent HBV without influencing cell proliferation and capsid assembly under these conditions. Furthermore, we found that Hsp60 did not encapsidate into nucleocapsids. Our results indicate that Hsp60 is important for HBV replication in vivo, presumably through activation of HBV pol before encapsidation of HBV pol into HBV core particle. In addition, A-ODNs specific for Hsp60 also inhibit replication of a mutant HBV strain that is resistant to the nucleoside analogue 3TC, which is the main drug used for HBV treatment, and we suggest that A-ODNs directed against Hsp60 are possible reagents as anti-HBV drugs. Conclusively, this report shows that the host factor, Hsp60, is essential for in vivo HBV replication and that mechanism of Hsp60 is probably through an activation of HBV pol by Hsp60.
Hepadnavirus is a small hepatotropic DNA-containing virus that replicates through an RNA intermediate (1). Human hepatitis B virus (HBV 1 ), a member of the hepadnavirus family, infects the liver acutely and chronically. Acute infections can produce serious illness, and ϳ0.5% of patients terminate with fatal, fulminant hepatitis. Chronic HBV infection causes liver diseases, such as liver cirrhosis, hepatocellular carcinoma, as well as other serious consequences. Nearly 25% of patients with chronic infections terminate in untreatable liver cancer (2). Annual deaths from liver cancer caused by HBV infection exceed one million worldwide. An effective vaccine has been available for 20 years, and attempts for universal vaccination are now underway in developed countries (3). However, vaccination is not effective for established infections and only prevents transmission, such as from mother to newborn (4).
Following infection of hepatocytes, the partially doublestranded DNA genome of hepadnavirus is converted into covalently closed circular DNA in the nucleus (5). Hepadnavirus produces four RNA transcripts for replication in the infected cells (1). Among the transcripts, pregenomic RNA (pgRNA) acts as an mRNA that encodes the HBV core protein and the polymerase (pol); pgRNA also acts as the template for genome synthesis after it is packaged into a capsid. Binding of pol to a stem-loop structure, ⑀, which is close to the 5Ј-end of pgRNA, is necessary for nucleocapsid assembly and replication initiation (6,7). This binding of pol to the 5Ј ⑀ stem-loop region is important because, after binding, to initiate replication of the HBV genome pol incorporates three to four nucleotides at tyrosine residue 63, a process called "priming" (8,9).
To study the biochemical aspects of HBV pol, many researchers have tried to produce in vitro HBV pol systems. However, HBV pol is not active in cell-free systems, and producing HBV pol in Escherichia coli is difficult. Only HBV pol fusions with large proteins, such as maltose binding protein, can be expressed in E. coli (10,11). However, pol is very unstable and highly degradable; therefore, reconstitution experiments of pol are complicated. Mechanistic questions have become tractable only after the establishment of a model system, the duck HBV model, which provides active pol in a cell-free system (12). During priming in vitro, duck HBV pol requires a chaperone complex, such as the Hsp90 complex, to change its conformational states (13)(14)(15). These changes enable duck HBV pol to bind to the ⑀ stem loop region in pgRNA, after which pol shows priming activity. Although much information has been obtained from the duck HBV pol model, many mechanistic details of HBV pol are still unknown.
Recently, HBV pol was shown to bind to Hsp60 and activate pol in vitro (16). In addition, HBV pol was shown to bind to Hsp60 in HepG2 cells, which are human hepatocytes (17). These results suggest that Hsp60 assists HBV pol in vivo. To probe the role of Hsp60 for HBV pol in vivo, we used antisense oligodeoxynucleotides (A-ODNs) to down-regulate Hsp60. Generally, A-ODNs targeting a specific gene are used to knock-out a gene without exerting other effects (18,19). For example, it was shown that protein kinase C ␣ and RAF1 are important in the translational regulation of lipoprotein lipase in adipocytes through down-regulation of protein kinase C ␣ and RAF1 by using A-ODNs (19). The most commonly used ODNs are phosphorothioate ODNs. These oligonucleotide analogues are very resistant to nucleases (20) but hybridize with a slightly lower efficiency than the binding efficiency of unmodified ODNs (21).
Using A-ODNs directed against Hsp60 (22), we reduced the level of cytoplasmic Hsp60 in HepG2 cells, thereby blocking HBV replication.
In this report, it was shown that Hsp60 is essential for HBV replication in vivo. This aspect is indicated by the loss of interaction between HBV pol and Hsp60 that is due directly to lowered Hsp60 levels by the A-ODNs directed against Hsp60 without the influence of other changes. In addition, Hsp60 can be targeted for anti-HBV drug development, because downregulation of Hsp60 in infected cells blocks replication of HBV and mutant HBV (M550V) that is resistant to the nucleoside analogue 2Ј-deoxy-3Ј-thiacytidine (3TC), the main drug for HBV treatment (23).

MATERIALS AND METHODS
Constructions and Oligonucleotides-The pHBV1.2ϫ construct is similar to a previously described construct (24) that supports HBV replication by providing all required HBV transcripts. Briefly, one copy of the HBV genome having sequences of subtype adr (25) was inserted into the pUC18 plasmid at the BamHI site (nucleotide 1397). A fragment spanning the BamHI site (nucleotide 1397) to the end of the pgRNA (nucleotide 1987) was added to this plasmid (Fig. 1A). The pCMV/Core plasmid was constructed by inserting the HBV core open reading frame into the pRc/CMV vector ( Cells and Transfections-HepG2 cells were cultured in minimal essential medium (Sigma) supplemented with 10% fetal bovine serum (Invitrogen). Cells were transfected by using the FuGENE 6 transfection reagent (Roche Applied Science) as instructed by the manufacturer. The transfection efficiency in each sample was normalized by cotransfecting the pCMV/␤-gal plasmid.
Detecting Cytoplasmic Capsids by the Endogenous Polymerase Assay-Transfected HepG2 cells were lysed by using 0.7 ml of lysis buffer containing 50 mM Tris, pH 7.5, 150 mM NaCl, 5 mM MgCl 2 , and 0.2% Nonidet P-40 per 6-cm dish. After 15 min on ice, the lysates were clarified by 20,000 ϫ g centrifugation at 4°C for 5 min. Nucleocapsids were immunoprecipitated from cleared lysates with rabbit anti-HBc antibody (Dako) and protein A-Sepharose CL4B (Sigma) for 4 h at 4°C. The beads were collected by a short spin, transferred to reaction tubes, and washed twice with 1 ml of 1ϫ PBS. For the endogenous polymerase assay, all liquid was removed, and 50 l of 50 mM Tris, pH 7.5, 75 mM NH 4 Cl, 1 mM EDTA, 20 mM MgCl 2 , 0.1% ␤-mercaptoethanol, 0.5% Nonidet P-40, 0.4 mM dATP, 0.4 mM dGTP, 0.4 mM dTTP, and 10 Ci of [␣-32 P]dCTP (3000 Ci/mmol, PerkinElmer Life Sciences) was added and then incubated at 37°C overnight. After the reaction, 20 l of 50 mM Tris, pH 7.5, 75 mM NH 4 Cl, 1 mM EDTA, and 0.25 mg/ml DNase I (Sigma) was added, and the mixture was incubated at 37°C for 30 min. The labeled viral genome was isolated by addition of 50 l of 1% SDS, 10 mM Tris, pH 7.5, 10 mM EDTA, 0.6 mg/ml proteinase K (Sigma), and 0.8 mg/ml tRNA, followed by incubation at 37°C for 30 min. This mixture was extracted once with 0.1 ml of phenol-chloroform (1:1). DNA was separated from unincorporated radioactive dCTP by precipitating samples with 25 l of 10 M ammonium acetate and 250 l of ethanol, incubating for 15 min at room temperature, and spinning for 15 min in a desktop microcentrifuge. The pellet was dissolved in 50 l of 10 mM Tris, pH 8.0, and 1 mM EDTA, and the DNA was precipitated again as described. The DNA pellet was dissolved in 10 l of Tris-EDTA buffer and applied to a 1% agarose-Tris acetate-EDTA gel for electrophoresis. The gel was transferred to Whatman paper, dried by vacuum, and exposed to x-ray film (Fuji).
Detecting Cytoplasmic Capsids by Agarose Gel Electrophoresis-Cleared lysates (2.5 ml) were layered on sucrose gradients (1 ml of 40% sucrose and 1.5 ml of 20% sucrose in 1ϫ PBS), and the capsids were sedimented by centrifugation at 20°C for 19 h at 240,000 ϫ g in a PST55Ti rotor (Hitachi). The pellets were resuspended in 50 l of 1ϫ PBS by sonication (three strokes, 1 s per stroke). Of each sample, 50 l was mixed with 6ϫ loading buffer containing 0.25% bromphenol blue and 30% glycerol and separated through 1% agarose in 40 mM Tris acetate and 1 mM EDTA. Following electrophoresis, gels were blotted overnight onto nitrocellulose membranes (PerkinElmer Life Sciences) by capillary transfer in 10ϫ SSC. HBV core particles on the membranes were analyzed by immunoblotting with the anti-HBc antibody (DAKO).
Quantifying Nucleocapsids by Quantifying Encapsidated HBV DNA-After separation, the capsid particles (50 l of resuspended samples) were mixed with 0.5 ml of immunoprecipitation buffer containing 50 mM Tris, pH 7.5, 150 mM NaCl, 0.2% Nonidet P-40, and the anti-HBcAg rabbit polyclonal antibody (DAKO). Immunoprecipitated capsid particles were washed three times in 1ϫ PBS. Of these samples, one half was dissolved in 1ϫ SDS sample buffer and analyzed by immunoblotting with the anti-Hsp60 (N-20) antibody. Encapsidated HBV nucleic acids were isolated from the other half of the immunoprecipitated capsid particles by treatment with 10 mM MgCl 2 and 500 g/ml DNase I for 1 h at 37°C. The isolated viral DNAs were quantified by using a "real-time" fluorescent-based PCR with primers (forward primer HBV1F: 5Ј-CCG TCT GTG CCT TCT CAT CTG-3Ј; reverse primer HBV1R: 5Ј-AGT CCA AGA GTY CTC TTA TGY AAG ACC TT-3Ј) and a fluorescent probe (HBV1TAQ: 5Ј-CCG TGT GCA CTT CGC TTC ACC TCT GC-3Ј) as previously described (26). As the negative control, HepG2 cells not transfected with pHBV1.2ϫ were used using the above methods. For each sample, the same volume was the same as that in the above nucleocapsid preparation steps.
Fluorescent Microscopy-HepG2 cells were seeded on coverslips coated with poly-L-lysine (Sigma), treated with 0.5 M FITC/A-ODN #1 or FITC/S-ODN, and incubated for 3 h. After incubation, coverslips were briefly washed twice with 1ϫ PBS and then incubated with 1ϫ PBS containing 5 M A-ODN #1 or S-ODN for 5 min at room temperature. The washed coverslips were fixed with 4% paraformaldehyde for 10 min at room temperature. The coverslips were washed for 10 min with quenching solution containing 50 mM Tris, pH 8.0, and 100 mM NaCl, followed by two brief washes in 1ϫ PBS. The washed coverslips were mounted on glass microscope slides with mounting medium (Sigma) and secured by rubber cement. Micrographs at 100ϫ magnification were acquired with a Nikon Eclipse E600 TM fluorescence microscope equipped with a fluorescein filter set (excitation filter 450 -490 nm, emission filter BP 520 -560) linked to an Olympus C4040 digital camera.
Subcellular Fractionation-HepG2 cells were fractionated by differential pelleting (27). The HepG2 cells were harvested and washed briefly with ice-cold 1ϫ PBS. After washing, the cells were resuspended in homogenization buffer containing 0.25 M sucrose, 10 mM Tris, pH 7.5, and 2 mM EDTA and then homogenized by using a Dounce homogenizer with a type B pestle (20 slow strokes). Cell debris and nuclei were removed from the lysed cells by centrifugation at 1,500 ϫ g for 15 min at 4°C. Postnuclear supernatants were separated by centrifugation at 30,000 ϫ g for 1 h into pellets (mitochondria fraction) and supernatants (postmitochondrial fraction). The postmitochondrial supernatants were separated by centrifugation at 100,000 ϫ g for 1 h into pellets (microsome fraction) and supernatants (cytosol fraction). The pellets were dissolved in homogenization buffer by sonication, and the amount of protein in each sample was determined by Protein Assay (Bio-Rad). The same amount of each fractionated protein sample was separated by 11% SDS-PAGE and analyzed by immunoblotting with the anti-Hsp60 (N-20) goat polyclonal antibody (Santa Cruz Biotechnology).
Metabolic Labeling-For metabolic labeling of HepG2 cells with L-[ 35 S]methionine, cells were cultured for 1.5 h in L-methionine-free minimal essential medium (Sigma) supplemented with 10% fetal bovine serum. After incubation, 0.1 mCi/ml L-[ 35 S]methionine (1000 Ci/mmol, PerkinElmer Life Sciences) was added to the culture media and incubated for 3 h. Cells were treated with 1 M A-ODN #1 or 1 M S-ODN at 24 h prior to L-methionine starvation, during L-methionine starvation, and during the metabolic labeling period.
Cell Proliferation and Apoptosis Assays-MTS assay for cell proliferation was performed with the CellTiter 96®AQ ueous Non-Radioactive Cell Proliferation Assay Kit (Promega). Caspase-3 assay for detection of cell apoptosis was performed with the CaspACE TM assay system, colorimetric kit (Promega). The above two assays were performed as instructed by the manufacturer.

RESULTS
Hsp60 Antisense ODNs Block HBV Replication-To determine whether Hsp60 is necessary for HBV pol function in vivo, we used A-ODN #1 for down-regulation of the Hsp60 level in HepG2 cells. The ODNs had been chemically modified to phosphorothioate ODNs by substituting the oxygen molecules of the phosphate backbone with sulfur for longer half-lives. To exclude nonspecific effects of the A-ODNs, we used two controls, S-ODN and A-ODN #2. The S-OND is against A-ODN #1 and is used to determine the effect of phosphorothioate ODNs on cells (Fig. 1B). A-ODN #2 (Fig. 1B) was directed against a sequence that has only four overlapping bases with A-ODN #1. It was used to test whether the sequence of A-ODN #1 nonspecifically affects the cells. Treatment of HepG2 cells with 1 M A-ODN #1 to down-regulate Hsp60 severely reduced the endogenous polymerase assay activity of HBV, similar to treatment with 1 M 3TC. However, treatment with 1 M S-ODN did not affect endogenous polymerase assay activity ( Fig. 2A). This concentration for A-ODN #1 was obtained during our experiment in which 1 M A-ODN #1 was sufficient for almost complete reduction of endogenous polymerase assay activity (Fig. 3A). Treatment of HepG2 cells with A-ODN #2 also resulted in reduced endogenous polymerase assay activity, but this reduction was not as efficient as the reduction seen with A-ODN #1. This result might be due to A-ODN #1 having a higher T m (48°C) than the T m (46°C) of A-ODN #2. In addition to the above result found after 2.5 days, HBV replication was also blocked by A-ODNs #1 treatment for 5 and 7.5 days (Fig. 2A).
Down-regulation of Hsp60 Does Not Affect Capsid Assembly-After A-ODN #1, A-ODN #2, and 3TC treatment, levels of the assembled HBV core particles were lower than that of the control (Fig. 2B, top panel). To test whether down-regulation of Hsp60 affected capsid formation, we examined capsid formation in HepG2 cells, where Hsp60 was down-regulated by treatment of A-ODN #1. To express the HBV core, we transfected HepG2 cells with the pCMV/Core plasmid and then treated the transfected with A-ODN #1. Assembled capsid particles were separated by ultracentrifugation 2.5 days after transfection. In Fig. 2B, the bottom panel clearly shows that, although Hsp60 was down-regulated, the level of HBV capsid formation was the same for both A-ODN #1-treated and untreated pCMV/Coretransfected cells. These data indicate that loss of replicationcompetent HBV nucleocapsids by A-ODN #1 treatment is not due to inhibition of HBV core assembly and HBV core protein expression, but that the loss might be due to inactivation of HBV pol. We also examined expression of HBV core proteins in insect cells and found that HBV core proteins did not bind to Hsp60 (data not shown).
Hsp60 Does Not Encapsidate in the Nucleocapsid-A previous report showed that ATP synergistically activates HBV pol (16). This result indicates that HBV pol might be liberated from the Hsp60 complex after activation, because the Hsp60 complex requires ATP for the release of the substrate (28). To determine whether Hsp60 associates with capsid particles, we quantified nucleocapsids where pol is found.
If one Hsp60 complex (composed of 14 subunits) associates with one nucleocapsid, 10 fmol of Hsp60 complexes (ϳ8 ng) should be present in 10 fmol of nucleocapsid (ϳ50 ng). Also, it is reported that HBV pol can be encapsidated into capsids without pgRNA (29). Even if Hsp60 associates with HBV pol in capsids without pgRNA, at least 10 fmol of Hsp60 complex should be present in the capsid. We were unable to detect Hsp60 in ϳ10 fmol of nucleocapsids, even though 0.6 fmol of FIG. 1. Constructions and A-ODN design. A, pHBV1.2ϫ has one copy of the HBV genome (nucleotides 1397-1397) plus 0.2ϫ HBV genome (nucleotides 1397-1987). pCMV/Core was constructed by inserting the HBV core open reading frame into pRc/CMV. B, design of A-ODNs specific for Hsp60. A-ODN #1 (5Ј-AGCATTTCTGCGGGG-3Ј) and A-ODN #2 (5Ј-GGGTAACCGAAGCAT-3Ј) are directed against nucleotides of Hsp60 from 5 to Ϫ10 and nucleotides from Ϫ8 to Ϫ22, respectively. The S-ODN (5Ј-CCCCGCAGAAATGCT-3Ј) is directed against nucleotides of Hsp60 from Ϫ10 to 5. FITC/A-ODN #1 and FITC/S-ODN are FITC-conjugated ODNs at the 5Ј-end. Analysis of the homology between the two A-ODNs and the primate sequences present in the GenBank TM data bases (release 131.0) revealed that the synthetic ODNs were fully complementary only to these two described regions of the Hsp60 gene.

FIG. 2. Inhibition of HBV replication by Hsp60-specific A-ODNs does not inhibit HBV core assembly.
A, 12 h after transfection, HepG2 cells harboring HBV1.2ϫ were treated with 1 M of A-ODN #1, A-ODN #2, S-ODN, or 3TC. Every 2.5 days, the medium were renewed and treated with the above. 2.5, 5, and 7.5 days (D) after treatment, replication-competent HBV nucleocapsids from each sample were estimated by endogenous polymerase assay, and assay products were analyzed by 1% agarose gel electrophoresis and autoradiography. B, HBV core assembly in HepG2 cells was tested by treatment with 1 M of A-ODN #1, A-ODN #2, S-ODN, or 3TC. After transfection of pHBV1.2ϫ (top panel) or pCMV/Core (bottom panel) into HepG2 cells, assembled and unassembled HBV core proteins were separated ultracentrifugation. The assembled HBV core protein was fractionated in 1% agarose and analyzed by immunoblotting with anti-HBcAg. In the case of pHBV1.2ϫ transfection (top panel), the samples were triplicated, and intensities of the immunostained core proteins were determined by 1D Image Analysis software (Kodak Digital Science). C, capsid particles were immunoprecipitated with anti-HBcAg after ultracentrifugation. One half of each sample was used for quantifying nucleocapsids, and the other half was used for detecting Hsp60 by immunoblot analysis. For immunoblot analysis, 10-fmol samples of nucleocapsids were fractionated by SDS-PAGE and analyzed by immunoblotting with the anti-Hsp60 (N-20) antibody. As controls, 60, 6, and 0.6 fmol of Hsp60 were used. D, after transfection with pHBV1.2ϫ (M550V), the transfected HepG2cells were treated with A-ODN #1 or 3TC. Replication-competent HBV nucleocapsids were estimated by endogenous polymerase assay, and assay products were analyzed by 1% agarose gel electrophoresis and autoradiography. the control Hsp60 complexes (ϳ0.5 ng) was detected in the analysis (Fig. 2C). In addition, we were unable to detect Hsp60 by an immunoblot analysis with anti-Hsp60 (N-20) antibodies on the stripped nylon membrane, which was used previously for detecting native nucleocapsid particles (about 10 fmol; data not shown). Therefore, we concluded that Hsp60 does not encapsidate in the nucleocapsid.
Down-regulation of Hsp60 Blocks Mutant HBV Resistant to 3TC-To test the potential of using A-ODN #1 as an anti-HBV drug, we tested A-ODN #1 in a mutant, 3TC-resistant HBV (M550V), which has mutations of HBV pol that change the YMDD motif to YVDD. This mutant virus produces severe problems during 3TC treatment, because it is highly resistant to 3TC and thus is the primary cause of treatment failure. To test the effect of down-regulation of Hsp60 by A-ODN #1 treatment in this mutant virus, we constructed the pHBV1.2ϫ (M550V) plasmid for producing a 3TC-resistant virus in HepG2 cells. In a previous report, the activity of this mutant HBV pol was shown to be lower than the activity of wild-type pol (30). As shown in Fig. 2D, the replication efficiency of the mutant virus was also lower than the efficiency of the wild-type virus (Fig.  2D). Treatment with A-ODN #1 blocked replication of the mutant virus, but treatment with 3TC did not block its replication. This result indicates that inhibition of HBV replication by A-ODN #1 appears to be effective against the 3TC-resistant mutant virus.

Reduction of HBV Replication by A-ODN #1 Treatment Is
Dose-dependent-HepG2 cells harboring the pHBV1.2ϫ plasmid were treated with serial dilutions of A-ODN #1 or 3TC. We estimated the level of replication-competent HBV nucleocapsids in these cells 2.5 days after treatment (Fig. 3, A and B). Furthermore, to quantify levels of secreted HBV, we harvested culture media from treated cells and estimated the presence of viral genome DNA. The results show that reduction of replication-competent HBV nucleocapsids in cells by treatment with A-ODN #1 is dose-dependent, similar to the result shown for 3TC treatment. Importantly, reduction of HBV secretion by A-ODN #1 treatment is dose-dependent, again similar to the result shown for 3TC. Fig. 3D indicates that A-ODN #1, in comparison to 3TC, inhibits HBV replication efficiently within a limited time range.
HepG2 Cells Uptake FITC-conjugated Hsp60 A-ODN #1-To test whether HepG2 cells uptake A-ODN #1, we used FITCconjugated A-ODN #1. Previously, FITC conjugation was shown to have no affect on uptake of ODNs in cells (31,32). HepG2 cells were treated with 0.5 M FITC-conjugated A-ODN #1 for 3 h. Fig. 4 clearly shows that HepG2 cells uptake both A-ODN #1 and S-ODN. For the control, HepG2 cells were treated with FITC-conjugated A-ODN #1 for 15 s. No signal was observed in this control (data not shown), indicating that the uptake shown in Fig. 4 is specific. Fig. 4 also shows that   FIG. 3. Dose response of A-ODNs against Hsp60. 12 h after transfection, HepG2 cells harboring pHBV1.2ϫ were treated with serial dilutions of A-ODN #1 (A) and 3TC as a control (B). And then 2.5 days after treatment, HBV replication efficiency was estimated by endogenous polymerase assay. C, comparison of HBV replication inhibition efficiency with 3TC. Intensities of endogenous polymerase assay products on x-ray film were determined by 1D Image Analysis software (Kodak Digital Science). ND, not determined. D, HepG2 cells harboring pHBV1.2ϫ were treated with serial dilutions of A-ODN #1 or 3TC, and secreted HBV was estimated by HBV DNA quantification. From samples of culture media, HBV nucleic acids were isolated after DNase I treatment (10 mM MgCl 2 and 500 g/ml DNase I) for 1 h at 37°C. HBV DNA was quantified by using "real-time" fluorescence-based PCR. This experiment shows the average values of data from duplicate or triplicate reactions.

Down-regulation of Cytoplasmic Hsp60 Levels by A-ODNs Is Not Mediated by Degradation of Hsp60 mRNA-Metabolic labeling of cells shows that treatment with A-ODN #1 inhibits
Hsp60 synthesis (Fig. 5A). The level of labeled ␤-actin protein was similar in the samples, which indicates that translation of ␤-actin was not affected by treatment with A-ODN #1. Also, ␤-galactosidase activities for normalizing transfection efficiency were similar in the samples (data not shown), suggesting that treatment with A-ODN #1 affects neither protein synthesis nor the function of other proteins in the cells.
To check Hsp60 levels in different regions of the cell, we treated extracts of cells with A-ODN #1 and fractionated treated extracts through differential pelleting by ultracentrifugation. Such methods have been well defined in many studies. Three fractions of mitochondria, microsome, and cytoplasm were fractionated by pelleting. Equal amounts of fractionated proteins were analyzed by SDS-PAGE and by immunoblotting with the anti-Hsp60 (N-20) antibody. Fig. 5B clearly shows that reduction of newly synthesized Hsp60 mainly affected the level of Hsp60 in cytoplasm, which is the site of HBV replication. This treatment did not affect the Hsp60 level in mitochondria, the main site of Hsp60 activity. These results suggest that down-regulation of Hsp60 in cytoplasm strongly affects HBV replication.
To test whether down-regulation of Hsp60 is due to mRNA degradation, we examined levels of Hsp60 mRNA by Northern blot analysis. Some A-ODNs are able to activate RNase H and induce target mRNA degradation (33,34). In this experiment, we found that the level of Hsp60 mRNAs in cells was not affected by treatment with A-ODN #1 (1, 0.1, and 0.01 M  A-ODN #1). This result indicates that down-regulation of the Hsp60 protein is not due to mRNA degradation by RNase H activation but due to inhibition of Hsp60 translation. Therefore, Hsp60 down-regulation is not irreversible, and the blocking of translation might be controlled by the concentration of A-ODNs.
Down-regulation of Hsp60 in the Cytoplasm Does Not Affect Cell Viability-Normally, Hsp60 is an abundant and essential protein for cell survival. Down-regulation of Hsp60 in cytoplasm, however, does not affect cell viability. To examine the viability of the cell, the MTS assay to check for cell proliferation and caspase-3 assay to check for cell apoptosis were performed as described under "Materials and Methods." In the MTS assay, treatment of serial concentration of A-ODNs #1 to HepG2 cells does not show a certain pattern (Fig. 6A). Even though treatment of A-ODNs #1 10-fold higher in concentration (10 M) than normally used (1 M) in this report, proliferation of the treated cells did not differ from the control (Fig. 6A). Caspase-3 assay shows that treatment of A-ODNs #1 (10 M) to HepG2 cells does not induce apoptosis as in treatment of S-ODNs (10 M), A-ODNs #2 (10 M), and 3TC (10 M) (Fig. 6B). The above results indicate that down-regulation of Hsp60 in cytoplasm does not affect cell functions during the limited times. DISCUSSION HBV pol participates in its genome replication, but mechanistic details have not yet been fully discovered. The obstacle for studying HBV pol has been its low expression level in HBV-infected hepatocytes and in heterologous expression systems. However, when pol was expressed in insect cells, it was very resistant to degradation and showed priming activity. Host factors for HBV pol in insect cells might be similar to the factors in hepatocytes, enabling stabilization and function of pol. Previous reports have indicated that Hsp60 activates HBV pol in insect cells and binds to HBV pol in hepatocytes (16,17). Such results suggest the possibility that Hsp60 might have an actual role in HBV replication. To probe the effects of Hsp60 on HBV replication in vivo, we used phosphorothioate A-ODNs to down-regulate the Hsp60 level in cells. With A-ODNs directed against Hsp60, we were able to show that Hsp60 participates in HBV replication and is essential for replication because downregulation of Hsp60 severely reduced the levels of replicationcompetent HBV nucleocapsids but did not inhibit capsid formation. Furthermore, Hsp60 synthesis was reduced by treatment with A-ODN #1, and Hsp60 levels were reduced mainly in the cytoplasm, which is the site of encapsidation of HBV pol and pgRNA into capsids.
To determine that the result is not caused by other effect such as viability and A-ODNs #1-cellular protein interaction, some experiments are carried. In these experiments, viability of cells treated with A-ODN #1 did not decrease, and apoptosis was not induced by treatment. Despite the requirement of Hsp60 for cell function, inhibition of Hsp60 synthesis did not affect cell viability, because Hsp60 localized in mitochondria is more than 95% in HepG2 cells and mitochondria are its main functional region. Even though HepG2 cells were treated with a high concentration of A-ODN #1 (10 M), viability was not affected. This result means that the reduction of replicationcompetent HBV nucleocapsids was not caused by an abnormality of cell functions. At each transfection, the transfection efficiencies that were normalized by measuring ␤-galactosidase activity were not affected by treatment with A-ODN #1. This result indicates that down-regulation of Hsp60 in the cytoplasm affects neither protein synthesis nor the function of other proteins. Sense phosphorothioate ODNs did not affect HBV replication. A-ODN #2, which is directed against another site of the Hsp60 gene, also blocks HBV replication, indicating that blocking is Hsp60-specific.
Under in vitro conditions, ATP synergizes Hsp60 function for HBV pol (16). Generally, ATP is required for release of substrate from the Hsp60 complex (28). This requirement indicates that Hsp60 might bind to HBV pol transiently and that bound Hsp60 might be liberated from the HBV pol-Hsp60 complex after activation. In this report, we found that Hsp60 is not encapsidated in the HBV nucleocapsid, meaning that HBV pol detaches from the Hsp60 complex before encapsidation, a result that coincides with our in vitro data.
Confirming Hsp60 function in vivo, our report also suggests A-ODN #1 as a drug for HBV treatment. Established drugs for HBV treatment have problems. Treatment of patients with chronic HBV infections with interferon ␣ (35) is limited by side effects, incomplete efficacy, restriction to patients with compensated disease, and the requirement for parenteral administration (36,37). Whereas direct administration of ODNs in vitro is not an effective delivery method, phosphorothioate ODNs administered intravenously without delivery reagents in animal models have shown effective and specific antisense inhibition. These surprising results helped revive antisense technology and have encouraged researchers to perform more clinical trials (38,39). The use of A-ODNs as antiviral agents has also emerged as a powerful new approach (40,41). Many A-ODN drugs are now being widely developed. Forvirsen, an A-ODN used against cytomegalovirus, is already approved by the Food and Drug Administration (42). In this report, replication inhibition of the HBV mutant by Hsp60-directed A-ODNs implicates Hsp60 as a possible novel target for combinational antiviral therapy with 3TC.
In conclusion, A-ODNs targeted against Hsp60 blocks HBV replication without HBV core assembly. This result shows that Hsp60 is required for HBV replication in vivo, possibly through activation of HBV pol. Our study also found that the Hsp60-HBV pol interaction is required before HBV pol encapsidation into HBV nucleocapsid.  (Promega). B, to test whether down-regulation of Hsp60 induces apoptosis, caspase-3 activity in HepG2 cells treated with A-ODN #1, A-ODN #2, S-ODN, or 3TC was estimated with the CaspACE TM assay system, colorimetric kit (Promega). These two assays were performed as described under "Materials and Methods."