INTRODUCTION

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Hepatitis B virus (HBV) is estimated to chronically infect approximately 300 million people worldwide. These individuals are at increased risk for the development of liver failure, cirrhosis, and hepatocellular carcinoma (3, 23). In addition, it is estimated that of those chronically infected, approximately 1 million die annually from HBV-induced liver disease (19).

At the present, interferon (IFN) is the only available treatment for chronic hepatitis in the United States. However, its efficacy is partial and of limited duration, with less than 30% of the chronic carriers being treated with IFN responding to treatment. In addition, approximately 50% of those who initially respond to IFN therapy experience a recurrence of viremia after the cessation of treatment (6, 29). In clinical trials, two nucleoside analogs, lamivudine [()--L-2',3'-dideoxy-3'-thiacytidine; 3TC] and ganciclovir, have proven to be effective in decreasing the levels of HBV DNA in the serum of chronically infected patients (4, 7-9). However, many patients relapsed shortly after the cessation of therapy. In addition, there are now reports of the isolation of 3TC-resistant variants of HBV from the serum of immunosuppressed patients undergoing 3TC therapy (2, 20, 30).

Here we report that AT-61, a member of a class of phenylpropenamide derivatives with antiviral activity against HBV replication (22), is a potent inhibitor of the replication of both wild-type and 3TC-resistant HBV in HepAD38, HepAD79, 2.2.15, and transiently transfected HepG2 cell lines. This compound does not inhibit the replication of duck HBV (DHBV), woodchuck HBV (WHBV), human immunodeficiency virus (HIV) type 1 (HIV-1), herpes simplex virus (HSV) type 1, (HSV-1), vesicular stomatitis virus (VSV), or Newcastle disease virus (NDV) and has very low toxicity in a number of cell lines. Moreover, when used in combination with 3TC, AT-61 acted synergistically to inhibit HBV replication in HepAD38 cells. Finally, the data suggest that this compound may exert its antiviral effect by interfering with the packaging of the pregenomic RNA into the immature core particle.

	MATERIALS AND METHODS

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Compounds. AT-61 was synthesized as described previously (22) (Fig. 1). 3TC and ()--L-2',3'-dideoxy-5-fluoro-3'-thiacytidine (FTC) were provided by Raymond Schinazi (Emory University, Atlanta, Ga.). Phosphonoformic acid (PFA) and tetracycline were purchased from Sigma Chemical Company (St. Louis, Mo.).

View larger version (10K): [in this window] [in a new window]   FIG. 1.   Chemical structure of AT-61.

Cell lines and culture conditions. Cells of the HepG2 (a human hepatocarcinoma cell line [12]); HepAD38 (a HepG2 cell line that produces HBV when it is grown in the absence of tetracycline [15]), HepAD79 (a HepG2 cell line that produces a 3TC-resistant variant of HBV when it is grown in the absence of tetracycline [16]), HepAD43 (a HepG2 cell line that produces -galactosidase when it is grown in the absence of tetracycline [11]), CA51 (a LMH cell line that produces duck HBV when it is grown at 33.5°C [25]), and Vero (a monkey kidney cell line) cell lines were maintained in Dulbecco's modified Eagle's/F-12 medium (DMEM/F-12; GIBCO BRL/Life Technologies, Gaithersburg, Md.) supplemented with 10% fetal bovine serum, 50 µg of penicillin per ml, 50 µg of streptomycin per ml, and 100 µg of kanamycin per ml (PSK) at 37°C in 5% carbon dioxide. In addition, HepAD38 and HepAD79 cells were grown in the presence of 400 µg of G418 (GIBCO BRL/Life Technologies) per ml and 0.3 µg of tetracycline per ml, and HepAD43 cells were grown in the presence of 100 µg of zeocin (Invitrogen, Carlsbad, Calif.) per ml, 400 µg of G418 per ml, and 0.3 µg of tetracycline per ml. Cells of the MT-2 cell line, a human T-cell line, and the 2.2.15 cell line, a HepG2 cell line which constitutively produces HBV (1, 26, 27), were maintained in RPMI 1640 medium supplemented with 10% fetal bovine serum and PSK. In addition, 2.2.15 cell maintenance medium contained 400 µg of G418 per ml. During the experimental procedures, the cells were grown in the media described above; however, the media did not contain penecillin, streptomycin, kanamycin, or G418.

HepAD38 and HepAD79 assays for inhibition of HBV replication. Inhibition of the production of extracellular virus in the HepAD38 cell line was determined as described previously (15). Briefly, HepAD38 cells, which express wild-type HBV, were plated onto 96-well microtiter plates in DMEM/F-12 medium supplemented with 0.3 µg of tetracycline per ml. Three days later, the medium was removed and was replaced with tetracycline-free medium containing compound. Three days after the addition of compound, the medium was removed, the cells were washed with warmed (37°C) phosphate-buffered saline (PBS), and fresh medium containing compound was added. Twenty-four hours later, the medium was collected, clarified by low-speed centrifugation, and assayed for the presence of HBV DNA by dot blot and phosphorimager analysis. The concentrations of compound that inhibited HBV replication by 50 and 90% (EC₅₀s and EC₉₀s, respectively) were determined by linear regression.

HepAD43 assay for inhibition of the tetracycline-responsive promoter. HepAD43 cells, which expresses the Escherichia coli -galactosidase gene under the control of a tetracycline-responsive promoter, were plated in 96-well microtiter plates (10⁵ cells/well) in the presence of tetracycline for 3 days. The medium was removed and replaced with medium without tetracycline but containing compound. Three days after commencing drug treatment, we removed the medium, washed the cells with warmed PBS, and added fresh medium containing compound to the cells. Twenty-four hours later, the medium was removed and the cells were washed with PBS and lysed by the addition of Reporter Lysis Buffer (-Galactosidase Enzyme Assay System; Promega Corp.). The concentration of -galactosidase was assayed as directed by the manufacturer (-Galactosidase Enzyme Assay System).

2.2.15 cell assay for inhibition of HBV replication. Compounds were tested for the ability to inhibit HBV replication in 2.2.15 cells as described previously (13, 14). Briefly, 2.2.15 cells were plated into 24-well plates and were grown at 37°C until confluent monolayers formed. The medium was removed and replaced with medium containing compound. Three and 6 days after the commencement of drug treatment, the medium was removed and replaced with fresh medium containing compound. Nine days after drug treatment began, the medium was collected and clarified by low-speed centrifugation. The supernatants were assayed for the presence of HBV DNA by dot blot and phosphorimager analysis.

Transiently transfected HepG2 assay for HBV and WHBV. Twenty-four hours after transfecting HepG2 cells with 4 µg of either pCMVhbv or pCMW82, we removed the medium, washed the cells with warmed PBS, and added medium containing compound. Three days later, the medium was removed and the cells were washed with warmed PBS. For HepG2 cells transfected with pCMVhbv, the cells were lysed and the HBV DNA replicative intermediates were isolated as described above. For HepG2 cells transfected with pCMW82, fresh medium with compound was added to the cells, which were then incubated for an additional 4 days. After a total of 7 days of drug treatment, the cells were lysed and the WHBV DNA replicative intermediates were isolated as described above for HBV DNA replicative intermediates. The quantities of HBV and WHBV DNA replicative intermediates were determined by Southern blot and phosphorimager analysis.

CA51 assay for DHBV. CA51 cells (CA51 is an LMH [chicken hepatocyte] cell line that produces DHBV when it is grown at 33.5°C but not at 39°C [25]) were plated (5 × 10⁵/60-mm plate) and incubated at 39°C. After 3 days, the medium was removed, the cells were washed with warmed PBS, and the medium was replaced with fresh medium containing compound. The cells were incubated at 33.5°C for 3 days, at which time the medium was again replaced with fresh medium containing compound. Seven days after the commencement of drug treatment, the medium was removed and the cells were washed with cold PBS and lysed with lysis buffer (50 mM Tris HCl [pH 8.0], 10 mM EDTA, 150 mM NaCl, 1% sodium dodecyl sulfate, 0.5 mg of proteinase K per ml). The DHBV DNA replicative intermediates were isolated as described above for HBV DNA replicative intermediates. DHBV DNA was quantified by Southern blot and phosphorimager analysis.

Yield reduction assay for inhibition of HIV-1 and HSV-1 replication. Compounds were screened for the ability to inhibit HIV replication as described previously (10, 21). Briefly, MT-2 cells were infected with HIV-1 (RF) at a multiplicity of infection of 0.02 in the presence of compound. Three days after infection, the medium was collected and clarified by low-speed centrifugation. The concentration of virus in each supernatant sample was determined by plaque assay.

Plaque reduction assay for NDV and VSV. Vero cells were infected with 200 and 1,000 PFU of either NDV or VSV in the presence of compound. Forty-eight hours later, the cells were stained with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT; Sigma Chemical Corp.) and the plaques were counted.

Cytotoxicity and antiproliferation assays. To determine if the compounds were cytotoxic to HepAD38 and HepG2 cells, the cells were plated in 96-well microtiter plates and were incubated at 37°C until a confluent monolayer formed. Various concentrations of compound were added to the wells and the cells were incubated for 3 days, at which time the medium was replaced with fresh medium containing compound. Twenty-four hours later, the cells were washed with warmed PBS and tested for viability with a cell proliferation assay kit as directed by the manufacturer (CellTiter 96 Non-Radioactive Cell Proliferation Assay; Promega Corp.). The concentrations of a compound that inhibited cell viability by 50 and 90% (TC₅₀s and TC₉₀s, respectively) were determined by linear regression.

HBV polymerase assays. Endogenous polymerase activity was assayed by a modified version of the protocol previously described by Sells et al. (27). HepAD38 cells were grown for 4 days in tetracycline-free medium. Medium was collected and HBV virions were precipitated in 6% polyethylene glycol 8000 (PEG 8000). The virus was resuspended in endogenous polymerase reaction buffer (10 mM Tris HCl [pH 7.5], dATP, dGTP, and TTP each at a concentration of 200 µM, 0.1% Triton X-100, 50 mM MgCl₂, and 100 µCi of [-³²P]dCTP) and compound for 16 h. The viral DNA was extracted with phenol and precipitated with ethanol. The DNA was separated by size by electrophoresis through a 1.5% agarose gel, and the quantity of radioactively labeled viral DNA in the sample was determined by Southern blot and phosphorimager analysis.

RNase protection assay. Viral RNA was isolated as described previously (16). To isolate cytoplasmic RNA, HepAD38 cells were induced and grown in the presence of AT-61 or FTC for 4 days. Cells were released from the plates with trypsin and were pelleted by low-speed centrifugation. Total cytoplasmic RNA was extracted from the cell pellets by using the RNeasy Kit as directed by the manufacturer (Qiagen, Santa Clarita, Calif.). To isolate core-associated RNA, HepAD38 cells were induced and grown in the presence of compound for 3 days and lysed with lysis buffer (50 mM Tris HCl [pH 8.0], 10 mM EDTA, 150 mM NaCl, 1% sodium dodecyl sulfate). The immature core particles were precipitated in 6% PEG 8000, and the viral RNA was isolated by using the RNeasy Kit. Viral RNA was quantified by the RNase protection assay with the RPA-II kit as described by the manufacturer (Ambion, Austin, Tex.). Plasmids, pBSCMVNBs (5) and pTRI--Actin (Ambion) were used to make riboprobes for HBV RNA (527 bases in length) and -actin (249 bases in length), respectively. The amount of HBV RNA found in the transfected cell cytoplasm was normalized to the amount of -actin RNA.

Synergy analysis with AT-61 and 3TC. HepAD38 cells were plated into 96-well microtiter plates in DMEM/F-12 medium supplemented with 0.3 µg of tetracycline per ml. Three days later, the medium was removed and replaced with tetracycline-free medium containing various concentrations of AT-61 and 3TC. Three days after the addition of compounds, the medium was removed, the cells were washed with warmed (37°C) PBS, and fresh medium containing the compounds was added. Twenty-four hours later, the medium was collected, clarified by low-speed centrifugation, and assayed for the presence of HBV DNA by dot blot and phosphorimager analysis. Six concentrations of each drug and drug combination were assayed in quadruplicate. For each molar ratio, eight datum points were analyzed for combination indices (CIs) by using the Calcsyn software package (Biosoft, Cambridge, United Kingdom). The CI values at 5, 15, 25, 35, 45, 55, 65, 75, 85, and 95% inhibition were used to determine if the drug combination was inhibiting HBV replication additively (CI = 1), synergistically (CI < 1), or antagonistically (CI > 1). Isobolograms of the data were plotted from the raw data by using the concentrations of compounds that would inhibit virus replication by 50 and 90%.

	RESULTS

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AT-61 selectively inhibits HBV replication. AT-61 belongs to a class of compounds that has an inhibitory effect on HBV replication in HepAD38 cells (22). To extend our studies on the antiviral activity of AT-61, we have tested its ability to inhibit HBV replication in two additional systems, as well as its ability to inhibit a 3TC-resistant strain of HBV, WHBV, DHBV, HIV-1, HSV-1, NDV, and VSV.

View larger version (18K): [in this window] [in a new window]   FIG. 2.   Inhibition of HBV replication by AT-61. HepAD38 cells were induced and treated with various concentrations of 3TC or AT-61. Three days after treatment commenced, the media were removed and replaced with fresh media containing compound. Twenty-four hours later, the media and cells were assayed for the presence of HBV DNA. HBV DNA was quantified by dot blot analysis as described in Materials and Methods. (A) Inhibition of the production of extracellular HBV DNA. Values are based on 18 independent experiments. , 3TC treatment; , AT-61 treatment. (B) Inhibition of the production of intracellular HBV DNA replicative intermediates by AT-61. Values are based on three independent experiments. , 3TC treatment; , AT-61 treatment.

AT-61 does not affect the tetracycline-responsive promoter. In HepAD38 and HepAD79 cells, transcription of HBV pgRNA is driven by a tetracycline-responsive CMV IE promoter. Since it was possible that some or all of the antiviral effect of AT-61 observed in these two cell lines may be caused by an inhibitory effect of the compound on this promoter, we created a cell line, HepAD43, that expresses the -galactosidase gene under the transcriptional control of the tetracycline-responsive CMV IE promoter. HepAD43 cells were induced and maintained in various concentrations of AT-61 or tetracycline for 4 days. AT-61 had no effect on the production of -galactosidase in HepAD43 cells, whereas tetracycline inhibited -galactosidase expression in a dose-responsive manner (Fig. 3).

View larger version (14K): [in this window] [in a new window]   FIG. 3.   Susceptibility of the tetracycline-responsive promoter to AT-61. HepAD43 cells were induced and treated with various concentrations of tetracycline or AT-61. Three days after treatment commenced, the media were removed and replaced with fresh media containing compound. Twenty-four hours later, the cells were collected and assayed for -galactosidase as described in Materials and Methods. , tetracycline treatment; , AT-61 treatment. Values are based on five independent experiments.

AT-61 has low toxicity in cells. In the HepAD38 cell line, a compound that does not specifically inhibit HBV replication but that is cytotoxic or has an antiproliferative effect on the cell could also score as a positive hit in the antiviral assay. To rule out the possibility that the anti-HBV activity of AT-61 was due to an anticellular activity, HepAD38 and HepG2 cells, either in confluent monolayers or in active replication, were grown for 4 to 7 days in the presence of various concentrations of AT-61 and tested for viability and changes in cell number. AT-61 at concentrations as high as 81 µM had no effect on the cell viability or number of HepAD38 and HepG2 cells (Table 2). In addition, we determined by microscopic examination that AT-61 at 81 µM had no cytopathic effect on HepAD43, 2.2.15, Vero, CA51, and MT-2 cells (Table 2).

AT-61 does not affect reverse transcriptase or DNA-dependent DNA polymerase activity. Since the DNA replicative intermediates are produced from the pgRNA by the reverse transcriptase and DNA-dependent DNA polymerase activities of the HBV polymerase, we wanted to determine if the dose-dependent reduction of the replicative intermediates in HepAD38 cells treated with AT-61 was due to the inhibition of either one of these activities by AT-61. To measure the reverse transcriptase activity of the HBV polymerase, a [³²P]TTP incorporation assay was performed with 0.1 µg of baculovirus-produced HBV polymerase in the presence of various concentrations of AT-61. We found that at 81 µM, AT-61 did not affect the ability of the HBV polymerase to incorporate radioactively labeled TTP into a newly synthesized strand of DNA; however, PFA at 1 mM inhibited reverse transcriptase activity, as expected (Table 3).

AT-61 reduces the number of immature core particles containing pgRNA. To determine if AT-61 affected the production of pgRNA or the packaging of pgRNA into immature core particles, we isolated both cytoplasmic and core-associated viral RNA from induced HepAD38 cells that were grown for 3 days in the presence of AT-61 or FTC at concentrations above their EC₉₀s (27 and 1 µM, respectively). Viral RNA was quantified by RNase protection assay and phosphorimager analysis. We found that 27 µM AT-61 reduced the amount of cytoplasmic viral RNA by approximately 50%, whereas FTC did not affect the concentration of cytoplasmic HBV RNA (Fig. 4A). Moreover, AT-61 at a concentration of 27 µM reduced the level of core-associated viral RNA to below the level of detection (Fig. 4B). As expected, FTC had no effect on the amount of HBV RNA associated with immature core particles.

View larger version (14K): [in this window] [in a new window]   FIG. 4.   Susceptibility of HBV RNA production and packaging to AT-61. (A) Production of HBV RNA. HepAD38 cells were induced and treated with AT-61 (27 µM) or FTC (1 µM) for 3 days. Cytoplasmic RNA was isolated as directed by the manufacturer (RNeasy kit; Qiagen). HBV and -actin RNAs were detected and quantified by an RNase protection assay (RPA-II kit; Ambion) with riboprobes for HBV RNA (527 bases) and -actin RNA (249 bases). Autoradiography exposure time was 16 h. Lane 1, uninduced; lane 2, induced; lane 3, induced and treated with AT-61 (27 µM); lane 4, induced and treated with FTC (1 µM). Note that lane 4 was loaded with four times more sample than the other lanes. (B) Packaging of HBV pgRNA. Induced HepAD38 cells were treated with AT-61 (27 µM) or FTC (1 µM) for 3 days. Core-associated viral RNA was isolated from intracellular immature core particles as described in Materials and Methods. Viral RNA was detected and quantified as described above. Autoradiography exposure time was 96 h. Lane 1, uninduced; lane 2, induced; lane 3, induced and treated with AT-61 (27 µM); lane 4, induced and treated with FTC (1 µM).

AT-61 inhibits the accumulation of cccDNA in the nucleus. Since HBV may remain latent in hepatocytes in the form of cccDNA, it is important to determine how any potential inhibitor of HBV replication affects the level of cccDNA in the nucleus of the cell. Since the nucleus of HepAD38 cells accumulates cccDNA only after tetracycline has been removed from the medium, the treatment of HepAD38 cells with a potential inhibitor of HBV replication allows one to determine only the effect of the inhibitor on the accumulation of new cccDNA and not on the fate of existing cccDNA.

View larger version (33K): [in this window] [in a new window]   FIG. 5.   Inhibition of cccDNA by AT-61. HepAD38 cells were induced and treated with AT-61 (27 µM) or FTC (1 µM). Three days after drug treatment started, the media were removed and replaced with fresh media containing compound. Twenty-four hours later, the cells were collected and lysed. The nuclear fractions of the cell lysates were assayed for the presence of cccDNA by Southern blot and phosphorimager analysis as described in Materials and Methods. Lane 1, uninduced; lane 2, induced; lane 3, induced and treated with AT-61 (27 µM); lane 4, induced and treated with FTC (1 µM). rc, relaxed circular.

Synergistic inhibition of HBV replication by AT-61 and 3TC. To determine if AT-61 acts synergistically with 3TC to inhibit HBV replication, several concentrations of AT-61 and 3TC were assayed for their ability to inhibit the release of HBV DNA from HepAD38 cells. AT-61 was tested from 0.1 to 30 µM, whereas 3TC was tested from 0.003 to 1 µM. The raw data were analyzed in two ways. The first method was to create isobolograms of the inhibition data by using the Calcsyn software package (Biosoft). The shift of the curve in the isobologram to the left of the additivity line indicates that these drugs are acting synergistically at their EC₅₀s and the EC₉₀s (Fig. 6A).

View larger version (13K): [in this window] [in a new window]   FIG. 6.   Synergistic inhibition of HBV replication by AT-61 and 3TC. HepAD38 cells were induced and treated with various concentrations of AT-61 and 3TC. Three days after treatment commenced, the media were removed and replaced with fresh media containing compounds. Twenty-four hours later, the media were removed and assayed for the presence of HBV DNA. HBV DNA was quantified by dot blot analysis as described in Materials and Methods. (A) Isobologram of the synergistic action between AT-61 and 3TC. Units for drug concentrations are micromolar. , concentrations required to inhibit HBV replication by 90%; , concentrations required to inhibit HBV replication by 50%. If the experimental line shifts to the left, the compounds are acting synergistically; if it shifts to the right, the compounds are acting antagonistically; and if it does not shift, the compounds are acting additively. (B) CI analysis of the combined antiviral activity of AT-61 and 3TC. A CI value of <1 would be indicative of synergy, a CI value of >1 would be indicative of antagonism, and a CI value of 1 would be indicative of additivity. Molar ratios of AT-61:3TC of 100:1 (), 10:1 (), and 1:1 () were used.

	DISCUSSION

Top Abstract Introduction Materials & Methods Results Discussion References

Currently, IFN is the only compound approved for use for the treatment of HBV-induced disease in the United States. In clinical trials, ganciclovir and 3TC, two nucleoside analogs, have shown much promise for the treatment of chronically infected patients (4, 7-9). However, the problems associated with IFN or nucleoside analog therapy have been well documented (2, 6, 20, 29, 30). Here we describe AT-61, a member of a novel class of potent and selective nonnucleoside inhibitors of HBV replication.

In HepAD38 cells, AT-61 had an EC₅₀ of 1.9 µM and a TC₅₀ of >81 µM, yielding a selectivity index of >43. In addition, the potency of AT-61, as measured by the EC₅₀, was similar when determined in the 2.2.15 cell line and transiently transfected HepG2 cells. However, when potencies were compared by using the EC₉₀s, the potency of AT-61 was five-fold less in the 2.2.15 cells than in HepAD38 cells or transiently transfected HepG2 cells. We have yet to explain the difference in the slope of the dose-response curve for AT-61 as determined in 2.2.15 cells versus HepAD38 cells and transiently transfected HepG2 cells, but it may be due to the fact that the former constitutively produces HBV, whereas in the latter systems, commencement of virus replication coincides with drug treatment.

Unlike most of the nucleoside analogs currently under development for the treatment of HBV infection, AT-61 is highly selective, inhibiting only the replication of human HBV but not the replication of WHBV, DHBV, HIV-1, HSV-1, NDV, or VSV. Since we have tested AT-61 only for antiviral activity against HBV, subtype ayw, we do not know if this compound will display similar potency against the other subtypes. Moreover, we have shown that the antiviral activity of AT-61 was not due to inhibition of the promoter responsible for controlling the transcription of pgRNA, as demonstrated by experiments performed with the HepAD43 cell line. Therefore, the inhibition of HBV replication by AT-61 must occur at an essential step in the replication cycle of human HBV.

With the initial observation that 3TC was quite effective at suppressing HBV DNA levels in the sera of chronically infected individuals and the subsequent appearance of 3TC-resistant variants in some of these patients, it was important to determine how AT-61 acted in combination with 3TC and if the potency of AT-61 would be affected by a mutation responsible for 3TC resistance. We observed that when AT-61 was used in combination with 3TC in HepAD38 cells, these two drugs acted synergistically to inhibit HBV replication. Moreover, experiments performed with the HepAD79 cell line demonstrated that a mutation in the HBV polymerase gene responsible for 3TC resistance did not affect the potency of AT-61.

Although we have yet to elucidate the mechanism of action by which AT-61 inhibits the replication of HBV, we have made great strides in its determination. Since AT-61 is equally effective at inhibiting both the production of the intracellular HBV DNA replicative intermediates and the release of virus, we can conclude that AT-61 does not block the egress of the virus. Likewise, AT-61 was not active in either the endogenous polymerase or the priming and reverse transcription assays, suggesting that the mechanism of action does not involve inhibition of the reverse transcriptase or the DNA-dependent DNA polymerase activities of the HBV polymerase.

Since AT-61 prevented the formation of core particles containing the expected DNA replicative intermediates but did not accomplish this by the inhibition of reverse transcription of pgRNA, it was imperative that we determine whether induced HepAD38 cells contained pgRNA. Western blot analysis and RNase protection assays demonstrated that HepAD38 cells that were grown in the absence of tetracycline but in the presence of AT-61 (27 µM) contained immature core particles which were devoid of viral RNA. RNase protection assays of the total cytoplasmic RNA from induced HepAD38 cells confirmed that HBV pgRNA was produced in these cells; however, its concentration was reduced approximately 50%. These data suggest that AT-61 acts to inhibit HBV replication by interfering with the packaging of pgRNA into the immature core particle.