ANTIMICROBIAL AGENTS AND CHEMOTHERAPY,0066-4804/01/$04.0010 DOI: 10.1128/AAC.45.6.1705–1713.2001

June 2001, p. 1705–1713 Vol. 45, No. 6

Copyright © 2001, American Society for Microbiology. All Rights Reserved.

Cross-Resistance Testing of Antihepadnaviral CompoundsUsing Novel Recombinant Baculoviruses Which Encode

Drug-Resistant Strains of Hepatitis B VirusWILLIAM E. DELANEY IV,1† ROS EDWARDS,1 DANNI COLLEDGE,1 TIM SHAW,1 JOSEPH TORRESI,1

THOMAS G. MILLER,2 HARRIET C. ISOM,2 C. THOMAS BOCK,3 MICHAEL P. MANNS,3

CHRISTIAN TRAUTWEIN,3 AND STEPHEN LOCARNINI1*

Victorian Infectious Diseases Reference Laboratory, North Melbourne, Victoria 3051, Australia1;The Pennsylvania State University College of Medicine, Hershey, Pennsylvania 170332;

and Medical School Hannover, 30625 Hannover, Germany3

Received 9 October 2000/Returned for modification 20 December 2000/Accepted 8 March 2001

Long-term nucleoside analog therapy for hepatitis B virus (HBV)-related disease frequently results in theselection of mutant HBV strains that are resistant to therapy. Molecular studies of such drug-resistantvariants are clearly warranted but have been difficult to do because of the lack of convenient and reliable invitro culture systems for HBV. We previously developed a novel in vitro system for studying HBV replicationthat relies on the use of recombinant baculoviruses to deliver greater than unit length copies of the HBVgenome to HepG2 cells. High levels of HBV replication can be achieved in this system, which has recently beenused to assess the effects of lamivudine on HBV replication and covalently closed circular DNA accumulation.The further development of this novel system and its application to determine the cross-resistance profiles ofdrug-resistant HBV strains are described here. For these studies, novel recombinant HBV baculoviruses whichencoded the L526M, M550I, and L526M M550V drug resistance mutations were generated and used to examinethe effects of these substitutions on viral sensitivity to lamivudine, penciclovir (the active form of famciclovir),and adefovir, three compounds of clinical importance. The following observations were made: (i) the L526Mmutation confers resistance to penciclovir and partial resistance to lamivudine, (ii) the YMDD mutationsM550I and L526M M550V confer high levels of resistance to lamivudine and penciclovir, and (iii) adefovir isactive against each of these mutants. These findings are supported by the limited amount of clinical datacurrently available and confirm the utility of the HBV-baculovirus system as an in vitro tool for the molecularcharacterization of clinically significant HBV strains.

Hepatitis B virus (HBV) is a small, partially double-strandedDNA (dsDNA) virus that causes acute and chronic hepatitis inhumans. More than 350 million people are persistently in-fected with HBV, making it a global public health concern.HBV is a leading cause of death in many parts of the world, aschronically infected individuals are at significantly increasedrisk for developing potentially fatal cirrhosis or hepatocellularcarcinoma (5, 35). Until recently, the only approved therapyfor chronic HBV infection was treatment with the cytokinealpha interferon. Only 30 to 40% of chronically infected indi-viduals with low-level viremia and evidence of active liver dis-ease (generally indicated by high alanine aminotransferase lev-els) respond to interferon with sustained elimination of HBV(46, 68). Apart from its limited efficacy, interferon is expensive,requires administration by subcutaneous injection, and maycause dose-limiting side effects. More effective therapies areneeded to treat the majority of chronically infected individuals,who do not respond to interferon. The recent approval of thedeoxycytidine analog lamivudine has provided an alternativetreatment option (16, 25, 31, 55). Other nucleoside and nucle-otide analogs, including famciclovir and adefovir, have recently

progressed to phase III clinical trials and may soon provideadditional options (9).

Lamivudine is a dideoxycytidine derivative which is activeagainst human immunodeficiency viruses (HIV) (8) and hep-adnaviruses (17, 25). It has several advantages as an antiviralagent, including high oral bioavailability, low toxicity, and po-tent and specific anti-HBV and anti-HIV activities (25). Lami-vudine was shown to suppress HBV replication very effectivelyin vitro (17), as well as in short-term preclinical (59) andclinical trials (16, 25, 31, 55). Lamivudine is phosphorylatedintracellularly by cellular kinases which generate lamivudinetriphosphate, a dCTP analog that competes with dCTP forrecognition by viral DNA polymerases. The addition of lami-vudine to the 39 end of nascent HBV DNA strands causesimmediate chain termination (57), which secondarily preventsthe maturation and release of infectious viral particles, thuslimiting the spread of the virus.

Famciclovir is a prodrug for the deoxyguanosine analog pen-ciclovir, a compound originally developed and approved forthe treatment of herpesvirus infections (65). Penciclovir wassubsequently shown to be an effective inhibitor of hepadnavi-rus replication in vitro (27, 58) and in duck and woodchuckmodels of HBV infection (59). Although clinical trials showedthat famciclovir treatment caused only modest suppression ofHBV replication in chronically infected patients (15a, 36), thistranslated into significant histological improvement (15a). The

* Corresponding author. Mailing address: Victorian Infectious Dis-eases Reference Laboratory (VIDRL), 10 Wreckyn St., North Mel-bourne, Victoria 3051, Australia. Phone: (61 3) 9342 2614. Fax: (61 3)9342 2666. E-mail: stephenlocarnini@compuserve.com.

† Present address: Gilead Sciences, Foster City, CA 94404.

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use of famciclovir to treat chronic HBV infection and as aprophylactic agent during liver transplantation is currently be-ing explored (2, 3, 54), as is its use in combination with lami-vudine and other agents (60).

Adefovir is an acyclic dAMP analog which shows broad-spectrum antiviral activity (12). The potent antihepadnaviralactivity of adefovir was identified initially in in vitro assays (73)and in animal models (21, 45) and later confirmed in phase IIand III clinical trials when patients who were chronically in-fected with HBV were treated with an orally available prodrug,adefovir dipivoxil (19).

Short-term monotherapy with either lamivudine, famciclo-vir, or adefovir causes a rapid decrease in viremia, which usu-ally returns to pretreatment levels after therapy is stopped (16,31, 43, 55). The most likely reason for this relapse is persis-tence of the covalently closed circular (CCC) form of viralDNA (47, 64) which exists as a minichromosome in the nucleiof infected cells (44). Viral mRNAs, which are both necessaryand sufficient to initiate HBV replication in the cell cytoplasm,continue to be transcribed from HBV CCC DNA even duringaggressive antiviral therapy (40, 47). Therapy with antiviraldeoxynucleoside or deoxynucleotide analogs inhibits cytoplas-mic HBV replication but has no direct effect on HBV CCCDNA, which is transcribed by cellular RNA polymerase II (42).For this reason, it is expected that long-term therapy will benecessary to maintain suppression of HBV replication.

Unfortunately, long-term monotherapy with nucleoside an-alogs, including lamivudine and famciclovir, has engenderedanother problem: the frequent emergence of drug-resistantstrains of HBV (1–4, 23, 32, 33, 41, 52–56, 70, 71). Drugresistance arises as a consequence of the inherently low fidelityof HBV replication and the high viral load and turnover whichcharacterize chronic HBV infection. In addition, immune pres-sure generates hypervariability in the major hydrophilic loop ofhepatitis B surface antigen (the main target for neutralizingantibodies) and consequently causes variability in the viralpolymerase, which is encoded by the same genome sequence inan overlapping reading frame (34, 42, 66).

The incidence of lamivudine resistance increases with treat-ment duration; it typically develops in 16 to 32% of patientswithin 1 year of therapy and rises to more than 50% within 2years (16, 32, 33, 55, 61). Sequencing of HBV DNA derivedfrom individuals who exhibit viral breakthrough during nucle-oside analog therapy has revealed a number of specific muta-tions (1–4, 24, 33, 62, 63). The majority of lamivudine-resistantHBV strains contain mutations in the C (catalytic) domain ofthe viral DNA polymerase which map to the sequence thatencodes the YMDD (tyrosine-methionine-aspartate-aspar-tate) motif. Based on homology to other polymerases, thistetrapeptide is believed to be involved in nucleotide bindingand catalysis of the DNA polymerization reaction. BothYVDD (M550V) and YIDD (M550I) changes are frequentlyobserved during lamivudine breakthrough; the former (M550V)almost invariably coexists with a second change (L526M) in theB (template binding) domain of the polymerase (24). TheL526M change and several others located in or near the Bdomain have been clinically correlated with resistance to fam-ciclovir (2, 3, 70). The emergence of such drug-resistant HBVstrains emphasizes the need for new antivirals and therapeuticstrategies. The phenotypes of drug-resistant HBV strains also

warrant further investigation both in vitro and in vivo, sincethey appear to differ substantially from wild-type (wt) HBV inreplication competence (29, 38, 48, 53) and this presumablyaffects pathogenesis in the host.

Characterization of both wt and drug-resistant HBV strainshas been hampered by the virus’ host specificity in vivo andpoor infectivity in vitro. Woodchuck hepatitis virus providesthe best opportunity for studying the selection of drug-resistantvirus populations in vivo. However, the mutations that conferlamivudine resistance on woodchuck hepatitis virus in thewoodchuck are different from those which confer resistance onHBV (37). Therefore, results obtained from woodchuck stud-ies must be interpreted with caution as they may not be pre-dictive for HBV. Whether recently developed transgenic mice(20) can be used for such studies remains to be established.Convenient in vitro cell culture systems are likewise required.Although viral replication may occur following delivery of full-length genomic HBV DNA to hepatic cell lines by transfection,most transfection methods are inefficient, making accuratequantitation of replication-deficient viral mutants extremelydifficult. We have previously reported the development of anovel in vitro system for studying HBV replication which uti-lizes recombinant baculoviruses to deliver replication-compe-tent HBV genomes to HepG2 cells (14). In contrast to othermethods of DNA delivery, baculovirus-mediated transductionof HBV is very efficient and leads to high levels of HBVreplication in the majority of cells. The increased levels ofHBV replication which can be achieved by this method makethe HBV-baculovirus model well suited for a variety of molec-ular studies, including antiviral testing. Recently, we have usedthis system to perform a detailed characterization of the effectsof lamivudine on wt HBV replication and CCC DNA accumu-lation in vitro (15). Based on the utility of the HBV-baculovi-rus system for studying wt virus, we sought to expand themodel to include novel baculovirus vectors to transfer HBVgenomes encoding mutations in the polymerase gene that arecommonly associated with clinical resistance to lamivudine andfamciclovir. The studies reported here therefore had two maingoals, namely, (i) to construct HBV-baculovirus vectors thatencode the L526M, M550I, and L526M M550V polymerasemutations and (ii) to use these vectors to compare the sensi-tivities of wt and mutant HBV to three antivirals of currentclinical importance: lamivudine, penciclovir (the active form offamciclovir), and adefovir.

MATERIALS AND METHODS

Cell culture. Sf21 insect cells (Invitrogen, Groningen, The Netherlands) weremaintained in a nonhumidified incubator at 28°C without CO2 and were grownin Grace’s insect medium (Gibco BRL Life Technologies, Grand Island, N.Y.)containing yeastolate, lactalbumin hydrolysate, and 10% heat-inactivated fetalbovine serum (15). HepG2 cells were maintained in humidified 37°C incubatorsat 5% CO2 and grown in minimal essential medium supplemented with 10%heat-inactivated fetal bovine serum.

HBV infectious clone and lamivudine-resistant mutants. A wt HBV clone of1.28 times genome length (genotype A, subtype adw2) previously shown to beinfectious in vitro was excised from the plasmid pHBV1.2 using the restrictionenzymes PvuII and BamHI and ligated into the baculovirus transfer vectorpBlueBac4.5 (Invitrogen) using SmaI and BamHI restriction sites (6). The re-sulting plasmid, pBBHBV1.28 (Fig. 1), was used to sequence both strands of theHBV construct to ensure that no sequences outside the published chronic-carrierconsensus sequence for wt HBV were present in the clone (3). Lamivudineresistance mutations were introduced into the wt pBSHBV1.28 plasmid by site-

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directed mutagenesis using the Quick change kit (Stratagene, La Jolla, Calif.).Introduced mutations caused the following amino acid changes in the polymer-ase protein: (i) a change from leucine to methionine at codon 526 (L526M) in theB domain, (ii) a change from methionine to isoleucine at codon 550 in the Cdomain (M550I), and (iii) both an L526M change and a change from methionineto valine at codon 550 (L526M M550V). Figure 2 illustrates these changes, aswell as the concomitant changes in the envelope protein (34).

The following primers were used during site-directed mutagenesis. TheL526M mutant was created using 59-CTACTAAACTGCGCCATGAGAAACGGACTGAGG-39 and 59-CCTCAGTCCGTTTCTCATGGCTCAGTTTACTAG-39, which changes codon 526 from TTG to ATG; M550I was created using59-GGCTTTCAGCTATATCGATATAGTGGTATTGGGGG-39 and 59-CCCCCAATACCACATCATCGATATAGCTGAAAGCC-39, which changes codon550 from ATG to ATC; and L526M M550V was created using 59-GGCTTTCAGCTATGTGGATGATGTGGTATTGGG-39 and 59-CCCAATACCACATCATCCACATAGCTGAAAGC-39 to mutate codon 550 from ATG to GTG in aplasmid in which the mutation responsible for the L526M change was alreadypresent. Following successful site-directed mutagenesis, 2.1-kb restriction frag-ments containing the resistance mutations were excised from the appropriateplasmids using the enzymes NsiI and BstX and cloned into the same sites in thebaculovirus transfer plasmid pBBHBV1.28. The polymerase regions of eachbaculovirus transfer plasmid were then sequenced to confirm that they harboredthe desired mutations.

Generation of recombinant baculovirus. HBV-baculovirus recombinants weregenerated by cotransfection of pBBHBV1.28 containing either wt HBV or therespective lamivudine- and famciclovir-resistant HBV and linear Bac-N-BlueAutographa californica nuclear polyhedrosis virus DNA (Invitrogen) into Sf21cells. Recombinant baculoviruses were isolated from the cotransfection super-natant by plaque purification in Sf21 cells. Baculovirus from individual plaqueswas amplified by inoculation into 100-mm-diameter dishes of Sf21 cells. Oneweek postinoculation, baculovirus was concentrated from the medium of infectedcells by centrifugation (14, 15) and viral DNA was extracted by incubation insodium dodecyl sulfate buffer with proteinase K (Roche Diagnostics AustraliaPty. Ltd., Rose Park, S.A., Australia), phenol-chloroform extraction, and ethanolprecipitation as previously described (50). DNA purified from recombinant viruswas analyzed by PCR amplification of the HBV polymerase region using theforward primer PCRBACF2 (59-ATTCTTTGTCCATTGATCGAAGCGAG-39) and the reverse primer OS1 (59-GCCTCATTTTGTGGGTCACCATA-39).PCR products were purified using the JetQuick DNA purification kit (Strat-agene), and both strands of the product were then sequenced using the primersTTS2 (59-TGCACGATTCCTGCTCAA-39 [plus strand]) and OS2 (59-TCTCTGACATACTTTCCAAT-39 [minus strand]) to ensure that the desired resistance

mutations were retained in each recombinant. Restriction enzyme digestion,agarose gel electrophoresis, and Southern blotting using a 32P-labeled HBVprobe were also performed as described previously (14) to verify that eachHBV-baculovirus recombinant contained an entire HBV insert.

Preparative baculovirus amplification and purification. Individual baculovirusisolates containing either wt HBV or the L526M, M550I, or L526M M550Vmutant were amplified by infecting suspension cultures of Sf21 cells in log phaseat a multiplicity of infection (MOI) of 0.5 PFU per cell. Infections were allowedto proceed until most of the cells showed visible signs of infection (after approx-imately 4 to 5 days). Baculovirus particles were concentrated and purified frominfected Sf21 medium as described previously (7, 50). Purified virus was titratedin quadruplicate in Sf21 cells by endpoint dilution (50). Baculovirus DNA waspurified from a small aliquot of each high-titer stock, and PCR and subsequentDNA sequencing were repeated as described above to confirm that each stockretained the intended mutations.

Baculovirus transduction and drug treatments. HepG2 cells were seeded intosix-well plates at a density of 2 3 106 to 3 3 106 cells per well and weretransduced with 50 PFU of HBV-baculovirus per cell 16 h postseeding as pre-viously described (14). HepG2 cells were fed medium containing the indicatedconcentrations of antiviral drugs immediately after transduction. Each antiviralwas tested against wt HBV and the L526M, M550I, and L526M M550V mutantsin parallel using the same stocks of drug-containing medium to ensure thatdifferent recombinants were exposed to identical drug concentrations. Trans-duced HepG2 cells were fed every other day with fresh drug for a total of 1 week,with the last drug treatment beginning 24 h prior to the analysis of HBV DNA.

Analysis of HBV DNA. Intracellular HBV replicative intermediates were iso-lated from cytoplasmic core particles essentially as previously described (22).Briefly, cell monolayers were lysed by incubation at 4°C for 20 min in phosphate-buffered saline containing 0.5% Nonidet P-40. Cell lysates were transferred tomicrocentrifuge tubes and spun for 5 min to pellet nuclei. Supernatants weretransferred to clean tubes, adjusted to 10 mM MgCl2, and incubated with 10 Uof DNase I (Roche Diagnostics) at 37°C. After 1 h, the digestion mixture was

FIG. 1. Diagrammatic representation of the HBV construct used togenerate HBV-baculovirus recombinants. HBV-baculovirus recombi-nants were constructed from a 1.28 times unit length HBV genome ofgenotype A, serotype adw2. This HBV clone has been fully sequencedand contains no nucleotides which lie outside a published wt consensussequence (3). Note that there is only one copy each of the precore,core, pre-S1–pre-S2–S, polymerase, and X open reading frames withinthe construct. The positions of the core (C), pre-S, S, and X promoters,enhancer I (EI), and enhancer II (EII), as well as the baculoviruspolyhedron promoter (PH), are indicated.

FIG. 2. Polymerase mutations in drug-resistant HBV. Drug resis-tance mutations in the 1.28 times unit length wt HBV construct (Fig.1) were generated by site-directed mutagenesis. The following aminoacid changes in the polymerase gene product resulted: (i) an L-to-Mamino acid substitution at position 526 (L526M) in domain B of thepolymerase, (ii) an M-to-I amino acid substitution at position 550(M550I) in domain C of the polymerase, and (iii) a double mutationwith the L526M amino acid substitution and a second, M-to-V, aminoacid substitution at position 550 (L526M M550V). The M550I andM550V mutations also resulted in changes to the deduced amino acidsequence of the surface antigen gene, as indicated. TP, terminal pro-tein; R., reverse; ORF, open reading frame; HBsAg, hepatitis B sur-face antigen.

VOL. 45, 2001 CROSS-RESISTANCE OF DRUG-RESISTANT HBV 1707

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adjusted to 50 mM EDTA–30 mM Tris–0.5% sodium dodecyl sulfate–500 mg ofproteinase K per ml, and incubation at 37°C was continued for an additional 4 h.Sequential extractions with Tris-saturated phenol and chloroform were per-formed, and nucleic acids were then recovered by precipitation with 1 volume ofisopropanol. Pellets containing viral DNA were redissolved in 10 mM Tris–10mM EDTA and digested with 20 U of DNase-free RNase (Roche Diagnostics)before analysis by electrophoresis and Southern blotting as described above.

Quantification of HBV DNA and data analysis. Intracellular HBV DNAreplicative intermediates were quantified by densitometery of suitable autora-diograms using a Bio-Rad GS-670 scanning densitometer and Molecular Analy-ist software (Bio-Rad). Densitometric data were analyzed using TableCurve2D,a graphics-statistics software package from Jandel Scientific (San Rafael, Calif.)as described previously (10, 11). Dose-dependent inhibition of the accumulationof intracellular HBV replicative intermediates, when it occurred, could be ex-pressed mathematically in nearly all cases by the following logistic dose-responseequation: y 5 a/(1 1 [x/b]c), which describes a sigmoid curve where y is theamount of HBV DNA detected compared to that in untreated controls (definedas 100%) and x is the drug concentration; a represents the curve’s amplitude, bis the x value at its transition center, and c is a parameter which defines itstransition width (Fig. 3). Values and confidence intervals for each parameterwere estimated from individual dose-response equations. When dose-dependentinhibition of intracellular HBV accumulation was incomplete over the range ofdrug concentrations used, an alternative procedure was adopted as described infootnote d to Table 1.

Nucleotide sequence accession number. The sequence of pBBHBV1.28 hasbeen submitted to the GenBank database and assigned accession numberAF305422.

RESULTS

Construction of recombinant baculoviruses. Recombinantbaculovirus containing wt HBV or the L526M, M550I, or

L526M M550V HBV variant was successfully generated andamplified in Sf21 cells. Southern blotting was used to confirmthat baculovirus stocks contained complete HBV constructs,the sequences of which were verified after PCR amplificationof both strands (data not shown). No mutations (other thanthose intentionally introduced by site-directed mutagenesis)were detected at any stage during baculovirus amplification,indicating that the baculoviruses maintained the sequences andstructural integrity of each HBV insert.

Sensitivity to lamivudine. To confirm that the point muta-tions which created the L526M, M550I, and L526M M550Vpolymerase mutants conferred phenotypic lamivudine resis-tance on the genotype A clone of HBV used in these experi-ments, the abilities of these viruses to replicate in the presenceof increasing concentrations of lamivudine were examined.HepG2 cells were transduced with baculovirus which con-tained either wt or mutant HBV at an MOI of 50 PFU per cell.The cells were then exposed continuously for 7 days to 0, 0.001,0.01, 0.1 1.0, or 50 mM lamivudine. HBV replicative interme-diates were extracted from the cytoplasm of transduced cells atthe end of the 7-day treatment period before analysis by South-ern blotting and autoradiography. Double-stranded replicativeintermediates were quantitated by densitometry of autoradio-graphs, and dose-response curves were fitted to the resultingdata with the aid of TableCurve2D (Fig. 3 and 4). Drug con-centrations which were required to reduce HBV replication by50% (IC50s) were estimated from the equations which de-scribed each curve (Table 1). The results verified that wt HBVwas extremely sensitive to lamivudine, whose IC50 was approx-imately 0.01 mM, as estimated from intracellular HBV dsDNA.In this experiment, the single L526M change conferred partialresistance to lamivudine, as demonstrated by a threefold in-crease in the estimated IC50. Both the M550I and L526MM550V changes conferred much greater resistance to lamivu-dine, as the altered dose-response plots (Fig. 4) and the in-creases in the estimated IC50s indicate. Table 1 presents theresults of a single experiment in which the levels of inhibitionof wt HBV and all of the mutants by all three analogs werecompared in parallel. For logistical reasons, replicate experi-ments compared either the sensitivities of wt and mutant HBVto individual drugs or the sensitivity of wt HBV or specificmutants to all of the drugs. For three consecutive experiments,the estimated IC50 of lamivudine as an inhibitor of wt HBVreplication was 0.10 6 0.10 mM (mean 6 standard deviation;range, 0.01 to 0.20 mM); in these experiments, 1 mM lamivu-dine inhibited wt HBV replication by 96.4% 6 4.1% (mean 6standard deviation; range, 91.8 to 99.6%. Despite interexperi-mental variation in the IC50s of the individual drugs, intraex-perimental variation was less than 20%. More importantly, theranking of IC50s was reproducible, showing sensitivity to lami-vudine consistently decreasing in the order wt . L526M .M550I . L526M M550V by factors of ,10 (L526M), .20(M550I), and .100 (L526M M550V), respectively.

Sensitivity to penciclovir. The sensitivities of wt and mutantHBV to penciclovir were compared next. Initial experimentsindicated that penciclovir was a relatively weak inhibitor of wtHBV replication in HepG2 cells and that it failed to inhibit wtHBV replication at concentrations as high as 500 mM in oneHepG2 subclone (data not shown). However, it was possible toreproduce dose-response relationships for wt HBV using high

FIG. 3. Inhibition of wt HBV replication by lamivudine, adefovir,or penciclovir. Double-stranded HBV replicative intermediates werequantified by densitometry of appropriately exposed autoradiographsof Southern blots (see Fig. 4 to 6). Image densities were expressed aspercentages of the mean density of untreated controls (defined as100%). The resulting data, which describe the dose-dependent effectsof drugs on wt HBV replication, were fitted to logistic dose-responseequations with the aid of TableCurve2D software. The 95% confidencelimits appear on either side of the fitted dose-response curves; theequation y 5 a/(1 1 [x/b]c) accurately describes the data. With theexceptions noted in footnote d to Table 1 and the legend to Fig. 5, alldata sets were also accurately described by this equation. As shown,the lowest concentrations of lamivudine and adefovir (0.001 and 0.01mM, respectively) appeared to stimulate wt HBV replication. This isalso reflected by .100 values for the a (amplitude) parameter listed inTable 1. The cause(s) of this phenomenon is not known but mayinclude (i) allosteric effects of (d)NTP analogs on intracellular nucle-otide pools and/or (ii) interference with virus secretion, which mightresult in accumulation of intracellular replicative intermediates.

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concentrations of penciclovir (Fig. 3 and 5). We performedcross-resistance testing by treating HepG2 cells transducedwith 50 PFU of baculovirus which encoded wt or drug-resistantHBV using penciclovir concentrations of 0, 1, 10, 50, 100, and500 mM. HBV replicative intermediates were extracted andassayed after 7 days of exposure to penciclovir (Fig. 5). Den-sitometric data were generated and analyzed as describedabove. Due to the high degree of penciclovir resistance shownby all of the mutants, it was not possible to fit logistic dose-response curves to the corresponding sets of data (see Table 1,footnote d). These results indicate that wt HBV replication wasinhibited only by relatively high concentrations of penciclovir,for which an IC50 of approximately 11.5 mM (95% confidencelimits, 8.2 to 16.2 mM) was estimated for the dsDNA species.The single L526M change, which has previously been associ-ated with clinical HBV breakthrough during famciclovir ther-apy (2, 3, 70), conferred significant resistance to penciclovir invitro, as indicated by the approximately almost 1-log increasein the estimated IC50. The M550I and L526M M550V mutantswere also penciclovir resistant. Interestingly, the degree ofresistance exhibited by both was greater than that shown by theL526M mutant, suggesting that mutations which affect nucle-otide binding (M550I and M550V) and template binding(L526M) may contribute independently to the resistance phe-notype (Table 1).

Sensitivity to adefovir. The sensitivities of wt and mutantHBV to adefovir, the active metabolite of the prodrug adefovirdipivoxil, were compared next. HepG2 cells transduced as de-scribed above with baculovirus which contained either wt or

mutant HBV were exposed for 7 days to 0, 0.01, 0.1, 1, 10, or100 mM adefovir. Intracellular HBV replicative intermediateswere extracted and analyzed as described above, with the re-sults shown in Fig. 3 and 6. In contrast to the results obtainedfollowing exposure to lamivudine or penciclovir, exposure toadefovir produced a dose-dependent inhibition of replicationof both wt and mutant HBV. Dose-response curves (Fig. 6)and IC50s derived from them (Table 1) indicated that wt HBVwas sensitive to adefovir, with an estimated IC50 of 0.08 mM(95% confidence limits, 0.06 to 0.10 mM). The correspondingIC50s of adefovir as an inhibitor of lamivudine-resistant mu-tants increased less than fourfold, suggesting that the L526M,M550I, and L526M M550V changes do not confer significantcross-resistance to adefovir (Table 1).

DISCUSSION

Many aspects of HBV research, including the investigationof potential antivirals, have been impeded by the lack of con-venient and reliable in vitro culture systems that are capable ofsupporting complete cycles of HBV replication. High levels ofin vitro HBV replication can be achieved using the recentlydescribed recombinant HBV-baculovirus system (14, 15),which makes it suitable for the study of HBV mutants thatreplicate poorly in other systems (29, 38, 48, 53). The higherlevel of replication and the ability to control the viral MOIfacilitate otherwise difficult comparisons of the sensitivities ofwt and mutant HBV to antiviral agents.

Data obtained in the present study confirm and extend con-

TABLE 1. Sensitivity of replication of wt and mutant HBV strains to inhibition by lamivudine, penciclovir, and adefovira

Drug and HBV strain

Equation parameters

Correlation (r2)

Sensitivity parameters

a b c % Inhibitionat 1 mM

IC50(mM)

Resistancefactorb

Lamividinewt 185 6 8 0.004 6 0.001 1.10 6 0.07 1.0 99.6 0.01 1L526M 102 6 18 0.027 6 0.04 0.51 6 0.27 0.89 85.8 0.03 3M550I 186 6 48 0.35 6 0.64 0.42 6 0.26 0.93 26.7 3.8 380L526M M550V 127 6 10 41.5 6 31 0.60 6 0.4 0.90 (215)c 86.2 8,620

Penciclovirwt 101 6 2 11.2 6 0.95 1.0 6 0.06 1.0 7.0 11.5 1L526M 100 6 7 103 6 12 4.8 6 7.3 0.93 (23.0) 103 9M550I 124 6 6 0.0007 —d 0.73 (224) 1,217e 106L526M M550V 111 6 7 0.002 —d 0.86 (210) 370 32

Adefovirwt 170 6 16 0.025 6 0.001 0.80 6 0.07 1.0 91.6 0.08 1L526M 203 6 49 0.014 6 0.01 0.61 6 0.08 1.0 86.2 0.09 1M550I 159 6 24 0.078 6 0.05 0.57 6 0.1 1.0 70.0 0.31 4L526M M550V 155 6 8 0.039 6 0.009 0.53 6 0.02 1.0 77.0 0.16 2

a The results presented here are derived from a single representative experiment in which all three analogs were assayed as inhibitors of all of the HBV strains tested(wt and three drug-resistant variants). The image densities of the HBV dsDNA bands in each autoradiograph were compared by densitometry, and the results wereexpressed as a percentage of the mean density of untreated controls for each set of assays.

b The resistance factor is the factor (to the nearest integer) by which the IC50 estimated for the mutant differs from the corresponding estimate for the wt. It wascalculated by dividing the IC50 for the mutant by IC50 for the wt. In replicate experiments, the ranking of resistance to each drug was consistent with the ranking shownhere.

c Negative values represent stimulation of replication relative to controls.d Logistic dose-response equations (10) did not accurately describe these data sets; instead, the a and b parameters are for a single exponential-decay equation, y 5

a(2bx). The values shown are means or means 6 standard errors. In some cases, low concentrations of inhibitor stimulated HBV replication; this is reflected by a valuesof .100% (compare tabulated a values with graphical illustrations in Fig. 3 to 6). The percentage of inhibition that occurred at a drug concentration of 1 mM wasestimated from each fitted plot.

e Extrapolated value.

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clusions from previous studies which used alternative assaysystems; in addition, they demonstrate the utility of the HBV-baculovirus system as a tool for the in vitro study of clinicallyrelevant drug-resistant variants of HBV. In summary, theyshow (i) that the L526M variant is resistant to penciclovirand partially resistant to lamivudine, (ii) that the M550I andL526M M550I variants are resistant to both lamivudine andpenciclovir, and (iii) that all of the variants remain sensitive toadefovir.

The differences in lamivudine sensitivity among the HBVmutants utilized in this study (Fig. 3 and 4 and Table 1) suggestthat each point mutation contributes independently to the re-sistance phenotype. The single L526M change in domain B ofthe HBV polymerase increased lamivudine resistance less than10-fold, while both the M550I and L526M M550V changesconferred much greater resistance (greater than 20- and 100-fold, respectively), consistent with previous observations (18,29, 30, 48).

Previous studies indicated that the anti-HBV activity of pen-ciclovir is more variable than that of lamivudine. Penciclovirwas initially found to be a potent inhibitor of wt HBV repli-cation in one clone of HepG2.2.15 cells, with a reported IC50

of 0.6 mM (27), but subsequent reports noted either lack ofactivity or IC50s which are 1 or 2 orders of magnitude greater(18, 48, 53, 72). The reason(s) for the differences is not clearbut may include poor and variable phosphorylation of penci-clovir, which seems to characterize most clones of hepatic celllines (T. Shaw and G. Civitio, unpublished data) and/or highintracellular concentrations of competing dGTP.

High concentrations of penciclovir produced reproducibledose-dependent inhibition of wt HBV replication (as reflectedby concentration-dependent reductions in the accumulation ofthe intracellular HBV dsDNA replicative intermediate) in theHepG2 clone used here. This made it possible to demonstratethat the L526M change alone was sufficient to significantlyincrease (almost 10-fold) penciclovir resistance. Both the dual(L526M M550V) and M550I mutants were also found to bepenciclovir resistant in this system. These results are consistentwith recent conclusions based on alternative assays (18, 55).While our observation that M550I in isolation is sufficient toconfer significant penciclovir resistance may seem inconsistentwith a report (based on data obtained using HepAD38 andHepAD79 cells which express wt or M550V mutant HBV,respectively) that M550V in isolation is not (72), the latterprobably has little relevance, since M550V has not been foundin clinical HBV isolates in the absence of L526M.

Our results suggest that development of the M550I andL526M M550V resistance mutations during lamivudine ther-apy would cause the failure of subsequent famciclovir therapy,a conclusion which is supported by the limited amount ofclinical data published to date (41, 67). Conversely, althoughthe L526M mutation which emerges during famciclovir treat-ment confers only partial resistance to lamivudine, it remainspossible that preselection of L526M as a result of prior expo-sure to famciclovir may predispose to failure of subsequentlamivudine treatment. Although L526M alone appears insuf-ficient to drive viral breakthrough during lamivudine treat-ment, its preexistence may affect replication fidelity in a waywhich increases the probability and/or rate of acquisition of anadditional mutation(s) (such as M550V) which confers greater

lamivudine resistance. This hypothesis is supported by at leasttwo reports, which describe the rapid replacement of theL526M mutant by an L526M M550V mutant after lamivudinereplaced or was added to famciclovir therapy (56, 62).

Adefovir was active against all three of the HBV mutantsused in the present study, in agreement with previous work,which has used either cell-based or enzyme-based assays (48,69, 70, 72). Differences between the adefovir sensitivities of wtand mutant HBVs were not significant (fourfold or less). Thisindicates (i) that the lamivudine and/or famciclovir resistanceconferred by the L526M, M550I, and L526M M550V polymer-ase changes does not confer cross-resistance to adefovir and

FIG. 4. Effects of lamivudine on wt and drug-resistant HBV.HepG2 cells were transduced at 50 PFU/cell with HBV-baculoviruswhich encoded either wt or mutant (L526M, M550I, or L526MM550V) polymerase. Immediately after transduction, cultures weretreated with the indicated concentrations of lamivudine (3TC). After 7days of treatment, HBV replicative intermediates were extracted fromcell lysates and analyzed by Southern blotting and autoradiography.(A) autoradiographs of Southern blots. After equivalent exposuretimes, image densities in (drug-free) control lanes were such as tosuggest that replication fitness decreased in the order wt . L526M .L526M M550V . M550I, assuming that transduction efficiencies wereequal and that the polymerase changes did not differentially affect virussecretion. The autoradiographs shown were exposed for different timesso that the image density in each control lane is approximately thesame. (B) results of graphical analysis of data from panel A. rc, relaxedcircular.

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(ii) that HBV mutants which are selected for by lamivudine arenot hypersensitive to adefovir, at least in the HBV-baculovirusassay system. Adefovir hypersensitivity is a phenomenon thathas been observed for the lamivudine-resistant M184V HIVmutant, which is analogous to the M550V single mutant ofHBV (39). These observations add to the already convincingevidence indicating that adefovir is active against lamivudine-resistant HBV strains in vitro, suggesting that adefovir dipiv-oxil treatment would be appropriate for patients in whomlamivudine therapy has failed. Indeed, two recent reports havedescribed the successful use of adefovir to rescue patients whoexperienced relapses after failure of lamivudine treatment dueto the emergence of resistant HBV (51, 52). While adefovirdipivoxil is an effective broad-spectrum antiviral that is poten-tially useful against lamivudine-resistant HBV, its clinical usehas been associated with nephrotoxicity during trials against

HIV. However, doses of adefovir which are sufficient to inhibitHBV replication in vivo (19) are lower than the doses usedduring clinical trials against HIV (13, 26). Clinical trials whichare currently under way will determine whether lower doses ofadefovir are sufficient to block HBV replication without caus-ing nephrotoxicity.

Although lamivudine, famciclovir, and adefovir have provedto be safe and efficacious suppressors of in vivo HBV replica-tion in the short term, there is little, if any, evidence that theyaffect HBV CCC DNA which persists in the nuclei of infectedcells. Elimination of viral CCC DNA is likely to require verylong-term therapy—probably longer than several half-lives ofthe infected cell population (9, 37, 40, 60). To achieve success-ful long-term control of HBV replication, additional antiviralagents will be required; fortunately, several potentially usefulagents, some of which have progressed to clinical trials, are

FIG. 5. Effects of penciclovir on wt and drug-resistant HBV.HepG2 cells were transduced at 50 PFU/cell with HBV-baculoviruswhich encoded either wt or mutant (L526M, M550I, or L526MM550V) polymerase. Immediately after transduction, cultures weretreated with the indicated concentrations of penciclovir (PCV). After7 days of treatment, HBV replicative intermediates were extractedfrom cell lysates and analyzed by Southern blotting and autoradiogra-phy. (A) autoradiographs of Southern blots (exposures were chosen togive approximately equal image densities, as noted in the legend to Fig.4). (B) results of graphical analysis of data from panel A. As noted inTable 1, footnote d, it was not possible to fit logistic dose-responsecurves to data for the M550I single and L526M M550V dual mutants.rc, relaxed circular.

FIG. 6. Effects of adefovir on wt and drug-resistant HBV. HepG2cells were transduced at 50 PFU/cell with HBV-baculovirus whichencoded either wt or mutant (L526M, M550I, or L526M M550V)polymerase. Immediately after transduction, cultures were treated withthe indicated concentrations of adefovir (PMEA). After 7 days oftreatment, HBV replicative intermediates were extracted from celllysates and analyzed by Southern blotting and autoradiography. (A)autoradiographs of Southern blots (exposures were chosen to giveapproximately equal image densities, as in Fig. 4 and 5). (B) results ofgraphical analysis of data from panel A. rc, relaxed circular.

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currently being developed (9, 60). In the future, these willprobably be used in combination(s), rather than alone, to min-imize the risk of development of viral resistance (10, 11, 28,60). Unfortunately, the fact that HBV resistance to adefovirhas not yet been observed is no guarantee that it will not occur.

The results presented here confirm and extend previousobservations and further validate the usefulness of the recom-binant HBV-baculovirus system, which offers several advan-tages, most notably the capacity to support high levels of rep-lication of both wt and mutant HBV. We anticipate that it willbecome a valuable asset for research into HBV and that therecombinants described here and others like them will be in-valuable not only for defining patterns of drug resistance butalso for testing the efficacy of different drug combinations. Thesystem also has potential for investigation of effects of antiviralagents on HBV CCC DNA amplification and maintenance,since this key replicative intermediate is amplified to readilydetectable levels (14, 15). Data thus obtained should contrib-ute to the rational design of new therapeutic strategies for themanagement of chronic HBV infection, especially in cases inwhich drug-resistant HBV strains are implicated.

ACKNOWLEDGMENTS

This work was partially supported by grants from the NationalHealth and Medical Research Council of Australia (to S.L.) and theNational Institutes of Health (CA73045 and CA23931 to H.C.I.). T.S.was partly supported by the Australian Government under a syndi-cated research and development scheme. Klaus Esser of SmithKlineBeecham, King of Prussia, Pa., kindly arranged the supply of lamivu-dine and penciclovir. Adefovir was a generous gift from Craig Gibbs ofGilead Sciences, Foster City, Calif.

We thank Scott Bowden for reviewing the manuscript.

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