10.1128/JVI.75.10.4771-4779.2001.

2001, 75(10):4771. DOI:J. Virol. ArnoldWestland, Craig S. Gibbs, Stefan G. Sarafianos and Edward Kalyan Das, Xiaofeng Xiong, Huiling Yang, Christopher E. Lamivudine (3TC) and Emtricitabine (FTC)Hepatitis B Virus Polymerase Resistance toCharacterization Reveal the Mechanism of Molecular Modeling and Biochemical

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JOURNAL OF VIROLOGY,0022-538X/01/$04.0010 DOI: 10.1128/JVI.75.10.4771–4779.2001

May 2001, p. 4771–4779 Vol. 75, No. 10

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

Molecular Modeling and Biochemical Characterization Revealthe Mechanism of Hepatitis B Virus Polymerase Resistance

to Lamivudine (3TC) and Emtricitabine (FTC)KALYAN DAS,1 XIAOFENG XIONG,2 HUILING YANG,2 CHRISTOPHER E. WESTLAND,2

CRAIG S. GIBBS,2 STEFAN G. SARAFIANOS,1 AND EDWARD ARNOLD1*

Center for Advanced Biotechnology and Medicine, Department of Chemistry, Rutgers University,Piscataway, New Jersey,1 and Gilead Sciences, Foster City, California2

Received 8 November 2000/Accepted 19 February 2001

Success in treating hepatitis B virus (HBV) infection with nucleoside analog drugs like lamivudine is limitedby the emergence of drug-resistant viral strains upon prolonged therapy. The predominant lamivudine resis-tance mutations in HBV-infected patients are Met552IIe and Met552Val (Met552Ile/Val), frequently in asso-ciation with a second mutation, Leu528Met. The effects of Leu528Met, Met552Ile, and Met552Val mutationson the binding of HBV polymerase inhibitors and the natural substrate dCTP were evaluated using an in vitroHBV polymerase assay. Susceptibility to lamivudine triphosphate (3TCTP), emtricitabine triphosphate(FTCTP), adefovir diphosphate, penciclovir triphosphate, and lobucavir triphosphate was assessed by deter-mination of inhibition constants (Ki). Recognition of the natural substrate, dCTP, was assessed by determi-nation of Km values. The results from the in vitro studies were as follows: (i) dCTP substrate binding waslargely unaffected by the mutations, with Km changing moderately, only in a range of 0.6 to 2.6-fold; (ii) Kis for3TCTP and FTCTP against Met552Ile/Val mutant HBV polymerases were increased 8- to 30-fold; and (iii) theLeu528Met mutation had a modest effect on direct binding of these b-L-oxathiolane ring-containing nucleotideanalogs. A three-dimensional homology model of the catalytic core of HBV polymerase was constructed viaextrapolation from retroviral reverse transcriptase structures. Molecular modeling studies using the HBVpolymerase homology model suggested that steric hindrance between the mutant amino acid side chain andlamivudine or emtricitabine could account for the resistance phenotype. Specifically, steric conflict between theCg2-methyl group of Ile or Val at position 552 in HBV polymerase and the sulfur atom in the oxathiolane ring(common to both b-L-nucleoside analogs lamivudine and emtricitabine) is proposed to account for theresistance observed upon Met552Ile/Val mutation. The effects of the Leu528Met mutation, which also occursnear the HBV polymerase active site, appeared to be less direct, potentially involving rearrangement of thedeoxynucleoside triphosphate-binding pocket residues. These modeling results suggest that nucleotide analogsthat are b-D-enantiomers, that have the sulfur replaced by a smaller atom, or that have modified or acyclic ringsystems may retain activity against lamivudine-resistant mutants, consistent with the observed susceptibilityof these mutants to adefovir, lobucavir, and penciclovir in vitro and adefovir in vivo.

Hepatitis B virus (HBV) infection is among the top 10 viralinfections, affecting an estimated 300 million people worldwideand over 1.5 million in the United States alone (10, 24).Chronic HBV infection can lead to cirrhosis, hepatocellularcarcinoma, and liver failure. Treatment of chronically HBV-infected patients with alpha interferon (28) is limited by sideeffects, incomplete efficacy, restriction to patients with com-pensated disease, and the requirement for parenteral admin-istration (8, 37). HBV, a hepadnavirus, replicates through anintermediate reverse transcription step carried out by the viralpolymerase (19, 33), which is functionally and structurally re-lated to human immunodeficiency virus (HIV) reverse tran-scriptase (RT). Some of the nucleoside analogs developed totreat HIV infection are highly potent against HBV infection (4,5) at concentrations below cytotoxic thresholds. Treatment ofchronically HBV-infected patients with nucleoside or nucleo-tide analogs (Fig. 1), like lamivudine (3TC), emtricitabine(FTC), famciclovir (the prodrug of penciclovir [PCV]), adefo-

vir dipivoxil (ADV [also called PMEA]), and lobucavir (LBV),leads to significant decreases in serum virus levels (26). Treat-ment with the nucleoside or nucleotide analogs has shownimmediate clinical benefits such as reduced viral load, suppres-sion of progression of liver disease, and induction of immuno-logical clearance or seroconversion (6, 18). Drug-resistantstrains of HBV containing specific polymerase mutationsemerge upon prolonged 3TC treatment (14, 23; H. Fontaine,V. Thiers, and S. Pol, Letter, Ann. Intern. Med. 131:716–717,1999) and are the primary cause of treatment failure. Treat-ment of HBV-infected patients with 3TC in phase III clinicalstudies showed a sequential increase in appearance of geno-typic resistance in HBV patients: 24% in the first year, 42% inthe second year, 52% in the third year, and 67% in the fourthyear (N. W. Y. Leung, C. L. Lai, J. Dienstag, G. Schiff,J. Heathcote, M. Atkins, C. Marr, and W. C. Maddrey, pre-sented at the Management of Hepatitis B Meeting, 8 to 10September 2000).

As with other nucleotide polymerases, the triphosphates ofthe nucleotide substrates or their analog inhibitors are thecatalytically active forms for polymerization by HBV polymer-ase, and the polymerization reaction has been shown to be

* Corresponding author. Mailing address: CABM and Rutgers Uni-versity, 679 Hoes Ln., Piscataway, NJ 08854. Phone: (732) 235-5323.Fax: (732) 235-5788. E-mail: arnold@cabm.rutgers.edu.

4771

Mg21 ion dependent (34). Two of three catalytically essentialaspartic acid residues are part of the highly conserved YMDDmotif at the active site of HBV polymerase and its close viralrelatives, including HIV type 1 (HIV-1) RT. The most com-mon 3TC resistance mutations, Met552Ile and Met552Val(Met552Ile/Val), appear at the Met (M) position in theYMDD motif of the HBV polymerase, analogous to the lami-vudine resistance mutations Met184Val/Ile of HIV-1 RT. In a

departure from the pattern observed with HIV, 3TC-resistantHBV frequently contains a second polymerase mutation,Leu528Met. Met552Ile/Val mutations alone and in combina-tion with the Leu528Met mutation confer a high degree ofresistance to 3TC triphosphate (3TCTP) in vitro (Table 1). Onthe other hand, ADV has been reported to be active against3TC-resistant HBV in vitro and in vivo (29, 30, 35). These dataindicate complementary drug resistance profiles for 3TC and

FIG. 1. Chemical structures of dCTP, 3TCTP, FTCTP, ADVDP, LBVTP, and PCVTP.

TABLE 1. Inhibition of HBV polymerases containing prototypic 3TC resistance mutations

Enzyme

dCTP 3TCTP FTCTP ADVDP PCVTP LBVTP

Km (mM)a Foldchangeb Ki (mM)c Fold

change Ki (mM)c Foldchange Ki (mM)c Fold

change Ki (mM)c Foldchange Ki (mM)c Fold

change

Wild type 0.14 6 0.01 1 0.25 6 0.03 1 0.32 6 0.09 1 0.10 6 0.01 1 4.8 6 0.1 1 0.078 6 0.012 1L528M 0.23 6 0.05 1.6 0.64 6 0.04 2.6 0.83 6 0.09 2.6 0.23 6 0.04 2.3 15.1 6 3.4 3.1 ND NDM5521 0.19 6 0.01 1.4 2.0 6 0.1 8.0 6.5 6 1.8 20.3 0.13 6 0.03 1.3 5.5 6 1.1 1.1 0.14 6 0.04 1.8M552V 0.36 6 0.03 2.6 4.9 6 0.4 19.6 4.9 6 0.9 15.3 0.22 6 0.02 2.2 14.7 6 3.8 3.1 0.14 6 0.02 1.8L528M1M552I 0.085 6 0.002 0.64 3.8 6 0.3 15.2 9.5 6 1.9 29.7 0.18 6 0.03 1.8 11.6 6 1.4 2.4 0.086 6 0.021 1.1L528M1M552V 0.34 6 0.01 2.4 6.3 6 2.4 25.2 4.2 6 0.6 13.1 0.08 6 0.02 0.8 11.9 6 1.7 2.5 0.074 6 0.09 0.9

a Means 6 standard deviations for two separate experiments.b Relative to wild-type value.c Means 6 standard deviations for three separate experiments.

4772 DAS ET AL. J. VIROL.

ADV against HBV, suggesting a potential advantage for com-bination therapy in treating chronic HBV infection where theemergence of resistance to either agent may be suppressed.

Knowledge of the structure of HBV polymerase would bevaluable for understanding the molecular basis of many of itsproperties, including mechanisms of polymerization, inhibi-tion, and drug resistance, and for interpretation of clinical andbiochemical data. Attempts to determine the structure of HBVpolymerase by various research groups have not yet been suc-cessful, as they have been limited by failure to obtain sufficientamounts of highly purified active protein.

The work presented here includes a molecular modelingstudy of HBV polymerase based on available retroviral RTstructures. The validity of the model developed in the presentstudy is supported by its ability to explain some of the keybiochemical data. The inhibition potencies of 3TCTP, FTCTP,ADV diphosphate (ADVDP), PCVTP, and LBVTP were eval-uated and compared with the Km for dCTP in in vitro enzymeassays for wild type HBV polymerase and a Leu528Met mu-tant, Met552Ile/Val mutants, and Leu528Met1Met552Ile/Valmutants. The results were analyzed at the atomic level usingthe modeled three-dimensional structure of HBV polymerase.dCTP, 3TCTP, FTCTP, and ADVDP were docked into themodeled enzyme so that the differential effects of Met552Ile/Val and Leu528Met mutations on different nucleotide analogscould be examined. Possible effects of these mutations on someother potent nucleotide inhibitors are addressed. Understand-ing of the roles of these drug resistance mutations might behelpful in achieving the broader goal of developing more ef-fective antiviral strategies for the treatment of chronic hepatitisB.

MATERIALS AND METHODS

Enzyme assay. (i) Inhibition of HBV polymerase. Recombinant HBV poly-merases were overexpressed and partially purified from insect cells as previouslydescribed (35). HBV polymerase activity was monitored by measurement of theincorporation of a-32P-labeled deoxynucleoside triphosphate (dNTP) into acid-precipitable products. Assays were performed in 40 ml of a solution containing100 mM Tris (pH 7.5), 10 mM MgCl2, 0.6 U of RNasin/ml, 5% glycerol, 0.2 mgof activated calf thymus DNA/ml, 100 mM unlabeled dNTPs (e.g., dATP, dGTP,and dTTP), various concentrations of a a-32P-labeled dNTP (;500 Ci/mmol),and various concentrations of inhibitors. a-32P-labeled dATP was used for thedetermination of the inhibition constants for ADVDP a-32P-labeled dGTP wasused for PCVTP and LBVTP, and a-32P-labeled dCTP was used for 3TCTP andFTCTP. HBV polymerase (5 ml, ;0.1 mg) was added to start the reaction.Aliquots (12 ml) were taken at various time points between 0 and 20 min andtransferred onto 3MM paper disks. The paper disks were washed three times in5% trichloroacetic acid plus 1% sodium pyrophosphate and once in 95% etha-nol. The incorporated radioactivity was measured in a Beckman scintillationcounter.

(ii) Enzyme kinetics. Kinetic constants were determined by fitting the initialrates to Lineweaver-Burk plots based on the algorithms described by Cleland (3).

Molecular modeling. (i) Sequence alignments. The protein segment fromposition 354 to 694 of the polypeptide chain translated from the HBV pol gene(Fig. 2) is responsible for the RT activity of HBV. A model was generated foramino acid residues 325 to 699 of the polypeptide chain, covering the entirepolymerase/RT region. The amino acid sequence identity is significant amongvarious HBV strains in the polymerase/RT region, but there is relatively lowsequence homology with other viral RTs and polymerases. The nearest relativesof HBV polymerase, in terms of sequence homology, for which crystal structuresare available are HIV-1 RT and murine leukemia virus (MuLV) RT, both withless than 25% sequence identity. Our sequence alignment (Fig. 2), however,indicates that the functionally important amino acid residues are highly con-served among the polymerases of HBV, HIV-1, and MuLV. The sequencealignments allowed us to derive a three-dimensional structural model for HBV

polymerase from the known structures of HIV-1 RT and Moloney MuLV(MMLV) RT.

(ii) Homology modeling, docking of substrates, and structure analysis. Crystalstructures of HIV-1 RT (7, 11, 12) and MuLV RT (9) were used as templates inthe modeling of the HBV polymerase domain. Multiple initial models for theHBV polymerase were obtained using the amino acid sequence alignment-basedthree-dimensional structure-generating program MODELLER-4 (31) and usingthe crystal structures of HIV-1 RT (PDB codes: 1 RTD, 2HMI, and 1DLO) andof MULV RT (PDB code: 1MML) as templates. The protein conformation ofthe model obtained by using the HIV-1 RT-DNA-dNTP complex structure(1RTD) was used as the initial scaffold for the HBV polymerase model. The lessconserved regions, insertions, and side chains were built by manual modelingusing the computer program O (15) and its reference to databases of knownmain-chain conformations and preferred side-chain rotamers. The other threemodels, derived from the HIV-1 RT-DNA-Fab complex (2HMI), unligandedHIV-1 RT (1DLO), and MULV RT (1MML), were used as additional guides inbuilding the molecular model of HBV polymerase. The secondary structure forthe model constructed as described above agreed very well with a sequence-based secondary structure assignment for the region using the program Homo-logue (21). Buried side chains were manually oriented to have favorable inter-actions with each other. The final model was minimized using the moleculargraphics and simulation program SYBYL, version 6.3 (Tripos, Inc.), and thequality of the geometrical parameters of the model was evaluated by PRO-CHECK (20); the overall G factor was 20.22, indicating that the moleculargeometry is stereochemically reasonable. Amino acid residue Met552 was mod-eled as part of an unusual type II9 turn, as observed in other RT structures. Themain-chain conformations for all other amino acids were within the favoredregions of the Ramachandran plot. The drug resistance mutations Met552Ile/Valand Leu528Met were modeled so that (i) their side chains occupied positionsthat had minimal steric conflict with neighboring amino acids; (ii) their side-chain torsion angles fell within statistically favored ranges; and (iii) the side chainof Val/Ile552 had an orientation similar to that of Ile184 in the Met184Ile mutantHIV-1 RT-DNA structure (32). The individual dNTP substrates and analoginhibitors were initially modeled using as a guide the conformation of dTTP inthe structure of the HIV-1 RT-DNA-dTTP complex (Fig. 3). After energyminimization, the substrates and nucleotide analog inhibitors were then dockedinto the active sites of the wild-type and mutant HBV polymerase models usingthe program SYBYL. The validity of the final model was further supported bythe proximity of the positions of some of the important drug resistance mutationsites with respect to substrates (Table 2).

RESULTS

In vitro assay. The effects of Leu528Met and Met552Val/Ilemutations on the binding of the natural substrate dCTP toHBV polymerase were evaluated by comparing the Km valuesfor dCTP for the mutant enzymes to those for the wild-typeenzyme. The Km values (Table 1) for the substrate dCTP were0.64- to 2.6-fold relative to those for the mutants of HBVpolymerase, indicating that the Leu528Met and Met552Val/Ilemutations do not significantly affect the binding of dCTP toHBV polymerase. In order to identify the desirable structuralfeatures for anti-HBV agents to be used to treat or prevent theemergence of 3TC resistance, five nucleotide analogs with dif-ferent structural characteristics were tested for their sensitivi-ties against 3TC-resistant HBV. ADVDP and PCVTP are acy-clic nucleotide analogs, 3TCTP and FTCTP are L-configurationnucleotide analogs with a b-oxathiolane ring, and LBVTPbears a cyclobutyl replacement for the sugar moiety in thenatural nucleotides. All of the structures are shown in Fig. 1.Susceptibility to these inhibitors was assessed by determiningKi in in vitro polymerase assays using recombinant wild-typeand mutant HBV polymerases.

The inhibition constants (Ki) for 3TCTP, FTCTP, ADVDP,PCVTP, and LBVTP against the Leu528Met mutant andMet552Val/Ile mutants are listed in Table 1. Our in vitroenzyme assay results showed that a single mutation, M552V or

VOL. 75, 2001 MODELING AND CHARACTERIZATION OF HBV POLYMERASE 4773

FIG. 2. Schematic representation of the HBV pol gene, an HBV polymerase homology model (amino acids 325 to 699), and the HBVpolymerase/HIV-1 RT sequence alignments used in constructing the model. The HBV polymerase is shown as a ribbon diagram (2) with the fingers(325 to 403 and 469 to 519), palm (404 to 440 and 520 to 613), and thumb (614 to 699) subdomains in blue, red, and green, respectively. The bounddouble-stranded DNA template primer is shown as a space-filled model in grey (with N and O atoms in blue and red, respectively), and dCTP isin gold. The four proposed disulfide links are represented by yellow lines. The HBV polymerase and HIV-1 RT sequence alignments are also colorcoded by subdomains, and sequence identities and amino acids functionally conserved between the two enzymes are in cyan.

4774 DAS ET AL. J. VIROL.

M552I, in the YMDD motif caused significant resistance to3TCTP and FTCTP, with the inhibition constants increased8- to 30-fold compared to that for the wild-type HBV poly-merase. The acyclic nucleotides ADVDP and PCVTP andthe D-nucleotide LBVTP, however, remained active againstall 3TC-resistant mutant enzymes, with the inhibition con-stants increased less than 3.1-fold (36). A moderate (2.6-fold) increase in Ki for 3TCTP and FTCTP against Leu528MetHBV polymerase is indicative of a minimal effect of the singleLeu528Met mutation on the nucleosides. Similar observationswere reported for 3TC resistance in various independent studies(17, 22).

Overview of the model. The final model (amino acids 325 to699) of HBV polymerase is shown in Fig. 2. Like HIV-1 RT,the modeled HBV polymerase has fingers (325 to 403 and 469to 519), palm (404 to 440 and 520 to 613), and thumb (614 to699) subdomains. The catalytic triad residues Asp431, Asp553,and Asp554 of HBV polymerase correspond to Asp110,Asp185, and Asp186 in HIV-1 RT. Many of the key protein-DNA interactions and protein-dNTP interactions are con-served (Table 2) between the HIV-1 RT structure and themodeled HBV polymerase. It is intriguing that HBV polymer-ase probably contains an element analogous to the “primergrip” of HIV-1 RT (13), including residues Met598 andGly599, which are equivalent to the conserved residues Met230and Gly231 of HIV-1 RT. Some major differences between theHBV polymerase model and HIV-1 RT structure include fourmodeled disulfide bonds in HBV polymerase compared tonone in HIV-1 RT, and a larger fingers region in HBV poly-merase than in HIV-1 RT. The differences in the fingers regionbetween HBV polymerase and HIV-1 RT may involve the

different primers used by retroviral RTs and HBV polymerase.Differences in the palm and thumb regions of the HBV poly-merase model and the HIV-1 RT structure are relatively smallbut significant. The DNA-binding cleft in the HBV polymerasemodel (Fig. 3) is well defined and more positively charged thanthe DNA-binding cleft in the HIV-1 RT-DNA-dTTP complexstructure (12). The dNTP-binding region, between the palmand fingers subdomain, appears to be partially filled by addi-tional amino acids in the HBV model, with the tip of its fingerstouching the base of its thumb. This part of the HBV polymer-ase model corresponds to the b3-b4 region of the HIV-1 RTstructure that contains some of the key HIV drug resistancemutation sites, where mutations can confer resistance to nu-cleoside drugs like zidovudine (AZT), dideoxyinosine (ddI),dideoxycytosine (ddC), and stavudine (d4T). These antiviraldrugs are not very potent against HBV. Some of the HIV-1 RTmutations, conferring resistance to the above drugs, are thenatural amino acids in the wild-type HBV polymerase. Thenucleoside resistance mutations Asp67Asn and Leu74Val ofHIV-1 RT correspond to Asn381 and Val391, respectively, ofthe HBV polymerase model. Two multidrug (AZT1d4T1ddI/ddC) HIV resistance mutations, Gln151Met and the insertionof three amino acids after Ser69, are found in wild-type HBV.Positions 151 and 69 of HIV-1 RT correspond to the positionsof Met519 and Pro382, respectively, in the modeled HBVpolymerase.

Positions of dNTP and nucleotide analog drugs. In the mod-eled HBV polymerase, the relative positions of the a-, b-, andg-phosphates of dCTP (and its analog inhibitors) with respectto the catalytic triad were assumed to occupy positions verysimilar to those of the dNTP in the crystal structure of the

FIG. 3. Electrostatic-potential surface diagrams of the modeled HBV polymerase (left) and of the HIV-1 RT-DNA-dNTP structure (right)plotted using the program GRASP (27). Regions in red and blue are charged negatively and positively, respectively. The locations of amino acidsinteracting with the DNA are labeled.

VOL. 75, 2001 MODELING AND CHARACTERIZATION OF HBV POLYMERASE 4775

HIV-1 RT-DNA-dNTP complex (12). The sugar and the basemoieties of the dCTP were oriented in their energy-minimizedconformations, which are constrained to base-pair with the firstDNA template overhang. The YMDD motif of the modeledenzyme interacts mostly with the sugar-phosphate portion ofthe docked dCTP. The Met552 side chain points towards thedeoxyribose ring of dCTP. The position and orientation of thisamino acid correspond to those of Met184 in HIV-1 RT.Leu528 of HBV polymerase, positionally equivalent to Phe160of HIV-1 RT, is part of a helix, and its side chain points tothe space between Met552 and Phe436. The aromatic ring ofPhe436, positionally equivalent to Tyr115 in HIV-1 RT,stacks almost in parallel with the sugar ring of the substrate.Unlike Met552, Leu528 of HBV polymerase does not haveclose interactions with the dNTP substrate. Upon mutation,however, residue 528 has the potential to affect the bindingof dNTP (or its analog inhibitor) by perturbing the sidechains of surrounding amino acids, particularly of Phe436and Met552.

DISCUSSION

Effects of the Met552Ile/Val mutation. The Met552Ile/Valmutations in HBV polymerase, in both the presence and theabsence of the Leu528Met mutation, conferred resistance to3TC and FTC, as indicated by significant increases of Ki in invitro polymerase assays (Table 1).

In our molecular modeling studies, the docked dCTP sub-strate was accommodated in a stereochemically feasible posi-tion and orientation (Fig. 4) in the wild-type HBV polymerasemodel. Residue Met552, which is part of the conserved YMDDmotif in RTs, is adjacent to the bound nucleotide substrate.The accessible surface area (Fig. 4) of the YMDD region ofHBV polymerase is complementary to the molecular surface ofthe dCTP. The Met552Ile/Val mutation limits the side-chainflexibility by introducing a branch, methyl group (Cg2), to itsCb atom. The most favorable conformation for a valine or anisoleucine at position 552 is with the Cg2 atom pointing to-wards the bound dNTP. The side chain of Ile184 in the crystalstructure of Met184Ile mutant HIV-1 RT-DNA (32), corre-sponding to position 552 of the HBV polymerase model, alsohad a similar conformation. Molecular modeling of theMet552Val mutation (Fig. 4) showed a decreased space be-tween the protein and the substrate. Consistent with the smallchanges observed in kinetic constants, the Met552Ile/Val mu-tation does not appear to interfere significantly with the pro-posed binding of the dNTP in its catalytically favorable con-formation, as shown in Fig. 4.

The nucleotide analog 3TCTP has an oxathiolane ring in ab-L configuration, replacing the b-D-deoxyribose ring of dCTP.Docking of 3TCTP into the active site of the wild-type HBVpolymerase model, with its triphosphate and base oriented asin dCTP, showed (Fig. 5) that the sulfur in the oxathiolane ringpoints towards the site of mutation (position 552). As a con-sequence of the b-L configuration of the oxathiolane ring,which is inverted with respect to the b-D configuration of thedeoxyribose ring of a natural substrate, the docked 3TCTPoccupies a larger volume extending towards the side chain ofMet552. The Met552Ile/Val mutation adds a methyl group atthe Cg2 position of the mutated amino acid, pointing towardthe sulfur atom of the oxathiolane ring of 3TCTP (Fig. 5). Ourmolecular modeling studies suggest that steric hindrance be-tween the Cg2-methyl group of Ile/Val552 and the oxathiolanering of 3TCTP may result from binding of 3TCTP to theMet552Ile/Val mutant HBV polymerase. A previous molecularmodeling study of HBV polymerase (1), based on less detailedinformation about HIV-1 RT structure, concluded that theMet552Ile/Val mutation leads to decreased protein-inhibitorinteractions. Subsequent biochemical and structural data, how-ever, strongly support our proposed mechanism involvingsteric hindrance with the Cg2-methyl group of Ile/Val552. Thismechanism may also apply more broadly to other L-nucleosideanalogs, like FTC, with anti-HBV activity.

Effects of Leu528Met mutation. In the wild-type HBV poly-merase model, Leu528 occupies a position between the sidechains of Phe436 and Met552. Although the Leu528 side chainpoints toward the sugar ring of the docked dCTP, a shortestdistance of about 4.5 A between them suggests that interac-tions between dCTP and Leu528 in wild-type HBV polymeraseare likely to be weak or indirect. The Leu528Met mutationintroduces a longer, yet more flexible, side chain. As a conse-quence of this mutation, the side chain of Met528 may interactdirectly with the docked 3TCTP or FTCTP but its greaterflexibility, unlike the Met552Ile/Val mutation, would disfavorsteric conflict of Met528 with the nucleoside inhibitor. Thishypothesis is in agreement with our results from in vitro studieson the effects of the Leu528Met mutation (Table 1) showing a

TABLE 2. Structurally conserved amino acid residues in HIV-1 RTand HBV polymerase

Description Structural element inHIV-1 RT

Equivalent inmodeled HBV

polymerase

Polymerase active site Asp110 Asp431catalytic triad Asp185 Asp553

Asp186 Asp554

Amino acids interacting Phe61 Phe376with template primer Asp76 Asp393

Gly152 Gly520Met230 Met598Gly231 Gly599Gln258 Gln636Val261 Val639Gly262 Gly640Trp266 Phe644YMDD (183–186) YMDD (551–554)

Conserved amino acids Lys65 Lys380interacting with Arg72 Arg389incoming dNTP Tyr115 Phe436substrates Phe116 Tyr437

Gln151 Met519

Nucleoside resistance Asp67Asn Asn384mutations in HIV-1 Leu74Val Val391RT

Multidrug Gln151Met Met519(AZT1d4T1ddI/ddC) 3-amino-acid Insertion isresistance mutations in insertion after present afterHIV-1 RT Ser69 Pro382

4776 DAS ET AL. J. VIROL.

modest increase in Ki for both 3TCTP and FTCTP of only2.6-fold at the maximum.

A possible role of Leu528Met mutation would be a confor-mational perturbation of the dNTP-binding region, in partic-ular the amino acids Phe436 and Met552. Phe436, whoseHIV-1 RT equivalent is Tyr115, is positioned below andstacked with the sugar ring of dCTP or its analog inhibitors. Asdiscussed above, Met552Ile/Val would introduce a rigid side

chain in the vicinity of the 3TCTP oxathiolane ring. Our invitro assays showed a higher degree of resistance of 3TCTPand FTCTP obtained with the double mutations Met552Ile/Val1Leu528Met than with the single Met552Ile/Val mutation(Table 1). A structural interpretation of this enhanced effect ofthe double mutation is the indirect involvement of Leu528Metmutation by reorienting the side chains of its surroundingamino acids, in particular of Phe436 and Ile/Val552. Such a

FIG. 4. The YMDD region of the modeled HBV polymerase with a docked dCTP substrate. Amino acids Met552 and Leu528 are mutated toconfer resistance to 3TC and FTC. The orange molecular surface (left) corresponds to the deoxyribose of the docked dCTP. The green molecularsurface of the protein around its YMDD region (left) indicated no steric hindrance between the protein and the substrate. The space between thetwo molecular surfaces is indicated by a white arrow. The space between the protein and substrate is reduced upon Met552Val1Leu528Metmutation (right).

FIG. 5. Binding of 3TCTP to wild-type (left) and Met552Val mutant (right) HBV polymerase. Molecular modeling suggests that sterichindrance (right), between 3TCTP and the mutated amino acid, Val552, is the primary cause of 3TCTP resistance. This steric conflict is notobserved in the binding of 3TCTP to the wild-type HBV polymerase.

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rearrangement might also be responsible for compensating forthe reduction of polymerase activity by the Met552Ile/Val mu-tation (22, 25).

Effects of Met552Ile/Val on other nucleoside inhibitors. Thenucleoside and nucleotide analog inhibitors ADV and PCVshow complementary in vivo drug resistance profiles (16, 29,35) with 3TC. An acyclic chain adds torsional flexibility toADV and PCV compared to 3TC or FTC, both of whichcontain a five-membered oxathiolane ring (Fig. 1). In addition,the chain connecting the base and the a-phosphonate group isshorter in ADV than in the oxathiolane analogs. This disparityin length (and volume) is illustrated in comparisons of molec-ular models of wild-type and Met552Val1Leu528Met mu-tant HBV polymerases in complex with double-stranded DNAand ADVDP. Our molecular modeling studies predict thatsmaller acyclic nucleotide analogs can be accommodated moreeffectively than the bulkier oxathiolanes in a more con-strained and “crowded” dNTP-binding pocket containing theMet552Val1Leu528Met mutations. This prediction is consis-tent with the resistance data that show that combinations ofMet552Val/Ile and Leu528Met mutations confer only 0.8- to2.3-fold resistance to ADVDP (35) and 0.9- to 1.8-fold resis-tance to LBVTP. Docking of LBVTP onto the modeled HBVpolymerase fragment suggested that the interaction betweenthe inhibitor and the mutating amino acids is qualitativelysimilar to that for ADVDP (Fig. 6). Furthermore, this modelcan explain why 3TC-resistant HBV mutants still retain sus-ceptibility to ADV, which has thus far not been reported toselect for resistance mutations in HBV (5, 29).

Summary and implications for drug design. Our molecularmodeling studies of HBV polymerase provide a plausi-ble structural basis for the effects of Met552Ile/Val andLeu528Met mutations on the susceptibility of the enzyme to3TCTP and FTCTP. Steric conflict between the b-branchedmutant amino acid side chains and the sulfur atom of theb-L-oxathiolane ring of the inhibitor is proposed as a structural

explanation for 3TC resistance by the Met552Ile/Val mutationin HBV polymerase. This explanation is in agreement with theproposed effects of the YMDD mutation Met184Ile in HIV-1RT based on the comparison of the Met184Ile mutant HIV-1RT-DNA (32) with wild-type HIV-1 RT-DNA-dTTP (12) andwild-type HIV-1 RT-DNA (7) structures. The Leu528Met mu-tation is proposed to have an indirect effect on substrate andinhibitor binding, potentially via rearrangement of its sur-rounding amino acids, particularly Phe436 and Met/Ile/Val552,although increased interaction between the side chain ofMet528 and an incoming nucleotide cannot be ruled out. Thismutation was reported to compensate for the decreased poly-merization by Met552Val (22, 25). The presence of aminoacids corresponding to drug resistance mutations in HIV-1 RTat the equivalent positions in the wild-type HBV polymerasemodel may explain the natural resistance of HBV to AZT anddideoxynucleoside inhibitors.

The structural explanation of the effects of the Met552Ile/Val mutation on inhibition by 3TCTP and FTCTP suggeststhat suitable modifications at the sugar ring of a dNTP analogcould lead to the design of inhibitors with increased potencyagainst the YMDD mutant strain. A similar suggestion wasmade for the design of HIV-1 RT inhibitors with reducedresistance due to a Met184Ile/Val mutation in the YMDDmotif (32). Our studies predicted stereochemically feasiblebinding of ADVDP at the active sites of both wild-type andMet552Ile/Val mutant HBV polymerase models, which is con-sistent with earlier favorable reports of the potency of ADVagainst Met552Ile mutant HBV strains. Also, nucleoside ana-log inhibitors with a smaller sugar ring (e.g., LBV) or withdifferent sugar ring conformational preferences might lead todevelopment of additional nucleotide analogs that would beeffective against Met552Ile/Val mutant HBV strains. Differ-ences in the mode of binding of nucleotide inhibitors to thedNTP-binding pocket of the HBV polymerase, as predictedfrom the current modeling studies, may account for the com-

FIG. 6. Both wild-type (left) and Met552Val1Leu528Met mutant (right) HBV polymerase appear to have no steric conflict with a dockedADVDP.

4778 DAS ET AL. J. VIROL.

plementary drug resistance profiles seen for different nucleo-tide analogs and support the concept that combination therapyagainst HBV may be more effective than monotherapy throughmutual suppression of the emergence of drug-resistant vari-ants.

ACKNOWLEDGMENTS

We gratefully acknowledge Gilead Sciences for support of this workand an NIH MERIT award (R29 AI27690) from the National Instituteof Allergy and Infectious Diseases to Edward Arnold for support ofHIV-1 RT structural studies.

We thank Stephen Hughes for helpful discussions.

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