hcv-796 susceptibility to a benzofuran inhibitor, replicon variants

Published Ahead of Print 16 June 2008. 10.1128/AAC.00238-08.

2008, 52(9):3327. DOI:Antimicrob. Agents Chemother. John O'ConnellRajiv Chopra, Girija Krishnamurthy, Tarek Mansour and

Bard,Mullen, Srinivas K. Chunduru, Dorothy C. Young, Joel Anita Y. M. Howe, Huiming Cheng, Stephen Johann, Stanley HCV-796Susceptibility to a Benzofuran Inhibitor,Replicon Variants with Reduced Molecular Mechanism of Hepatitis C Virus

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ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, Sept. 2008, p. 3327–3338 Vol. 52, No. 90066-4804/08/$08.00�0 doi:10.1128/AAC.00238-08Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Molecular Mechanism of Hepatitis C Virus Replicon Variants withReduced Susceptibility to a Benzofuran Inhibitor, HCV-796�

Anita Y. M. Howe,1* Huiming Cheng,1 Stephen Johann,1 Stanley Mullen,1 Srinivas K. Chunduru,3†Dorothy C. Young,3 Joel Bard,2 Rajiv Chopra,2 Girija Krishnamurthy,2

Tarek Mansour,2 and John O’Connell1

Antivral Discovery, Wyeth Research, 500 Arcola Road, Collegeville, Pennsylvania 194261; Chemical Screening Sciences, Wyeth Research,Cambridge, Massachusetts 021402; and ViroPharma Incorporated, 405 Eagleview Boulevard, Exton, Pennsylvania 193413

Received 20 February 2008/Returned for modification 2 April 2008/Accepted 4 June 2008

HCV-796 selectively inhibits hepatitis C virus (HCV) NS5B RNA-dependent RNA polymerase. In hepatomacells containing a genotype 1b HCV replicon, HCV-796 reduced HCV RNA levels by 3 to 4 log10 HCV copies/�gtotal RNA (the concentration of the compound that inhibited 50% of the HCV RNA level was 9 nM). Cellsbearing replicon variants with reduced susceptibility to HCV-796 were generated in the presence of HCV-796,followed by G418 selection. Sequence analysis of the NS5B gene derived from the replicon variants revealedseveral amino acid changes within 5 Å of the drug-binding pocket. Specifically, mutations were observed atLeu314, Cys316, Ile363, Ser365, and Met414 of NS5B, which directly interact with HCV-796. The impacts of theamino acid substitutions on viral fitness and drug susceptibility were examined in recombinant replicons andNS5B enzymes with the single-amino-acid mutations. The replicon variants were 10- to 1,000-fold less efficientin forming colonies in cells than the wild-type replicon; the S365L variant failed to establish a stable cell line.Other variants (L314F, I363V, and M414V) had four- to ninefold-lower steady-state HCV RNA levels. Reducedbinding affinity with HCV-796 was demonstrated in an enzyme harboring the C316Y mutation. The effects ofthese resistance mutations were structurally rationalized using X-ray crystallography data. While differentlevels of resistance to HCV-796 were observed in the replicon and enzyme variants, these variants retainedtheir susceptibilities to pegylated interferon, ribavirin, and other HCV-specific inhibitors. The combinedvirological, biochemical, biophysical, and structural approaches revealed the mechanism of resistance in thevariants selected by the potent polymerase inhibitor HCV-796.

Hepatitis C virus (HCV) is an enveloped, positive-sense,single-stranded RNA virus of approximately 9.6 kb that pos-sesses an RNA-dependent RNA polymerase (RdRp), NS5B.Like that in many RNA viruses, this RNA replicase lacks aproofreading mechanism. The mutation rate of the HCVRdRp is estimated to be 10�3 to 10�4 mutations/nucleotide, or1 mutation per genome replication (13a, 27). Genetic hetero-geneity is further amplified by robust viral production of �1 �1012 virions per day. As a consequence, quasispecies of viralvariants have been found in HCV-infected patients (4, 6, 8).During chemotherapy, the high rates of viral replication andthe high frequency of mutation lead to rapid generation ofdrug-resistant mutants. Emergence of resistant viruses is amajor challenge in developing effective antiviral therapiesagainst HCV infection.

NS5B RdRp, the principal catalytic enzyme for HCV repli-cation, is a viable target for anti-HCV therapeutics (44). Re-cent research efforts have led to the discovery of many inhib-itors that specifically target this enzyme (5, 7, 11, 19, 34, 35).Among all polymerase inhibitors reported to date, HCV-796(Fig. 1) represents one of the most potent and selective anti-viral agents demonstrating in vitro and in vivo activities

(A. Y. M. Howe, S. K. Chunduru, D. C. Young, H. Cheng, D.Pevear, M. Collett, C. Burns, A. Del Vecchio, T. Bailey, B.Kulkarni, T. Faitg, S. Rippin, C. Blackledge, D. Rys, T. Lessen,J. Swestock, Y. Deng, T. Nitz, J. Reinhardt, H. Feng, A. Saha,T. Herbertz, T. Mansour, and J. F. O’Connell, presented at the13th International Meeting on Hepatitis C Virus and RelatedViruses, Cairns, Australia, 27 to 31 August 2006). Because ofthe high mutation rate during HCV replication, amino acidchanges may accumulate in the NS5B RdRp, leading to de-creased sensitivity to the polymerase inhibitors. Resistancestudies using tissue culture systems help to validate the enzymetarget, delineate the mechanism of action, and provide impor-tant information for optimizing second-generation inhibitorsagainst HCV. In addition, mutations identified in the resistantviruses may serve as diagnostic and prognostic markers toscreen for drug susceptibility in patients.

Currently, cell cultures that support whole-virus replicationare restricted to a few isolates, including a genotype 2a JFH-1isolate derived from an individual with fulminant hepatitis (43,49), a genotype 1a H77-S isolate that harbors five tissue cul-ture-adaptive mutations (47), and chimeric constructs derivedfrom genotype 1 and 2a JFH-1 (17). These infectious culturesystems have not been widely used for resistance studies be-cause of their restricted genotype specificities and limited in-fectivity after multiple cell passages. Several studies success-fully demonstrated the HCV subgenomic replicon in selectingvariants resistant to antiviral inhibitors (12, 16, 38, 46). Areplicon is a subgenomic RNA that contains all essential ele-

* Corresponding author. Present address: Tibotec, Gen. De Witte-laan L11 B3 2800 Mechelen, Belgium. Phone: 32 15 46 1035. Fax: 3215 46 1936. E-mail: [email protected].

† Present address: TetraLogic Pharmaceuticals, 365 PhoenixvillePike, Malvern, PA 19355.

� Published ahead of print on 16 June 2008.

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ments and genes required for replication in the absence ofstructural genes (3, 18). The HCV replicon also contains aforeign gene encoding a drug-selectable marker (neomycinphosphotransferase) to allow antibiotic selection of cells thatcontain a functional replicon. Transfection of the HCV repli-con into human hepatoma (Huh-7) cells leads to autonomousHCV RNA replication. This report describes the selection andcharacterization of replicon variants that have reduced suscep-tibility to HCV-796. Mapping of the amino acid changes en-coded by the NS5B gene derived from the replicon variantsidentified the key mutations within the drug-binding pocket.Mutations in this pocket were responsible for the reducedsusceptibilities of recombinant replicons and enzymes molec-ularly engineered with the single mutations to HCV-796. Thedrug susceptibilities of these replicon variants were evaluatedin comparison to a panel of antiviral agents, including pegy-lated interferon and ribavirin.

MATERIALS AND METHODS

Materials. All tissue culture reagents were purchased from Gibco BRL andHyClone. Clone A cells (licensed from Apath, LLC) were derived from Huh-7cells, a human hepatoma cell line. The clone A cells contain approximately 1,000genome copies of HCV genotype 1b replicon per cell when maintained in asubconfluent monolayer in the presence of 1 mg/ml geneticin (G418 sulfate;Gibco BRL). The sequence of the replicon in clone A cells is similar to that ofthe genotype 1b Con 1 strain of HCV (GenBank accession no. AJ238799), withthe exception of two mutations in NS3 (Q1112R) and NS5A (S2204I). Clone Acells were propagated in Dulbecco’s minimal essential medium (Gibco BRL)containing 10% fetal bovine serum (HyClone) supplemented with 1% penicillin/streptomycin (Gibco BRL), 1% nonessential amino acids (Gibco BRL), 1 mg/mlG418, and 0.66 mM HEPES buffer, pH 7.5.

The plasmid pBB7 containing the HCV genotype 1b BB7 replicon cDNA wasalso licensed from Apath, LLC. The coding sequence of pBB7 is similar to thatof the genotype 1b Con 1 strain of HCV except for one nucleotide mutationresulting in an amino acid change of S2204I within NS5A. All other molecularbiology reagents were obtained from suppliers as indicated.

Selection of drug-resistant HCV replicons in clone A cells. Approximately 3 �105 clone A cells were seeded in a T-25 tissue culture flask in triplicate andcultured in medium containing 2% fetal bovine serum without G418 and with 0.1or 1 �M HCV-796 dissolved in dimethyl sulfoxide (DMSO) (final concentration,0.5% [vol/vol]). As a control, clone A cells were passaged in parallel in the samemedium containing 0.5% DMSO without compound. When the cell densityreached approximately 80% confluence (about 2 to 3 days), the cells were split(1:3) in fresh medium containing HCV-796. An aliquot of the cells from eachpassage was collected to monitor HCV RNA levels. As the intracellular HCVload was reduced and reached a plateau (about 16 days), fresh medium contain-ing HCV-796 and 0.5 mg/ml G418 was added to select for cells containing afunctional replicon variant. Approximately 20 days after the selection, smallcolonies of cells resistant to the inhibitor and the antibiotic became visible andwere pooled. The resistant cells (796R) generated from 0.1 and 1 �M HCV-796were named 796R(0.1 ��) and 796R(1 ��), respectively. To generate resistantcells at a high compound concentration, cells from 796R(0.1 ��) and 796R(1��), respectively, were further incubated with 10 �� HCV-796 and 0.5 mg/mlG418 to generate pools of 796R(10 ��) cells. The selection of 796R(10 ��)cells from the 796R(0.1 ��) and the 796R(1 ��) pools required another 20 days

after the addition of the drug. All resistant cells were cultured at the indicateddrug concentrations in the presence of 0.5 mg/ml G418 for at least 3 weeks beforeanalysis. To ascertain the reproducibility of the selection, genotype 1b (BB7isolate) replicon-containing cells were cultured in the presence of 0.1 �M or 0.2�M of HCV-796 with 0.5 mg/ml or 1 mg/ml G418, respectively, for six passages.As a control, genotype 1b (BB7 isolate) replicon-containing cells were passagedin parallel without HCV-796.

Isolation and sequencing of the NS5B gene from the replicon-containing cells.Total cellular RNA was extracted from the replicon-containing cells using aMicro-to-Midi total-RNA purification system (Invitrogen). The NS5B-contain-ing cDNA was generated in a two-step reverse transcription (RT)-PCR. Thefirst-strand cDNA was generated by RT in a 10-�l reaction mixture containing0.1 to 0.3 �g of total cellular RNA, 2 pmol of primer (7761R, 5�-CGTTCATCGGTTGGGGAGTA-3�) and 10 nmol each of deoxynucleoside triphosphatesusing the SuperScript first-strand synthesis system for RT-PCR (Invitrogen). Thereaction mixture was mixed, heated at 65°C for 5 min, and placed on ice to allowthe primer to anneal to the template RNA. Ten microliters of the RNA-primermixture was added to 9 �l of the SuperScript II reaction mixture, which con-tained 10 mM dithiothreitol, 5 �M MgCl2, and 40 units of RNaseOut RNaseinhibitor. After the reaction mixture (19 �l) was incubated at 42°C for 2 min, theRT reaction was initiated by adding 1 �l of the SuperScript II reverse transcrip-tase (50 units), followed by incubation at 42°C for 50 min. The reaction wasterminated at 70°C for 15 min, followed by digestion with RNase H at 37°C for20 min. To amplify the NS5B gene, 2 to 4 �l of the RT reaction products wasmixed with 10 pmol each of the primers (5919F, 5�-GATCTCAGCGACGGGTCTT-3�, and 7761R), 10 nmol each of deoxynucleoside triphosphates, 2 units ofTaq DNA polymerase, and 1� buffer supplemented with 1.5 mM MgCl2 pro-vided by the supplier (Invitrogen). The reaction (final volume, 50 �l) was carriedout at 95°C for 1 min, followed by 25 cycles of 95°C for 30 s, 60°C for 30 s, and72°C for 2 min and an extension at 72°C for 7 min. The PCR products wereevaluated by agarose gel electrophoresis. The 1.8-kb fragment was excised andpurified from the gel. The cDNA was ligated with the PCR4-TOPO vector, andthe resulting recombinant DNA plasmid was transformed into One Shot chem-ical-competent Escherichia coli according to the manufacturer’s instructions(Topo TA cloning kit for sequencing; Invitrogen). The presence of the HCVNS5B insert in the plasmids was verified by EcoRI digestion. Plasmids containingthe HCV NS5B inserts were subjected to nucleotide sequencing using an ABIPrism BigDye terminator cycle-sequencing ready-reaction kit v3.0 (Applied Bio-systems) according to the manufacturer’s instructions. The sequenced productswere gel purified using a DyeEx 96 kit (Qiagen), dried down, denatured withformaldehyde, and separated by electrophoresis using an ABI Prism 3700 DNAsequencer. Sequence data were analyzed using Sequencher v4.0.

Cloning and mutagenesis. Standard recombinant DNA technology was used toconstruct and purify pBB7 replicon variant plasmids. All NS5B variants wereinitially generated using the plasmid NS5B-BB7dCT21-His as the input template(11). Single-nucleotide changes were introduced using the QuikChange XL SiteDirected Mutagenesis kit (Stratagene) according to the manufacturer’s proce-dure. Amino acid mutations were designated by the single-letter code of theparental amino acid, the amino acid position within NS5B, and the altered aminoacid in the mutant constructs (e.g., L314F). The change in the nucleotide se-quence in each mutant construct is indicated in Table 1. Individual clones weresequenced to confirm the presence of the desired mutations and the lack of otherchanges. To prepare the pBB7 replicon variant plasmids, the Bsu36I fragmentsfrom the mutant plasmid NS5B-BB7dCT21-His were cloned into pHCVrep1b.BB7(licensed from Apath, LLC) backbones digested with Bsu36I. The pBB7 plasmidswere sequenced to confirm the expected single-nucleotide changes in the codingsequence for NS5B.

RNA transcription and electroporation of cultured cells. pBB7 replicon vari-ant DNAs were linearized with ScaI, and in vitro transcription was performedusing Ambion’s Megascript T7 High Yield Transcription kit. Purified RNAtranscripts were electroporated into Huh-7 cells in quadruplicate using a Bio-Rad Gene Pulsar Electroporation System (settings, 270 V and 950 �F). Stablytransfected replicon variant cell lines were initially selected with 0.25 mg/mlG418 and stepped up to 1 mg/ml before further testing. One cell plate wasstained with crystal violet to visualize the number of colonies and to determinethe colony formation efficiency. Individual cell clones from each plate werepooled and expanded for drug susceptibility testing. The NS5B gene of anearly-passaged replicon variant was sequenced to confirm the presence of theexpected nucleotide changes in the coding region for NS5B. No other changesaffecting the amino acid sequence of NS5B were detected.

Expression and purification of NS5B enzyme variants. All NS5B enzymeswere expressed and purified according to the protocol for NS5B-BB7dCT21-Hisas previously described (11). Briefly, recombinant plasmids were transformed

FIG. 1. Structure of HCV-796, 5-cyclopropyl-2-(4-fluorophenyl)-6-[(2-hydroxyethyl)(methanesulfonyl)amino]-N-methyl-1-benzofuran-3-carboxamide.

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into E. coli cells, and the NS5B expression was initiated by the addition of IPTG(isopropyl-�-D-thiogalactopyranoside). After 4 to 6 h of incubation, the cellswere harvested and lysed. NS5B enzymes were purified by chromatography usinga nickel affinity column (Talon; BD Biosciences Clontech), followed by a cation-exchange column (Poros HS; Applied Biosystems).

Evaluation of antiviral agents in replicon variants. The drug susceptibility ofthe replicon-containing cells was evaluated as described previously (11). Briefly,the cells were treated with increasing concentrations of compounds in mediumcontaining 2% fetal calf serum without G418 for 3 days at 37°C and 5% CO2.After incubation, total RNA from the replicon-containing cells was isolated. Thelevels of HCV, GAPDH (glyceraldehyde 3-phosphate dehydrogenase), andrRNAs were quantified using TaqMan reverse transcriptase PCRs. The amountsof HCV, 18S rRNA, and GAPDH RNA in each sample were estimated bycomparing the number of cycles during the exponential phase of the PCRamplification with those in the corresponding standard curves. HCV RNA stan-dards used for the construction of the standard curve were prepared by extractingthe total RNA from clone A cells. The RNA sample was sent to the NationalGenetics Institute to quantify HCV RNA. The total RNA extracted from cloneA cells was quantified by measurement of the optical density at 260 nm and usedfor construction of the standard curves of rRNA and GAPDH. The levels ofHCV RNA and GAPDH were expressed as HCV RNA (copies) and GAPDH(ng) per �g of total RNA using rRNA as a marker for total-RNA measurement.The concentrations of the compounds that inhibited 50% of the HCV RNA level(EC50) were determined using the MDL LSW data analysis software in MicrosoftExcel.

Binding of HCV-796 to NS5B. Changes in the intrinsic fluorescence of HCV-796 were monitored by the excitation at 320 nm in the presence and absence ofthe NS5B polymerase. The emission maximum of the inhibitor occurred at 395nm when free in solution and at 375 nm when bound to the enzyme. The shift inthe emission maximum was accompanied by an increase in the fluorescenceintensity of the bound inhibitor. To determine the binding affinity using theseintensity changes, increasing concentrations of either the recombinant NS5Benzyme or the C316Y mutant enzyme were added to 200 nM of the inhibitor.The fluorescence spectra were recorded after incubation for about 40 min. Therecombinant enzymes used in this study lacked 21 amino acids at the C terminusand were tagged with six histidines (11). In the control experiments, bufferinstead of the enzyme was added to the inhibitor. The resulting changes in thefluorescence intensity at the emission maximum of 375 nm were used to generatebinding isotherms and estimates of the affinity. The fluorescence spectra wererecorded on a Fluoromax-3 (Jobin-Yvon, Edison, NJ). The fluorescence intensitychanges were fitted to a single binding-site model to estimate KD values, usingthe following equation:

F � �KD � �Pt � �It � � ��KD � �Pt � �It �2 � 4�Pt �It �/2c,

where F is the fluorescence intensity change at a fixed wavelength, KD is thebinding affinity of the inhibitor for the protein, [Pt] is the total protein concen-tration, [It] is the total inhibitor concentration, and c is a constant that relates thefluorescence intensity to the concentration.

RESULTS

Selection of replicon variants with reduced susceptibility toHCV-796. To select for HCV-796-associated replicon variants,cells bearing a genotype 1b HCV replicon were treated multi-ple times with 0.1 and 1 �M HCV-796 (an equivalent of 10-and 100-fold EC50, respectively, in a 3-day assay). At the end ofthe 16-day treatment, about 3.6 log10 and 4.2 log10 reductionsof HCV RNA levels were observed in cells treated with 0.1 and1 �M HCV-796, respectively (Fig. 2). The mRNA level of ahousekeeping gene, the GAPDH gene, remained essentiallyunchanged throughout the 16-day period (data not shown).These results suggested that HCV-796 had a direct antiviraleffect on HCV replication and that the compound was welltolerated by the cells.

The HCV replicon encodes a drug-selectable (neomycinphosphotransferase) gene that allows selection of cells with afunctional replicon in the presence of G418. During the courseof drug selection, only cells that contained replicon variantswith reduced susceptibility to HCV-796 survived and gave riseto colonies. These colonies of variant cells (796R), designated796R(0.1 �M) and 796R(1 �M) cells, were pooled and ex-panded. A third pool of resistant cells [796R(10 �M)] wasgenerated by further treating the 796R(0.1 �M) and 796R(1�M) cells with 10 �M HCV-796.

The susceptibility of the variant cells to HCV-796 was mea-sured after treating the cells in the presence or absence ofincreasing concentrations of the compound for 72 h (Fig. 3).The levels of HCV RNA were determined using a quantitativeTaqMan RT-PCR assay. All three 796R variant cell popula-tions had similar steady-state HCV RNA levels compared towild-type clone A cells (Table 2) and displayed different ex-tents of susceptibility to the compound (Fig. 3). At the solu-bility limit (56 �M) of the compound in cell culture medium,HCV-796 reduced HCV RNA levels by 1.4 log10, 0.7 log10, and0.5 log10 units in the 796R(0.1 �M), 796R(1 �M), and 796R(10

FIG. 2. Multiple treatments of clone A cells with HCV-796. CloneA cells were treated with 0.1 �M and 1 �M of HCV-796 in Dulbecco’sminimal essential medium containing 2% fetal calf serum, 0.5%DMSO, and no G418. Control cells were grown in the same mediumwithout HCV-796. When the cell density reached about 80% conflu-ence, the cells were split, and aliquots of cells were harvested for totalcellular RNA extraction. The amounts of HCV RNA and rRNA weredetermined in a quantitative duplex TaqMan RT-PCR assay. The yaxis represents HCV copies per �g of total cellular RNA (using rRNAas a marker for quantification). Each data point represents an averageof three cell replicates. The error bars indicate standard deviations.

TABLE 1. Mutations in NS5B

Mutation in 1bBB7 NS5Ba

Nucleotidepositions

Nucleotidechange

L314F 940–942 CTC3TTCC316F 947–949 TGC3TTCC316Y 947–949 TGC3TACC316N 947–949 TGC3AACC316S 947–949 TGC3AGCI363V 1087–2089 ATA3GTAS365A 1094–1096 TCA3GCAS365T 1094–1096 TCA3ACAS365L 1094–1096 TCA3TTAS368F 1102–1104 TCC3TTCM414I 1240–1242 ATG3ATCM414T 1240–1242 ATG3ACGM414V 1240–1242 ATG3GTGN316Cb 947–949 AAC3TGC

a The numbering system for the amino acids and their corresponding codingnucleotide are based on the 1b, BB7 NS5B.

b This construct was derived from the 1b BK isolate.

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�M) cells, respectively. Control cells had a 2.1 log10 reductionin HCV RNA levels (Table 2). Comparison of the EC50 valuesfor HCV-796 in the 796R cells with those in the control cellsindicated that the replicon variants had 23- to �6,812-fold-reduced susceptibility to HCV-796 (Fig. 3 and Table 2). Theresistant phenotypes were also confirmed by selecting repliconvariants in the presence of 0.1 and 0.2 �M HCV-796. About25- to 65-fold-reduced susceptibilities were observed amongthe variant cells in the second study (data not shown).

Mapping of amino acid changes in HCV NS5B. HCV-796 isa potent and selective inhibitor of HCV NS5B RdRp (A. Y. M.Howe, S. K. Chunduru, D. C. Young, H. Cheng, D. Pevear, M.Collett, C. Burns, A. Del Vecchio, T. Bailey, B. Kulkarni, T.Faitg, S. Rippin, C. Blackledge, D. Rys, T. Lessen, J. Swestock,Y. Deng, T. Nitz, J. Reinhardt, H. Feng, A. Saha, T. Herbertz,T. Mansour, and J. F. O’Connell, presented at the 13th Inter-national Meeting on Hepatitis C Virus and Related Viruses,Cairns, Australia, 27 to 31 August 2006). The crystal structureof NS5B in complex with HCV-796 showed that HCV-796

binds near the catalytic site in the palm domain of the enzyme(Fig. 4A). Therefore, it is likely that the resistance observed inthe 796R cells was due to mutations within the NS5B gene. Tomap the nucleotide changes within NS5B, total cellular RNAwas extracted from the 796R cells. The gene segment encodingNS5B was amplified by RT-PCR, followed by cloning andtransformation into E. coli. Ninety-three bacterial clones con-taining a full-length NS5B gene derived from the pools of 796Rcells were sequenced. In addition, 11 clones containing theNS5B gene derived from the control clone A cells were used ascomparators.

As shown in Fig. 5A, the NS5B genetic sequence from thecontrol cells contained random amino acid changes with nospecific patterns. A total of 32 amino acid changes were ob-served among the 11 clones, with an average of 3 amino acidchanges per clone. All amino acid changes contained one nu-cleotide change per amino acid, resulting in a mutation rate of1.6 � 10�3 mutations per nucleotide for the HCV replicon.

Several unique mutations within NS5B not found in thecontrol cells were observed in the 93 clones derived from the796R cells (Fig. 5B and C). Our attention was focused onchanges in the vicinity of the HCV-796 binding site, whichincluded C316Y, C316F, C316S, I363V, S365L, S365A, S365T,S368F, M414I, and M414T. An additional change, L314F, wasobserved in the second study. Cysteine 445 is located approx-imately 14 Å from the HCV-796 binding pocket. The C445Fsubstitution was also frequently detected in replicon variantsselected from other classes of HCV polymerase inhibitors. Aluciferase replicon bearing the C445F mutation had over 10-fold-reduced susceptibility to HCV-796 (data not shown).

As illustrated in Fig. 4A and B, the key amino acid substi-tutions are distributed among four structural componentswithin the drug-binding pocket. Amino acids Leu314 and Cys316

are within the active-site loop; Ile363, Ser365, and Ser368 are inthe serine-rich loop; and M414 is in the �-helix M. All of theseamino acids have direct interactions with HCV-796, as identi-fied in the crystal structure of the NS5B-HCV-796 complex(Fig. 4B).

To assess if there was any pattern of mutations in the NS5Benzyme of replicon variants, combinations of amino acid sub-stitutions were evaluated. Amino acid substitutions detectedonce in the DMSO-treated control cells were considered ran-dom mutations and were not included in the evaluation. Usingthese criteria, a total of 24 amino acid changes within NS5Bwere observed (Fig. 5B and C). Close examination of the

FIG. 3. Effect of HCV-796 on variant cells selected by HCV-796.Seven thousand clone A or 796R cells were seeded per well in a 96-welltissue culture dish and treated with increasing concentrations of HCV-796 in the absence of G418. Cells were harvested 3 days after treat-ment and analyzed for HCV and rRNAs using a quantitative duplexTaqMan RT-PCR. The numbers of HCV RNA copies per �g totalRNA were compared with those in the control cells. The data shownin the graph are the results from 1 of the 12 independent experiments.Each point represents an average of four replicates. The EC50 in thereplicon-containing cells is indicated. The error bars indicate standarddeviations.

TABLE 2. Activities of HCV-796 against replicon variants

Cellsa EC50 (�M) � SEb Reduced susceptibility(n-fold)

Viral load (copies/�gtotal RNA) � SEc

Mean log10 � SE viralreductiond

Clone A 0.013 � 0.013 (n � 8) 3.7 � 108 � 2.4 � 108 2.1 � 0.4 (0.7)796R(0.1 �M) 0.3 � 0.2 (n � 4) 23 2.9 � 108 � 0.5 � 108 1.4 � 0.3 (56)796R(1 �M) 8.0 � 5.2 (n � 12) 618 2.4 � 108 � 2.0 � 108 0.7 � 0.2 (56)796R(10 �M) �56.0 (n � 4) �6,812 4.1 � 108 � 1.7 � 108 0.5 � 0.3 (56)

a 796R represents cells that are less susceptible to HCV-796.b EC50 values were determined using MDL LSW data analysis. Inhibitory activity is expressed as mean EC50 � standard error. n indicates the number of independent

experiments.c Steady-state levels of HCV RNA after 3 days of incubation in tissue culture medium. The results represent at least three independent determinations.d Viral load reduction was determined at the indicated compound concentrations (�M, in parentheses) in a 3-day assay. The results represent at least three

independent determinations.



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amino acid changes revealed seven patterns of mutations. Ofthese, the double mutations (K355R plus C445F) were foundin all three pools of 796R cells, and V85L plus F162Y plusC316F with or without T19P and C316S/Y plus C445F werefound in replicon variants selected from 1 and 10 �M HCV-796. The rest of the combinations, P197A plus C445F plusV581A, C316Y plus M414I, and S365L plus T390I, were de-tected in either 796R(1 �M) or 796R(10 �M) variant cells. Insome replicon variants, C445F and S365L existed as the soleamino acid change (Fig. 5B and C).

Characterization of the amino acid substitutions in repliconvariants. The contributions of amino acid substitutions locatedwithin the drug-binding pocket to drug susceptibility were as-sessed in replicon variants containing single-amino-acid muta-tions in NS5B in the background of the genotype 1b BB7adaptive replicon (3). The replicon variants were tested in thepresence or absence of increasing concentrations of HCV-796in a 3-day assay. Within the active-site loop, the substitution ofamino acid L314F did not alter the susceptibility of the repli-con to HCV-796 (Table 3). In contrast, the replacement ofcysteine 316 with phenylalanine, tyrosine, or serine (C316F/Y/S) resulted in EC50 values that were 130-, 166-, and 10-fold,respectively, greater than that of the wild-type 1b BB7 replicon(Table 3). Amino acid 316 within NS5B is highly polymorphicamong natural isolates of genotype 1b (see Discussion). Al-though the C316N replicon variant was not detected in thereplicon resistance selection, 316N makes up 40% of the NS5Bsequences of natural isolates, as estimated from 249 sequencesin the NIH genetic-sequence database (GenBank). A mutantreplicon bearing the C316N substitution displayed over 26-fold-reduced susceptibility to HCV-796 (Table 3).

While changes in residues 363 (I363V) and 368 (S368F)within the serine-rich loop had a modest effect on susceptibilityto HCV-796, replacement of serine 365 with alanine or threo-nine (S365A/T) led to 41- and 212-fold, respectively, reducedsusceptibility to the compound (Table 3).

In �-helix M, the replacement of methionine 414 with iso-leucine or valine (M414I/V) resulted in low to moderate in-creases in replicon EC50 values leading to three- to eightfold-reduced susceptibility to HCV-796 (Table 3). The change ofmethionine 414 to threonine did not change susceptibility toHCV-796 in the replicon.

The effects of the amino acid substitutions beyond the vicin-ity of the drug-binding pocket have not been evaluated. Giventhat the pools of 796R cells displayed a substantially higherresistance than the individual mutations examined (compareTable 2 and Table 3), these additional mutations might serveimportant functions, such as adaptation to cell culture replica-tion or compensation for the impaired fitness induced by theprimary mutations (see below), in addition to direct effects ondrug susceptibility.

The impact of amino acid substitutions on viral fitness andgrowth kinetics was estimated based on the colony formationefficiency and steady-state HCV RNA levels in the replicon-containing cells. Transfection of the replicon RNAs into Huh-7cells resulted in colony formation in the presence of G418within 20 days after transfection. No colonies were obtainedfrom Huh-7 cells transfected with the RNAs containing a GAA(polymerase-negative) mutation of NS5B or mock transfected(result not shown). As shown in Table 4, the colony formationefficiencies for the replicon variants were 10- to 1,000-fold lessthan for the wild-type BB7 replicon, suggesting that the aminoacid substitution in NS5B adversely affected viral fitness. Inparticular, the amino acid substitutions within the serine-richloop (I363V and S365T/L) produced the most severe impair-ment of colony formation (Table 4). The replicon variant bear-ing S365L formed small colonies initially but could not sustainG418 selection and failed to establish a stable cell line. Re-duced fitness was not as apparent once the cell lines wereestablished. HCV RNA levels comparable to those of the wild-

FIG. 4. Crystal structure showing HCV-796-associated amino acidmutations. (A) Overview of the HCV NS5b protein bound to HCV-796. The compound is shown with yellow carbons. The thumb domainis shown in blue, the palm in gold, and the fingers in green. Theserine-rich loop is shown in orange (just in front of the compound), theactive-site loop is shown in cyan, helix G is shown in salmon, and helixM is shown in dark blue. (B) Details of the HCV-796 binding site. Theimage is rotated 90° clockwise from panel A. HCV-796 is shown inyellow in the center of the model. The serine-rich loop is shown in orange,the active-site loop is shown in cyan, and helix G is shown in salmon. Thehydrogen bond between the amide nitrogen of HCV-796 and the OH ofSer 365 is shown in blue. The positions of key wild-type amino acids arehighlighted.



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type replicon were observed in the replicon variants L314F,I363V, and M414V (compare Table 4 to Table 3). Even thoughthe S365L/T replicon variants exhibited impaired colony for-mation efficiencies (Table 4), the replicon RNA level in theS365T variant was comparable to that in the control 1b BB7cells (Table 3). It should be noted that the HCV RNA levels inthe pools of 796R cells were comparable to that in the wild-type clone A cells (Table 2). It is possible that compensatory

mutations present in NS5B and/or other regions of the replicongenome might have restored the viral RNA to wild-type levels.

Inhibitory activity of HCV-796 in mutant NS5B enzymes. Toassess the effects of HCV-796 on polymerase activity in thereplicon variants, recombinant genotype 1b BB7 NS5B en-zymes molecularly engineered with single mutations werecloned and expressed in E. coli. The polymerase activities ofthe purified mutant enzymes were evaluated in a biochemical

FIG. 5. (A) Amino acid changes in NS5B derived from clone A control cells. (B) Amino acid changes in NS5B derived from 796R(0.1 �M)and 796R(1 �M) cells. (C) Amino acid changes in 796R(10 �M) cells. Some amino acids in the replicons are colored to illustrate the linkage ofmutations. Replicon variants with only one mutation within NS5B are shaded in grey.



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assay in the presence or absence of increasing concentrationsof HCV-796. Similar to the replicon variants, the polymerasevariants displayed reduced susceptibility to HCV-796 com-pared with the wild-type enzyme; although the levels of resis-tance were substantially attenuated (Table 5). Among the en-zyme variants, the C316N/Y/F substitution resulted in 2- to124-fold-reduced susceptibility to HCV-796, whereas theM414V/I and the I363V substitutions showed little differencein susceptibility to the compound (Table 5).

In the biochemical assay, the recombinant HCV NS5B en-zymes derived from the natural genotype 1b isolates BK and J4(26), which contain asparagine at position 316, are less suscep-tible to HCV-796 than enzymes with a cysteine at this position(A. Y. M Howe, S. K. Chunduru, and D. C. Young, presentedat the 13th International Meeting on Hepatitis C Virus andRelated Viruses, Cairns, Australia, 27 to 31 August, 2006). Toascertain the effects of asparagine and cysteine at position 316in NS5B on susceptibility to HCV-796, the NS5B enzyme de-rived from the genotype 1b BK isolate (BK N316) was engi-neered with a single asparagine-to-cysteine change at aminoacid 316 (BK-N316C). This enzyme variant was 4.5-fold moresusceptible to HCV-796 than the wild-type BK enzyme (Table5), confirming the importance of this residue for drug suscep-tibility in HCV-796.

Biophysical binding of HCV-796 to mutant NS5B protein.Sequence analysis of the NS5B gene derived from the 796Rcells led to the identification of several amino acid changeswithin NS5B, including L314F, C316Y/F/S, I363V, S365L/A/T,S368F, and M414I/T/V. The X-ray crystal structure of HCV-796 in complex with HCV NS5B revealed that all these aminoacids have direct interactions with HCV-796 (Fig. 4B). Themutations at cysteine 316 and serine 365 exhibited the stron-

gest impact on drug susceptibility in both the replicon and theenzyme assays. To understand the mechanism of resistance ofone of these mutations, the binding affinities of HCV-796 withthe wild-type 1b BB7 NS5B and the mutant NS5B bearing theC316Y substitution were evaluated. The changes in the intrin-sic fluorescence of HCV-796 were used to evaluate the bindingaffinity of the compound with the two enzymes. The emissionmaximum of HCV-796 occurred at 395 nm when free insolution and 375 nm when bound to enzyme. This emissionmaximum shift was accompanied by an increase in the flu-orescence intensity of the bound inhibitor. Titration ofHCV-796 with increasing concentrations of the enzymesresulted in an increase in the intrinsic fluorescence of thecompound, which was used to generate the binding iso-therms and to estimate the binding constants (Fig. 6). Theresults of the binding studies showed that the NS5B 1b BB7enzyme bound HCV-796 tightly with a KD value of 180 nM.The mutant enzyme containing the C316Y substitutionbound weakly with no evidence of saturation within thesame enzyme concentration range, suggesting that the bind-ing affinity was at least 1 log unit weaker. These resultsdemonstrated that in the absence of RNA, NS5B with atyrosine at position 316 binds HCV-796 much more weaklythan with a cysteine at position 316, consistent with thereduced susceptibility in the C316Y replicon variant.

Activities of antiviral agents in HCV-796-resistant replicon-containing cells. The antiviral activities of a panel of antiviralagents, including two broad-spectrum antiviral agents and fourHCV-specific inhibitors, were evaluated with the C316Y rep-licon variant and a pool of variant cells selected from HCV-796. Pegylated interferon and ribavirin both have demon-strated antiviral activities against many viruses (2, 10, 15, 22,

FIG. 5—Continued.



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23, 40, 48). Pegylated interferon inhibited the HCV replicationin the C316Y replicon variant and the 796R cells as efficientlyas in the wild-type replicon (Table 6). Ribavirin had similarpotencies against the C316Y and the wild-type replicons andwas fivefold more active in the pool of resistant cells selectedfrom 10 �M of HCV-796. The reasons for hypersensitivityagainst 796R are not clear; it might be due to metabolic vari-ability in different cell lines and/or direct antiviral activities.Ribavirin also inhibited the replicon variants bearing the otherHCV-796-associated mutations listed in Table 3 (data notshown).

The antiviral activities of four inhibitors that bind to differ-ent sites of the NS5B polymerase were evaluated. HCV-086, abenzofuran inhibitor structurally similar to HCV-796, showedreduced inhibitory activities against the C316Y replicon and796R cells, suggesting a general resistance of the mutations tothis series of inhibitors (Table 6). Interestingly, an anthranilatecompound, an allosteric inhibitor that binds to the palm sub-domain of the polymerase (25), also displayed reduced activi-ties in the HCV-796R cells (Table 6). HCV-371, a pyranoin-

dole inhibitor that binds in the lower thumb domain of thepolymerase (11), inhibited both the wild-type and HCV-796-associated resistant replicons with similar activities (Table 6).2�-C-Methylcytidine, an active nucleoside inhibitor of valopic-itabine (NM 283) (39), which has demonstrated clinical effi-cacy, inhibited HCV-796-resistant replicons as effectively asthe wild-type replicon.

DISCUSSION

HCV infection is the leading cause of cirrhosis and livercancer affecting 170 million people worldwide (9, 45). Thecurrent standard of care, a combination of interferon productsand ribavirin, has limited efficacy in less than 50% of patientsinfected with genotype 1 of the virus. Severe side effects lead-ing to �10% to 20% treatment discontinuation further limit itsutility in the clinic. Several HCV-specific protease and poly-merase inhibitors, including BILN2061, telaprevir and boce-previr, valopicitabine, and HCV-796 (14, 21, 28, 29), have beenevaluated in the clinic. In monotherapy studies, these agents

TABLE 3. Activities of HCV-796 against HCV-796 replicon variants

Replicon varianta HCV RNA EC50(nM) � SDb

Resistance(n-fold)

Viral load(HCV copies/�g) � SD

Viral loadreductionc

1b BB7 3.0 � 1.0 (n � 11) 1.8 � 108 � 1.1 � 108 1.9 � 0.31b BB7-L314F 4 � 2 (n � 4) 1 0.3 � 108 � 0.1 � 108 1.6 � 0.31b BB7-C316F 392 � 209 (n � 4) 130 1.0 � 108 � 0.2 � 108 0.8 � 0.51b BB7-C316Y 501 � 291 (n � 4) 166 1.3 � 108 � 0.6 � 108 0.9 � 0.21b BB7-C316Nd 220 � 110 (n � 4) 26d NAe NA1b BB7-C316S 30 � 4 (n � 4) 10 1.3 � 108 � 0.7 � 108 1.3 � 0.11b BB7-I363V 16 � 5 (n � 3) 5 0.2 � 108 � 0.1 � 108 1.4 � 0.11b BB7-S365A 124 � 41 (n � 4) 41 1.2 � 108 � 0.3 � 108 1.7 � 0.11b BB7-S365T 643 � 168 (n � 4) 212 1.3 � 108 � 0.6 � 108 0.6 � 0.11b BB7-S365Lf NA NA NA NA1b BB7-S368F 5 � 2 (n � 4) 2 2.6 � 108 � 1.2 � 108 1.4 � 0.31b BB7-M414I 23 � 3 (n � 5) 8 1.3 � 108 � 0.5 � 108 1.5 � 0.21b BB7-M414T 3 � 1 (n � 4) 1 1.5 � 108 � 0.7 � 108 2.0 � 0.21b BB7-M414V 8 � 1 (n � 3) 3 0.4 � 108 � 0.1 � 108 1.5 � 0.1

a 1b BB7 represents the HCV genotype 1b BB7 isolate. The nomenclature of the replicon NS5B variants (e.g., L314F) is expressed as the amino acid of the inputreplicon, the amino acid position, and the amino acid substitution.

b EC50 values were determined using MDL LSW data analysis. Inhibitory activity is expressed as mean EC50 � standard deviation. n indicates the number ofdeterminations.

c The viral load reduction was determined at 2,240 nM HCV-796 in a 3-day assay. The data represent the mean log reduction of viral RNA � standard deviation.The results represent at least three determinations.

d The evaluation of 1b BB7-C316N was performed in a separate laboratory. The EC50 for HCV-796 in 1b BB7 was 8.6 � 4 (n � 14), which was used to calculatethe resistance for 1b BB7-C316N.

e NA, not applicable.f Replicon variant S365L failed to establish a stable cell line upon selection with G418.

TABLE 4. Colony formation efficiencies of replicon variants inHuh-7 cells

Replicon variant No. of CFU/�g RNA

1b BB7 control .................................................................. 20,0001b BB7-L314F.................................................................... 1,5001b BB7-C316F ...................................................................3,000–5,0001b BB7-C316S....................................................................3,000–5,0001b BB7-I363V .................................................................... 1001b BB7-S365A ................................................................... 1,0001b BB7-S365T.................................................................... 1201b BB7-S365L.................................................................... 20a

1b BB7-M414V.................................................................. 5001b BB7-M414T .................................................................. 3,000

a Did not survive G418 selection.

TABLE 5. Activities of HCV-796 on HCV NS5B enzyme variants

Enzyme IC50a (nM) � SD Susceptibility relative

to wild-type enzymeb

1b BB7 (C316) 40 � 20 (n � 35)1b BB7-C316N 81 � 42 (n � 4) �21b BB7-C316Y 320 � 10 (n � 3) �81b BB7-C316F 1,508 � 419 (n � 3) �1241b BB7-M414V 28 � 2 (n � 3) �1.41b BB7-M414I 24 � 6 (n � 3) �1.71b BB7-I363V 60 � 10 (n � 3) �1.51b BK (N316) 140 � 50 (n � 33)1b BK-N316C 31 � 4 (n � 3) �4.5

a IC50, 50% inhibitory concentration.b n-fold more (�) or less (�).



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reduced HCV RNA levels in patients chronically infected withHCV by 1 to 4 log units (1, 14, 31, 33, 39, 42). Resistant viruseswere isolated in patients treated with all these agents (32, 41).Some mutations present in resistant clinical isolates were pre-viously selected in the replicon system, underscoring the im-portant utility of the replicon for preclinical resistance studies(16, 38).

In anticipation of resistance that might arise in the clinic, wesought to identify and characterize the HCV-796 resistanceprofile in vitro. We have done so using a comprehensive rangeof assays, from a cell-based replicon system to protein biophys-

ical assays. Replicon variants, selected after exposure to HCV-796, displayed 23- to 6,812-fold-reduced susceptibility to thecompound (Table 2). In a total of 93 clones that were se-quenced in the NS5B region, several key mutations were iden-tified. The frequencies of mutations varied, with C316Y/F/S,S365A/T/L, and C445F the most prevalent substitutions ob-served (Fig. 5B and C). The resistant phenotype of the repliconvariants suggested that these amino acids played an importantrole in determining susceptibility to HCV-796 (Table 3). Basedon low colony formation efficiency (Table 4) and the reducedHCV RNA levels in the established replicon cell lines (Table3), these variants were less fit than the wild-type replicon. Thebinding of HCV-796 to apo-HCV NS5B has been character-ized by an X-ray crystallography study of the complex (R.Chopra, G. Krishnamurthy and A. Y. M. Howe, unpublishedobservations). Using this information, the sites of mutationswere mapped to the three-dimensional model, and their effectswere evaluated. Ser365 forms a hydrogen bond from the sidechain oxygen to the amide nitrogen of HCV-796. The S365A,S365T, and S365L mutations perturb this key hydrogen bond,providing a structural understanding of the 41- to 212-fold-reduced susceptibility to HCV-796 in the S365A/T/L repliconvariants (Table 3). Similarly, the mutation on �-helix M atposition 414 results in a direct perturbation of a side chaininteraction with the compound HCV-796 and highlights a re-gion of the protein that has been observed to generate resis-tance to other inhibitor classes that are located in close prox-imity to the HCV-796 binding site (20). Cys316 is immediatelyadjacent to the catalytic triad (GDD motif; G317, D318, andD319) of the NS5B RdRp, which coordinates the metal ionsand nucleotide triphosphate during HCV RNA synthesis (18).Replacement of Cys316 with a larger hydrophobic side chain,

FIG. 6. Binding of HCV-796 with NS5B. Shown is the binding isotherm of the interaction of HCV-796 with the wild-type NS5B and mutantNS5B bearing the C316Y substitution (NS5B C316Y). HCV-796 was incubated with the enzyme according to the procedure described in Materialsand Methods. The change in the intrinsic fluorescence of HCV-796 at 375 nm, F0-F, in the presence of increasing concentrations of either thewild-type or mutant NS5B enzyme is proportional to the bound inhibitor concentration. The solid line corresponds to the curve fit to the quadraticequation.

TABLE 6. Activities of antiviral agents against HCV-796-associatedresistant replicon variants

Compound Replicon EC50 (�M orpg/ml) � SD

Resistance(n-fold)

Pegylated IFN-�-2ba 1b BB7 (WT)b 22 � 29 (n � 3)796R(1 �M) 15 � 13 (n � 7) 0.7

Ribavirin 1b BB7 (WT) 123 � 41 (n � 5)796R(10 �M) 29 � 17 (n � 3) 0.2C316Y 201 � 37 (n � 3) 1.6

HCV-086 1b BB7 (WT) 0.3 � 0.1 (n � 4)796R(10 �M) �80 (n � 4) �40C316Y �80 (n � 2) �40

Anthranilate 1b BB7 (WT) 0.7 (n � 2)796R(10 �M) 13 � 1 (n � 3) �19

HCV-371 1b BB7 (WT) 12 � 2 (n � 2)796R(10 �M) 12 � 5 (n � 3) 1C316Y 11 � 1 (n � 2) 0.8

2�-C-methylcytidine 1b BB7 (WT) 1.0 (n � 2)796R(10 �M) 0.4 � 0.1 (n � 4) 0.4

a IFN, interferon. EC50 is expressed in pg/ml.b WT, wild type.



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such as phenylalanine or tyrosine, would result in a substan-tially different binding mode for HCV-796. Indeed, measureddirect binding of HCV-796 to the mutant C316Y NS5B proteinrevealed an order-of-magnitude-weaker binding relative towild-type NS5B (Fig. 6). Cys316 in NS5B is highly conserved inHCV genotype 1a isolates, whereas the position is rather poly-morphic among isolates in genotypes 1b and 4. Of 117 geno-type 1b sequences reported in GenBank, 40% contain aspar-agine, 57% contain cysteine, and 4% contain tyrosine at aminoacid 316 of NS5B. Five percent of the natural isolates in ge-notype 4 contain asparagine at this position. In the 1b rep-licon, the C316Y mutation was selected after multiple treat-ments with HCV-796; however, the Cys316Asn mutation wasnot. The C316Y mutation requires one nucleotide change(underlined) (TGC3TAC), whereas the mutation of C316Nrequires two simultaneous or sequential substitutions(TGC3AAC) (Table 1). Although the C316N mutationmight be more difficult to generate due to two nucleotidesubstitutions, the presence of Asn316 variants in the naturalgenotype 1b isolates might affect the efficacy of HCV-796.Both the Tyr316 and Asn316 replicon variants have reducedsusceptibility to HCV-796 (Table 3).

A combination cocktail containing HCV-796 and other an-tiviral agents might help to combat viral resistance. The com-bination of a polymerase inhibitor, HCV-796, and a proteaseinhibitor, boceprevir, has been demonstrated to enhance anti-viral activities in replicons and reduce the emergence of resis-tant variants (13). In addition to the nucleoside analogues, allnonnucleoside inhibitors reported to date were shown to bindat one of the four regions in the HCV NS5B: two at the thumbsubdomain (36) and two at the palm subdomain. With theexception of anthranilate, the resistant variants selected byHCV-796 remained sensitive to HCV polymerase inhibitorsthat bind to different sites of the polymerase (Table 6), sup-porting the use of different polymerase inhibitors in a combi-nation therapy. The reason for cross-resistance between thebenzofuran and anthranilate classes of inhibitors is unclear. Astructural comparison of the anthranilate and HCV-796 bind-ing sites showed an overlap of amino acid residues in the palmsubdomain of the polymerase (unpublished data). Several in-hibitors from different chemical classes were shown to bindNS5B at or proximal to the anthranilate binding site (20, 24,37); it is prudent to examine the potential cross-resistances ofthese inhibitors before combination therapy.

In vitro resistance data have been relatively easy to gen-erate but difficult to interpret. We have delineated the im-portant amino acids (Cys316, Ser365, and Met414) as markersfor HCV-796 resistance generated either by exposure to thecompound or by selection from preexisting pools of repli-cons. In addition to identifying and characterizing resistantvariants selected by HCV-796 in the replicon and enzymesystems, the studies described here were unique in relatingthe HCV polymerase resistance to the site of inhibitionthrough structural and biophysical analyses. At present, it isunclear if the resistant replicons selected by HCV-796 invitro predict selection of resistant viruses in vivo. Somereplicon variants with reduced replicative capacities werestabilized only under selective pressure from G418. Thesevariants may not survive or may represent a minority of theHCV population in vivo. Nevertheless, the selection pres-

sure exerted by an immune response in vivo is predicted tosignificantly affect the genetic evolution of the virus. Toassess the impacts of resistance on chemotherapy, the mu-tation frequency, population size, temporal profile, and rep-lication fitness of the resistant variants in patients should beevaluated or monitored. In the phase 1b proof-of-conceptstudies, patients treated with HCV-796 alone or in combi-nation with pegylated interferon had mean viral-load reduc-tions of 1.4 and 3.3 log10 units, respectively. The clinicaltesting of HCV-796 was halted due to the liver enzymeelevation observed in some patients after 8 to 10 weeks ofdosing during the phase 2 studies. Population sequencing ofthe clinical isolates derived from the phase 1 patient samplesshowed that C316Y was the predominant mutation associ-ated with viral rebound. At present, we do not know if othermutations identified in the replicon selection might alsoexist as minor variants in these clinical samples. The com-bined use of virological, biochemical, and structural meth-ods has enhanced our understanding of the molecular mech-anism of resistance, which may help us to design moresuccessful treatment strategies and develop second-genera-tion inhibitors with improved potencies and resistance pro-files. In spite of a setback in clinical development, HCV-796represents the only compound at this binding site that hasdemonstrated clinical efficacy. Efforts to eliminate the clin-ical side effects in this class of inhibitors will likely yield anoptimal compound that will prove to have clinical utility.

ACKNOWLEDGMENTS

We thank Anne Deatly and Mike Flint for their critical review andhelpful discussion of the manuscript. Special thanks are extended tothe Wyeth Core Sequencing Facility for their technical assistance. Weare also grateful to Susan Nastasee for editorial support with manu-script preparation.

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hcv-796 susceptibility to a benzofuran inhibitor, replicon variants

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