Structural Basis for Resistance of the Genotype 2b Hepatitis C Virus NS5B Polymerase to Site A...

doi:10.1016/j.jmb.2009.06.012 J. Mol. Biol. (2009) 390, 1048–1059

Available online at www.sciencedirect.com

Structural Basis for Resistance of the Genotype 2bHepatitis C Virus NS5B Polymerase to Site ANon-Nucleoside Inhibitors

Edwin H. Rydberg1, Antonella Cellucci1, Linda Bartholomew1,Marco Mattu1, Gaetano Barbato1, Steven W. Ludmerer2,Donald J. Graham2, Sergio Altamura1, Giacomo Paonessa1,Raffaele De Francesco1, Giovanni Migliaccio1 and Andrea Carfí1⁎
1Istituto di Ricerca di BiologiaMolecolare, P. Angeletti, ViaPontina Km 30600, I-00040Pomezia, Rome, Italy2Department of AntiviralResearch, Merck ResearchLaboratories, P.O. Box 4,Sumneytown Pike, West Point,PA 19486, USA
Received 27 March 2009;received in revised form29 May 2009;accepted 3 June 2009Available online6 June 2009

⁎Corresponding author. E-mail [email protected] address: R. De Francesco,

Molecular Biology Unit, Via FrancesMilano, Italy.Abbreviations used: 1bdc21, HCV

containing a C-terminal His6 tag; 1bNS5B ΔC55; 2adc21, HCV 2a NS5BC-terminal His6 tag; 2bdc21, HCV 2containing a C-terminal His6 tag; Ninhibitor; HCV, hepatitis C virus; Nprotein 5B; RdRp, RNA-dependent

0022-2836/$ - see front matter © 2009 E

Hepatitis C virus (HCV) exists in six major genotypes. Compared with the1b enzyme, genotype 2b HCV polymerase exhibits a more than 100-foldreduction in sensitivity to the indole-N-acetamide class of non-nucleosideinhibitors. These compounds have been shown to bind in a pocket occupiedby helix A of the mobile Λ1 loop in the apoenzyme. The three-dimensionalstructure of the HCV polymerase from genotype 2b was determined to 1.9-Å resolution and compared with the genotype 1b enzyme. This structuralanalysis suggests that genotypic variants result in a different shape of theinhibitor binding site. Mutants of the inhibitor binding pocket weregenerated in a 1b enzyme and evaluated for their binding affinity andsensitivity to inhibition by indole-N-acetamides. Most of the point mutantsshowed little variation in activity and IC50, with the exception of 15- and 7-fold increases in IC50 for Leu392Ile and Val494Ala mutants (1b→2b),respectively. Furthermore, a 1b replicon with 20-fold resistance to this classof inhibitors was selected and shown to contain the Leu392Ile mutation.Chimeric enzymes, where the 2b fingertip Λ1 loop, pocket or both replacedthe corresponding regions of the 1b enzyme, were also generated. Thefingertip chimera retained 1b-like inhibitor binding affinity, whereas theother two chimeric constructs and the 2b enzyme displayed between 50-and 100-fold reduction in binding affinity. Together, these data suggest thatdifferences in the amino acid composition and shape of the indole-N-acetamide binding pocket are responsible for the resistance of the 2bpolymerase to this class of inhibitors.

© 2009 Elsevier Ltd. All rights reserved.

Keywords: hepatitis C virus; polymerase; resistance; non-nucleosideinhibitor binding sites
Edited by K. Morikawa
ress:

INGM, Genomics andco Sforza 28, 20122

1b-con1 NS5B ΔC21dc55, HCV 1b-bkΔC21 containing ab NS5B ΔC21NI, non-nucleosideS5B, non-structuralRNA polymerase.

lsevier Ltd. All rights reserve

Introduction

The hepatitis C virus (HCV) is thought to haveinfected 180 million people worldwide, an estimated130 million of whom are at risk of developingchronic liver disease, cirrhosis and hepatocellularcarcinoma. Three to four million people are believedto be newly infected each year, and it is estimatedthat 50%–80% of infections are asymptomatic.1 Thecurrently approved standard of care for chronicHCV infection is based on pegylated interferon-α,either as monotherapy or in combination withribavirin. However, this therapy is poorly toleratedand of limited efficacy.2

d.

Fig. 1. Location of NNI binding sites on HCV NS5BΔC55-1b polymerase. The three sites are localized inpockets formed by helices O, Q, S and T. Sites A and B areeach located on the surface of the thumb domain. Site A isoccupied by helix A, the tip of the fingertip Λ1 loop, in theinhibitor-free NS5B polymerase structures. Site C islocated near the enzyme active site.

1049Structure of HCV Polymerase 2b

HCV exists in six major genotypes, varying by30%–35% in sequence over the entire genome. In turn,each genotype can be divided in several subtypes,differing between 20% and 25%.3 In addition, thehigh rate of HCV mutation quickly generates adiverse population of quasi-species from a singlesubtype.4 Worldwide, genotypes 1 (a and b) and 2(a and b) are the most prevalent forms, accountingfor approximately 13 million cases of disease in theUnited States and Europe combined. HCV geno-type is a major determinant of treatment outcome.HCV belongs to the Flaviviridae family of envel-

oped viruses; its positive-stranded RNA genome is9.6 kb long and codes for a single open-readingframe flanked by conserved 3′- and 5′-untranslatedregions. A combination of host- and virus-mediatedproteolytic processing of the single polyproteinresults in at least three structural proteins andseven non-structural proteins (reviewed by Reedand Rice5). Non-structural protein 5B (NS5B), theRNA-dependent RNA polymerase (RdRp) at thecore of the HCV replicative complex, has beendemonstrated to be crucial for viral infectivity and isa validated target for the development of therapeu-tics against HCV.The three-dimensional structures of C-terminally

truncated forms of NS5B (ΔC21 and ΔC55) fromgenotypes 1b6–11 and 2a12 have been reported. Theyshare the samegeneral right-handed finger, thumbandpalm domain organization and the same catalyticmachinery with other single-subunit polynucleotidepolymerases (Fig. 1). However, the HCV polymerasehas an elongated loop (Λ1), which protrudes from thepalm-distal region of the finger domain (the so-calledfingertip), also observed in the RdRp's of other viruses,such as bacteriophage Φ6,13 bovine viral diarrheavirus14 and foot-and-mouth disease virus.15 Thus, thefingertip extends from the finger domain to contact thethumb domain, enclosing the active-site cleft andresulting in a relatively closed, spherical appearancefor the enzyme. This active-site enclosure also suggeststhe need for a conformational change to affect thecomplete catalytic function.7,9

Screening of inhibitor libraries has resulted in thediscovery of several classes of non-nucleosideinhibitors (NNIs). Although all these moleculesinhibit the polymerase at a stage prior to elongationand non-competitively with RNA template andNTPs, at least three distinct binding sites (Fig. 1)have been identified:16 site A17,18 and site B10,19,20 arelocated on two opposite sides of helix Q and helix Ton the surface of the thumb domain at 30 and 35 Åfrom the active site, respectively; site C is located inproximity of the enzyme active-site cavity.21,22

Of interest, structural and biochemical studieshave shown that site A inhibitors17,18,23 bind to apocket masked in the apoenzyme by the Λ1 loophelix A (Fig. 1). Further evidence for mobility of thisloop was observed for NS5B from the 2a genotype,where both a “closed” form (similar to the apo-1bstructure) and an “open” form where helix A hasmoved away from the thumb domain and adopted aβ-hairpin-like structure were observed.12 Moreover,

in the same study, a site B inhibitor was reported tobind only to the closed form of the enzyme and tocause a transition to the open form. These data areevidence for crucial interdomain communicationwithin the polymerase that can be interfered at leastby both site A and site B inhibitors.In addition to resistance mutations at each inhibitor

binding site,17,24,25 it has been found that inherentgenotypic differences are often responsible for drama-tically different sensitivities to various inhibitors(reviewed by De Francesco & Carfi,16 Beaulieu &Tsantrizos,26 Li et al.27 and Ma et al.28). During thecharacterization of HCV polymerase NNIs, we havediscovered that the genotype 2b polymerase is morethan 100-fold less sensitive to inhibition by the indole-N-acetamides than is the genotype 1b enzyme(Table 1). To understand the molecular basis forthis genotypic heterogeneity in NS5B inhibition, wehave determined the three-dimensional structure ofthe C-terminally truncated (ΔC21)HCV 2b.2NS5B.29

Furthermore, a series of 1bNS5Bvariants of the indole-N-acetamides binding site, site A, were produced, andtheir binding and inhibition by this class of moleculeswere assessed. Our structural, biochemical and bind-ing data suggest that differences in the shape of the siteA binding pocket are in a large part responsible for theresistance of the 2b polymerase to the indole-N-acetamide inhibitors. Furthermore, our data showthat variations at positions 392 and 494 in genotype 2bNS5B have themost significant effect on the sensitivityof the HCV NS5B to this class of compounds.

Results and Discussion

Protein expression and purification

The genotype 2b ΔC21 HCV polymerase (2bdc21)was expressed and purified in soluble form with

Table 1. Genotypic sensitivity to indole-N-acetamides

Table 2. Crystallographic data and refinement statisticsfor HCV 2b NS5B

2b

Space group C2Unit cell parametersa (Å) 153.25b (Å) 64.50c (Å) 135.58β (°) 90.16

Data collectionBeamline European Synchrotron

Radiation Facility, ID14-2Wavelength (Å) 0.933Resolution (Å) 1.90High-resolution shell (Å) 1.97–1.90Total observations 328,676Unique reflections 104,470Completeness (%) 97.3 (88.3)a

RefinementRefinement resolution 20–1.90Unique reflections (working/test) 99,343/5127Rworking/Rfree 16.6/21.0Number of atomsb 8745/—/830r.m.s. deviation bond length (Å) 0.024r.m.s. deviation bond angle (Å) 1.87Mean B-factor (overall, Å2) 21.0Ramachandran analysis (%)Most favored 81.5Additionally favored 6.5a The values in parentheses refer to the highest-resolution shell.b Non-hydrogen protein atoms/non-hydrogen inhibitor

atoms/water molecules.

1050 Structure of HCV Polymerase 2b

truncation of the 21-amino-acid C-terminal region,corresponding to the highly hydrophobic mem-brane anchor. The expression level was substantiallylower than that of the corresponding NS5B ΔC21enzyme from genotype 1b-bk (1bdc21), and thepurified yield of 2bdc21 was 2.5 mg/L. In addition,upon concentration, it was apparent that thesolubility of 2bdc21 was also significantly lower(ca 7 mg/mL) than that of the 1bdc21 (greater than20 mg/mL). Nevertheless, the purified 2bdc21protein could be crystallized, and its structurewas solved by molecular replacement and refinedto 1.9-Å resolution (Table 2).

Overall structure of 2bdc21

The structure of 2bdc21 is highly similar to that ofthe HCV NS5B ΔC21 enzymes from genotypes 1b(1bdc21)6,7,9 and 2a (2adc21) in the “closed” form.12 Ifthe palm domains are superimposed, the overall Cα r.m.s. deviations between 2bdc21 and 1bdc21 andbetween 2bdc12 and 2adc21 are 0.42 and 0.25 Å,respectively. The largest variations were observed inthe N-terminal region of the fingertip Λ1 loop(residues 21–27) and in the C-terminal tail, suggest-ing some flexibility in these regions of the protein(Fig. 2a and b). However, despite shifts of 1.5–3.2 Å inthe Cα atom positions of the C-terminal tail regions,the side chains of the hydrophobic L547W550F551motif30 are generally spatially conserved (Fig. 2b).

NNI site C

The internal allosteric NNI pocket is located inthe active-site cavity, 8.0 Å away from both theactive site and helix O (Fig. 1). Several classes ofcompounds are known to bind this site, including

benzothiadiazines,22 phenyl-phenoxy acrylicacids21,22,31 and the reversible covalent arylsulfonylrhodanines.32 X-ray structures of 1bdc21 with inhi-bitors from each class have been reported.21,22,31–33

Most of the residues forming this pocket areinvariant across HCV genotypes, with the exceptionof Met414 (Gln in genotype 2, Val in genotype 4a)and Tyr415 (the conservative Phe mutation is presentin several genotypes, such as 1a/c and 6a). Inaddition, two other mutations are located in proxi-mity of this site and may affect inhibitor binding.Cys316 and Ile447 in genotype 1b are mutated inAsn and Met, respectively, in genotype 2. TheCys316Asn mutation may partially affect the posi-tion of Arg200 side chain, whereas the larger sidechain of Met447 in the β-flap may constrain theposition of the adjacent Gln414 side chain, prevent-ing optimal positioning of the inhibitor (Fig. 3).Previous studies have shown that the mutationMet414Thr is sufficient to confer resistance to thebenzothiadiazines in both the isolated polymeraseand the replicon system,34 demonstrating that theamino acid in this position plays an important rolefor binding of site C inhibitors.

NNI site B

Site B is on the surface of the thumb and is formedby helices Q and T and the beginning of the C-terminal tail (Fig. 1). It is a long, narrow cleft 35 Å

Fig. 2. Structural comparison between NS5B ΔC21-1b(PDB ID 2FVC) and ΔC21-2b. (a) The Cα traces of theΔC21-1b and ΔC21-2b fingertip Λ1 loops are shown ascoils in yellow and cyan, respectively. The side chains ofsome of the 1b and 2b amino acids are shown in stickrepresentation. A transparent gray surface covers theΔC21-2b polymerase thumb domain. (b) The Cα trace ofthe ΔC21-2b polymerase, except the C-terminal residues(540–561), is shown as a ribbon in gray. The C-termini ofthe two ΔC21-2b molecules in the asymmetric unit of thecrystal and the C-terminal residues of the ΔC21-1b areshown in blue, red and yellow, respectively. Regionsimmediately up- and downstream the C-terminalL547W550F551 motif are poorly ordered in most of theHCV ΔC21 polymerase structures determined so far, butthe motif itself is well defined and makes the sameinteractions with the thumb domain in all knownstructures. Figures 2, 3 and 5 were produced and renderedin PyMOL (DeLano Scientific).

Fig. 3. Structural comparison between ΔC21-1b (PDBID 2FVC)22 and ΔC21-2b at the internal site C. The Cα

trace is shown only for the 1b enzyme in a ribbonrepresentation. The side chains of some of the key residuesforming the inhibitor binding site C are shown in stickrepresentation. The Cα position of F551, from theL547W550F551 motif, is indicated by a small black circle.Amino acids from the NS5B ΔC21-2b enzyme are shownin cyan, whereas the equivalent residues from the NS5BΔC21-1b enzyme are in green. TheΔC21-2b polymerase isresistant to compounds that inhibit the 1b enzyme bybinding to site C due to the Met414Gln mutation.


from the catalytic site. Known inhibitors of this siteare dihydropyranone,20 thiophene carboxylicacids10,12 and some cyclopentylethyl-phenoxyacrylic acids.31 In the 2b enzyme, there are fourconservative variations with respect to genotype 1b(this site is identical in genotypes 2a and 2b) in the

cleft. However, these mutations are in solvent-exposed positions of the structure and do notappear to result in important structural differencesbetween the two NS5B pockets.

Site A: the indole-N-acetamide NNI binding site

Site A, where the NNIs with the related benzimi-dazole and indole-N-acetamide scaffolds bind, islocated in a pocket in the thumb domain that in theapoenzyme is filled by helix A of the mobile Λ1fingertip loop. The site consists of two components,the fingertip Λ1 loop and the pocket. The structureof the complex between compound 1 and the1bdc5518 revealed that the inhibitor establishesseveral contacts with amino acids forming the siteA pocket (Fig. 4). Resistant replicons have beenisolated only with mutations of Pro495, Pro496 or, toa lesser extent, Val499 to Ala or Ser.17,34,35 BothPro495 and Pro496 are strictly conserved across allgenotypes.There are few mutations between genotypes 1b

and 2b/a at site A (Fig. 4), in addition to the“nearby” mutation Val499Ala. Superposition of the2bdc21 structure here reported with that of the1bdc55 [Protein Data Bank (PDB) ID 2BRL] showsthat these mutations significantly alter the shape ofthe inhibitor binding site (compare Fig. 5a and b).Ala393Thr and Leu392Ile would affect the region inproximity of the inhibitor phenyl ring. In the 1benzyme, Ala393 provides a lipophilic environmentfor the para-position of the inhibitor phenyl moiety.This is more favorable than that generated by thepolar Thr393 oxydryl atom, which would also be in

Fig. 4. Schematic representation of the interaction between compound 1 and the ΔC55-1b polymerase (PDB ID2BRL).18 Red, black and blue spheres represent oxygen, carbon and nitrogen atoms, respectively. The letter R on theinhibitor indicates the piperidine substituent (Table 1). Residues from the 1b polymerase involved in hydrophobiccontacts with the inhibitor are represented by red semicircles. Blue semicircles indicate the location of amino acidvariations between the 1b and 2b polymerases. The same mutations, with the exception of A393T, are also present in thegenotype 2a polymerase. Hydrogen bonds are shown as dotted black lines. With the exception of the piperidinesubstituent, all the atoms of the inhibitor are within 4.0-Å distance from ΔC55-1b site A pocket residues.


closer contact to the phenyl ring para-C atom (Fig.5c). Interestingly, genotypes 2a and 2b have most ofthe residues in the indole binding site in common,with the exception of position 393, which is Ala in 2aas it is in 1b (Fig. 4). This suggests that Ala393Thrmutation could be responsible for the difference inresistance to these compounds between genotypes2a and 2b. Another change that likely would affectthe interaction with the inhibitor is Leu392Ile. In the2bdc21 structure, the β-methyl group of Ile392 isoriented toward the pocket, thus reducing the spaceavailable for the inhibitor (Fig. 5a). In particular, theIle392 Cβ2 would likely form several unfavorablecontacts with both the inhibitor phenyl and cyclo-hexyl moieties (Fig. 5a and c). Leu425Ile, at the“bottom” of the cyclohexyl pocket, where Leu29interacts in the apo structure, has the most subtleeffect of the five variants, producing a slightly deeperpocket in the 2bdc21 enzyme. Finally, Leu424Val andVal494Ala interact with the indole moiety, and theseconcomitant variations result in a deeper pocket inthe 2b structure, significantly reducing the favorablevan der Waals contacts between the indole and theprotein (Fig. 5a–c). The influence of Val499 inresistance to the related benzimidazole inhibitors(see above) is likely played out through its role inorienting Arg503, which may interact with substi-tuents on the carboxylic acid, and in part constrain-ing the Pro493–Gly496 loop (Fig. 5d). Thus, theinfluence of the Ala variant on inhibitor bindingwould be expected to be variable and dependent onthe specific indole substituent.

Enzymatic and kinetic evaluation of variants

To clarify the contribution of the pocket mutationsto resistance to site A NNI, we generated a series ofvariants with the 2b mutations introduced into the1b-con1 scaffold. Point variants for each of thegenotypic differences in the pocket (Fig. 4) andVal499Ala in addition to three chimeras, fingertip(Ala15Gly, Ala16Pro, Ser19Glu, Ala25Pro, Leu31-Met, His33Phe, Met36Lys), pocket (Leu392Ile,Ala393Thr, Ile424Val, Leu425Ile, Val494Ala,Val499Ala) and full site A (fingertip+pocket), wereproduced. The fingertip Λ1 loop, although disor-dered in the structure of the 1bdc55 in complex withthe indole-like inhibitor18 and thus unlikely to makeany strong contacts with the inhibitor, couldconceivably attenuate inhibitor accessibility to thebinding site via altered flexibility in different geno-types. The high degree of sequence and structureconservation of the region surrounding site A suggeststhat the six variants in the pocket chimera shouldprovide a strong approximation of the complete 2bpocket. The only other mutations in the regionsurrounding the site A pocket are the “second sphere”variants Ala400Val, Ala421Val and Cys488Thr. How-ever, superposition of the 1bdc21 and 2bdc21 struc-tures suggests that these mutations do not affect theposition of the main and side chains of residuesdirectly contributing to the inhibitor binding pocket.All point variants had enzyme activities within 3-

fold of the wild-type enzymes, with the exception ofAla393Thr (10-fold decrease in activity). On the

Fig. 5. Differences in the shape of the site A pocket between the HCVNS5B 1b (PDB ID 2BRL) and 2b polymerases. (a)Surface of the ΔC55-1b site A pocket with 1b and 2b amino acid side chains underneath in yellow and cyan, respectively.The Cδ of Ile392 side chain and the hydroxyl group of Thr393 side chain in the 2b enzyme stick out from the 1b site Asurface, thus reducing the space available for the inhibitor. (b) Surface of the NS5B ΔC21-2b site A pocket with 2b and 1bamino acid side chains underneath represented as in (a). The Cγ of the NS5B 1b Val494 side chain sticks out from the 2bsite A surface, indicating a larger 2b site A pocket. In addition, two pockets can be observed in proximity of the Leu392side chain, which are absent in the 1b polymerase site A surface, pointing to a deeper site A pocket in the 2b polymerase[see panel (a)]. (c) Compound 1 modeled in the ΔC21-2b site A pocket: amino acids that may affect the binding affinity ofindole-N-acetamide inhibitors are shown in red. (d) Val499 in the 1b polymerase may constrain the position of Arg503 andthe 493–496 loop. The Cα positions of Pro496 and Gly493 are indicated by black circles.


contrary, the chimera enzymes were more than 500-fold less active (Fig. 6a). It is likely that themutations introduced in these molecules perturbedthe interaction between the fingertip loop and thethumb domain, resulting in catalytically defective

polymerases. The IC50 value for compound 1 wasdetermined on each of the point variants and the 1b-con1 and 2b enzymes. The results, as shown inFig. 6b, are normalized to the IC50 of 1b-con1. Threeof the point variants caused an increase in resistance,

Fig. 6. Characterization of site A 1b→2b mutants. (a) Enzymatic activity of the ΔC21-1b site A point mutants andfingertip, thumb and entire site A chimera mutants. (b) Resistance of the 1b→2b mutants to inhibition by compound 1.Red asterisks indicate the NS5B 1b polymerase point mutants for which compound 1 has an increased IC50 comparedwiththe wild-type 1b enzyme.

Table 3. Binding constants of compound 1 for the NS5B1b, 2b, and 1b → 2b mutant enzymes

Variant kon (1/ms) koff (1/s)KD(μM)

Half-life(min)

A393T 2.51e+04 2.69e−03 0.11 4.29L425I 1.27e+04 5.86e−03 0.46 1.97V499A 9.81e+03 6.78e−03 0.69 1.701b 0.18L392I 0.85V494A 1.602b 9.40“Fingertip” 0.08“Pocket” 3.20“Full site A” 8.60


and three others caused an increase in susceptibility.Leu392Ile and Val494Ala were 15- and 7-fold moreresistant, respectively, while Val499Ala was approxi-mately the same as wild-type enzyme (1.5-fold moreresistant). Ile424Val and Leu425Ile were equivalenttowild type (1.5- and 2-foldmore sensitive), whereasAla393Thr was 4-fold more sensitive to inhibition.With the notable exception of the Ala393Thrmutants, these results are in agreement with thepredictions from the structural analysis.Using the above data, we constructed a triple

mutant incorporating the mutations that desensi-tized the enzyme to the inhibitor (Leu392Ile,Val494Ala and Val499Ala). Although the increasein IC50 for Val499Ala against compound 1 wasmarginal, the point variant was included as asignificant contribution to resistance due to theobservation of different effects on resistance againstalternatively substituted indoles.17 The activity ofthe triple mutant was within 3-fold of the wild type,and the IC50 was 52-fold higher than that of 1b-con1(Fig. 6a and b). Thus, these three mutations weresufficient to account for more than half of theresistance of the 2b enzyme.

Inhibitor binding studies

To gain further insights into the resistance of 2bNS5B to indole-N-acetamides, we characterizedwild-type 1b and 2b enzymes as well as the chimeric1b→2b proteins for binding to compound 1 usingsurface plasmon resonance analysis. Determinationof the kinetic binding constants kon and koff was notpossible for some of the enzymes (1b, 2b, Leu392Ile

and Val494Ala) because the sensorgrams exhibited amultiexponential character, indicating that morethan a single kinetic event was being monitored;for these mutants, only the equilibrium measure-ments were used to determine the Kd values. ForAla393Thr, Leu425Ile and Val499Ala, kinetic andequilibrium evaluations were performed, resultingin very similar Kd values (Table 3). The bindingstudies showed that the 2b enzyme has amuch loweraffinity for the inhibitor than the 1b enzyme.Importantly, this difference appeared to be duemainly to an exceedingly fast dissociation while thesensorgram association phase remains comparable(Fig. 7), demonstrating that resistance is not causedby an impaired accessibility to the inhibitor bindingpocket. Similar behavior was observed for the“pocket” and “entire site A” chimera, which showeda fast dissociation as for wild-type 2b. Finally, the“fingertip” chimera bound the inhibitor as

Fig. 7. Indole-N-acetamide inhibitor binding to the 1b,2b and 1b/2b polymerase chimeras measured by Biacore.Biacore sensorgrams show the response over time(resonance units) during the binding of compound 1 towild-type NS5B ΔC21-1b (green), ΔC21-1b finger chimera(blue), ΔC21-1b thumb chimera (red), ΔC21-1b pocketchimera (dark gray) and wild-type ΔC21-2b (cyan)enzymes. Binding of compound 1 to the ΔC21-2b andΔC21-1b pocket chimera enzymes shows a very fast koff.Instead, binding of compound 1 to the NS5B ΔC21-1bfingertip chimera and wild-type 1b polymerase showsvery similar koff and kon values.


effectively as did the wild-type 1b enzyme (Fig. 7),confirming that the difference in the shape of theindole binding pocket rather than accessibility tothe site or structure of the fingertip is responsiblefor the reduced affinity and consequently for theresistance of the 2b enzyme to this class of inhibitors.Analysis of the Kd values for the single-pocket

mutants is in agreement with the structural analysisand the initial inhibition studies, with Val494Alaand Leu392Ile being the mutants most impaired inbinding (Table 3).

Selection of Leu392Ile-resistant 1b replicon

Our biochemical data suggest that HCV 1bpolymerase with mutations in the site A pocketmight be resistant to the indole-N-acetamide NNIs.Thus, we used the replicon system to identify HCV1b polymerase mutants conferring resistance toindole-N-acetamide compounds. Replicon-harboringcells were incubated with G418 and escalatingconcentrations of a cell-permeable indole-N-aceta-mide structural analogue of compound 1 (see alsoMaterials and Methods). This protocol allowed theselection of about 30 drug-resistant clones that werethen isolated, expanded and further characterized.Most of these clones exhibited reduced susceptibilityto compound 1, with EC50 values that were at leastthree to four times higher than those of the wild-typereplicon cells. The RNA of the clones with at least afour-fold shift in inhibitor susceptibility was isolated,and a PCR fragment corresponding to the NS5Bcoding region was sequenced. Sequence analysesshowed that replicons isolated from resistant clonescontained mutations that were not present in thereplicon of cells not treated with the inhibitor.

Some of the mutations that were detected matchedpreviously reported mutations, namely in V494,P495 and P496. However, one of the resistant clonesharbored the new mutation Leu392Ile in NS5B. Therole of this mutation in HCV 1b polymeraseresistance to the indole-N-acetamide inhibitors wasconfirmed by a reverse genetic experiment where thesingle Leu392Ile mutation was introduced in thewild-type RNA replicon and transiently transfectedin Huh-7 cells. This mutant replicon resulted in 20-fold more resistance to compound 1 than the wild-type replicon clone.

Conclusions

In this article, we report the X-ray crystal structureof the ΔC21 HCV polymerase from genotype 2b andthat of a panel of 1b→2b single mutants andchimera enzymes that were characterized for theirsusceptibility to inhibition and their binding affi-nities to an indole-N-acetamide NNI. The structurewas found in a closed conformation, as previouslyobserved for the genotype 1b polymerase and for the2a polymerase. Slight changes were observed in theconformation of the fingertip and in the C-terminalregion, confirming the flexibility of these regions ofthe molecule. No large rearrangement of α-helix A ofthe Λ1 loop, as observed in the 2a polymerase incrystal form 2, was observed although comparison ofthe two independent molecules present in theasymmetric unit of the crystal and that with other1b polymerase structures suggest that this region isindeed conformationally flexible.The structure of the HCV 2b polymerase provides

a plausible explanation for the observed resistanceof the 2b enzyme to the indole-based NNIs.Comparison of the 1b and 2b indole-N-acetamideNNI binding pockets reveals that, together, theconservative mutations in the amino acids formingthe binding site result in an altered pocket shape. Inparticular, mutations of Leu392Ile and Ala393Thrcause a narrowing of the pocket in proximity of thephenyl ring of the inhibitor, whereas mutations ofVal494Ala and Leu425Ile result in a deeper pocket,thus decreasing the number of van der Waalscontacts with the indole core of the inhibitor. Thestructural analysis is supported by the results of ourmutagenesis and resistance-to-inhibition studies.Among all the mutants, Leu392Ile and Val494AlaNS5B enzymes were found to be the most resistant,15- and 7-fold, respectively. Furthermore, we foundthat the Leu392Ile mutation in the 1b repliconconfers 20-fold resistance to inhibition by anindole-N-acetamide NNI without affecting NS5Breplication efficiency. Combination of the Leu392Ileand Val494Ala mutations with Val499Ala, a muta-tion that only marginally affected resistance in vitro,resulted in an NS5B enzyme with a 50-fold reductionin sensitivity to inhibition by the indole-N-acetamideNNIs. Thus, these three mutations together mayaccount for a large fraction of the resistance of theHCV 2b polymerase to this class of molecules.


Notably, Thr393Ala resulted in a 4-fold greatersusceptibility to inhibition instead of being resistant,as suggested by the structural analysis. However, ithas to be noted that this mutant was 10-fold lessactive in our activity assay. It is plausible that theincreased susceptibility to the indole inhibitors ofthis mutant may be due to changes in its enzymaticmechanism. For instance, this mutation mightdisfavor the elongation step compared with theinitiation step, resulting in an overall increasedsusceptibility to these inhibitors.The affinities of wild type, mutants and chimera

enzymes for the inhibitor were then measured bysurface plasmon resonance. This study confirmedthat the decrease in inhibitor susceptibility wasassociated with a decrease in binding affinity.Analysis of the binding curves showed that, withthe exception of Leu392Ile, the kon of binding lieswithin 1 order of magnitude for most of the mutantstested and for both the 1b and 2b enzymes,demonstrating that resistance is not caused by adecreased mobility of the fingertip α-helix A and/ora lower accessibility to the binding pocket. Rather,the binding studies show that dissociation (i.e.,stability of the complex) is faster for the 2b enzymeand for the “pocket” and “entire site A” chimerapolymerases, lowering the complex half-life (Fig. 7).In conclusion, the present study suggests that the

conservative mutations in the NNI site A bindingpocket are at the basis of the observed resistance ofthe 2b HCV polymerase to the indole-N-acetamideclass of inhibitors. Although we identified residuesin the inhibitor binding site that contribute toresistance, we cannot exclude that other mutationsin other regions of the enzyme, farther away fromthe binding pocket, might also partially contributeto resistance (e.g., by affecting the mobility of thehelices containing residues that participate in theformation of the pocket). Finally, it is important tonote that the mutations we identified can arise in the1b polymerase by simply one base change in theHCV 1b genome and therefore may easily emergeupon treatment with this class of inhibitors.

Materials and Methods

Cloning and mutagenesis

The cloning of 1Bcon1 and 2B.2 NS5B ΔC21⋅His6-pT7(2bdc21) has been described previously.29,35 All pointvariants and chimeras were generated using eitherQuikChange XLII or QuikChange Multi kits (Stratagene)as per the manufacturer's instructions.

Protein expression and purification

The 2bdc21 enzyme was expressed from Escherichia colistrain BL21 Codon Plus (Novagen). After growth to anoptical density at 600 nm of 0.8, the cells were inducedwith 0.4 mM IPTG and incubated at 18 °C for 23 h beforeharvesting by centrifugation. Following resuspension ofthe pellet in buffer A [10 mM Tris–HCl, pH 8.0, 300 mM

NaCl, 10% (v/v) glycerol, 10 mM β-mercaptoethanol andprotease inhibitor cocktail (Roche)], the cells were lysed bya microfluidizer. The suspension was then stirred withDNase I (5 U/mL) and MgCl2 (10 mM) for 30 min at 4 °Cbefore centrifuging at 18,000g for 45 min. The clarifiedsolution was then subjected to Ni-nitrilotriacetic acidchromatography (5 mL; Qiagen). Protein was eluted witha step gradient of imidazole (20, 40, 200 and 500 mM), andfractions containing NS5B, as judged by SDS-PAGE andWestern blotting, were pooled and dialyzed overnightagainst buffer B [10 mM Hepes, pH 8.0, 400 mM NaCl,10% (v/v) glycerol, 5 mM DTT and 1 mM ethylenediami-netetraacetic acid]. The dialyzed protein was furthersubjected to heparin chromatography (5-mL HiTrapHeparin column, Amersham). The bound protein waseluted with a linear NaCl gradient (400 mM to 1.0 MNaClover 5 column volumes), and the fractions containing2bdc21 (as judged by SDS-PAGE and Western blotting)were pooled and concentrated before applying to aSuperdex-200 gel-filtration column (Amersham) equili-brated in buffer B containing 600 mM NaCl. The peakfractions, now N95% pure as judged by SDS-PAGE, werepooled and concentrated to 6.5 mg/mL.Expression of 1Bcon1 NS5B ΔC21⋅His6 wild type and

variants was performed in a 3-L fermentor (BioFlo-3000,New Brunswick Scientific) under batch conditions. Anovernight culture was used to inoculate (1:100 v/v) aminimal medium [50 mM KPi, 25 mM (NH3)2SO4, 0.5 mMCaCl2, 1 mM MgSO4, 30 g/L of glucose, supplementedwith trace metals]. The fermentor culture was grown at30 °C to an optical density at 600 nmof 10 before expressionwas induced at 18 °C with 0.4 mM IPTG. The cells werefurther incubated for 23 h before harvesting. Purificationwas performed similarly to the 2bdc21 NS5B with thefollowing exceptions: lysis was performed by cell disruptorafter freeze-thawing, DNase I digestion was carried out for2 h and no gel filtration was performed.Expression levels of the 1b-con1 variants varied greatly

in yield; for example, the Ftip and Pocket chimerasyielded 2 mg of purified protein from a 3-L fermentorculture, while V494A had a 10-fold higher yield.Solubility of most variants was not a concern; however,during purification of the Ftip chimera, precipitate wasobvious in the peak fractions from Ni-nitrilotriacetic acidchromatography. Nevertheless, no difference in aggrega-tion was observed between the clarified Ftip chimera andwild-type 2b as judged by dynamic light scattering (datanot shown).

RdRp assay and IC50 determinations

The standard HCV RdRp assay used here has beenpreviously reported.24,36 Briefly, between 2 and 100 nMenzyme (dependent on the activity of the variant) waspreincubated with template/primer (polyA/oligoU) for10 min at room temperature prior to addition of 3H-UTP.The 50-μL reaction was further incubated for 90 min atroom temperature with shaking. An equal volume of 20%trichloroacetic acid/20 mM sodium pyrophosphate wasadded, followed by 5-min incubation on ice, to stop thereaction. The samples were then filtered using UnifilterGF/B plates (Packard), and the scintillation was read in aTOPCOUNT (Packard) after addition of 50 μL ofMicroscint(Packard).For inhibition assays, the same protocol was followed

with the addition of a 10-min preincubation of enzymewith inhibitor (compound 1 in Table 1). Synthesis ofcompound 1 has been previously described.37 Data forIC50 curves were produced by plotting the percentage


inhibition from serial dilutions of compound 1 againstcompound concentration and fitting to a four-parameterlogistic equation using KaleidaGraph software.

Protein crystallization and data collection

The 2bdc21 protein was crystallized by the sitting-dropvapor-diffusion method combining 2 μL of enzymesolution (6.5 mg/mL) and 2 μL of well solution(100 mM Na citrate, pH 6.1, 200 mM NaCl, 10%polyethylene glycol 4000 and 5 mM DTT). Large, thinplates (300 μM×200 μM×20 μM) grew in 1 day. For datacollection, crystals were transferred to cryo-protectantsolution (mother liquor containing 20% glycerol) and wereflash frozen in liquid nitrogen. X-ray diffraction data to1.9-Å resolution were collected at the European Synchro-tron Radiation Facility (Grenoble, France) at 100 K with anADSC Q4 CCD detector. The crystals belonged to themonoclinic C2 space group with a=153.25 Å, b=64.49 Å,c=135.58 Å, β=90.16° and two molecules per asymmetricunit. DENZO and SCALEPACK38 were used to integrateand scale the data, respectively. The CCP4 suite ofprograms39 was used for further data processing. Finaldata collection statistics are summarized in Table 3.

Structure determination and refinement

The structure of 2bdc21 was solved by molecularreplacement with MOLREP.40 A monomer of the 1bHis6-HCV NS5B ΔC219 was used as search model (PDBID 1C2P). All non-conserved residues and the residues ofNNI binding site A were replaced by alanine and theentire Λ1 loop (amino acids 20–40) was omitted from theinitial model to reduce model bias for initial electrondensity map calculation. Model building and correctionwere performed in O41 using σA-weighted electrondensity maps as calculated by Arp/wArp in Molrepmode.42 Positional refinement was performed in Crystal-lography & NMR System43 using a maximum-likelihoodtarget.44 A bulk solvent correction and anisotropic B-factortensor were applied throughout the refinement. Near theend of refinement, water molecules were added andindividual B-factors were refined. The final model includes1116 of the total 1152 residues in the asymmetric unit and1300 water molecules. Weak or no electron density wasobserved for residues 149–153, 564–570 and the C-terminalHis6 tag in both molecules of the asymmetric unit. Noresidue was located in disallowed regions of the Rama-chandran plot as assessed with PROCHECK.45 Refinementstatistics are presented in Table 3.

Surface plasmon resonance measurements

Surface plasmon resonance analysis was performedusing BIACORE 3000 optical biosensor with research-grade CM5 sensor chips (Biacore AB, Uppsala, Sweden).The protein was immobilized using standard amine-coupling chemistry as described by the chip supplier(Biacore, Inc.). The protein at a concentration of 50 nM in10 mM 4-morpholineethanesulfonic acid, pH 6.4, wasinjected for 7 min, resulting in immobilized densitiesaveraging 1000±300 RU (resonance units), which wassufficient to bind a range of 10–100 RU of inhibitors. In allcases, the surfaces were blocked with a 7-min injection of1.0 M ethanolamine, pH 8.0. Flow rates were 40 μL/minduringtheassociation–dissociationphase.Foreachmutant,tests were performed to verify that mass transfer was

negligible. All measurementsweremade at 25 °C in 10mMHepes, pH 7.4, containing 150mMNaCl, 3.4mM ethylene-diaminetetraacetic acid and 0.005% surfactant P-20. Inhi-bitor concentrations used were between 10 nM and 10 μM,dependent on thepolymerasemutant immobilized.Experi-ments used for kinetic constant evaluationwere performedallowing180sof association-phase contact timeand700 sofdissociation-phase contact time, while experiments per-formed at equilibrium times were performed allowing 360and 700 s, respectively. Dissociation–regeneration stepswere obtained using short pulses of 15 s of NaCl at500 mM. Parameter evaluation was obtained using theBiacore Biaevaluation software. Baseline correction wasobtained using the double-reference method, whereas thereference-corrected curves were further subtracted by ablank injection of buffer. The mathematical model usedin the fitting was the 1:1 model with no mass transfer; Kdvalues were obtained either by simultaneous fit of bothkon and koff values or by equilibrium analysis.

Selection of indole-N-acetamide-resistant replicons

Replicon-harboringHBI10A cellswere cultured and clonesresistant to indole-N-acetamide were selected as describedpreviously.46 An indole-N-acetamide inhibitor was dissolvedin dimethyl sulfoxide and serially diluted such that the finaldimethyl sulfoxide concentration was below 1%. HBI10Acells were plated in 15-cm tissue culture dishes at a density of3×103/cm2 and cultured in the presence of 0.8 mg/mL ofG418 and inhibitor at concentrations increasing from 0.2 to1μM.Approximately 15 days after the beginning of selection,small colonies of cells resistant to inhibitor and antibioticbecame visible and were isolated. The sequence of HCVreplicons present in drug-resistant clones was obtainedfollowing extraction of replicon RNA from resistant clones,reverse-transcriptase PCR and direct sequencing of PCRproducts.46 The effect of indole-N-acetamide on viral replica-tion and the replication proficiency of the mutant repliconswere estimated by monitoring expression of the NS3 proteinby cell ELISA with the anti-NS3 Mab 10E5/24 as describedpreviously.46 For reverse genetic experiments, the selectedmutation was introduced in the wild-type replicon and RNAtransfected by electroporation in 10AIFN cells as describedpreviously.46 Cells were supplemented with the compoundsbetween 1 and 4 h after transfection.

Accession number

Coordinates and the structure factors have beendeposited in the PDB with accession number 3GSZ.

Acknowledgements

We thank Stefania Di Marco, Licia Tomei andMatthew Bottomley for discussions and the staff atbeamline ID14-2 of the European SynchrotronRadiation Facility for help during data collection.This paper is dedicated to the memory of our belovedcolleague and friend Giovanni Migliaccio.Dedication. This paper is dedicated to the memory

of our beloved colleague and friend GiovanniMigliaccio.


References

1. WHO (January2006, postingdate). Initiative forVaccineResearch (IVR): hepatitis C [Online]. http://www.who.int/vaccine_research/diseases/viral_cancers/en/index2.html.

2. Feld, J. J. & Hoofnagle, J. H. (2005). Mechanism ofaction of interferon and ribavirin in treatment ofhepatitis C. Nature, 436, 967–972.

3. Simmonds, P. (2004). Genetic diversity and evolutionof hepatitis C virus—15 years on. J. Gen. Virol. 85,3173–3188.

4. Bowen, D. G. & Walker, C. M. (2005). The origin ofquasispecies: cause or consequence of chronic hepati-tis C viral infection? J. Hepatol. 42, 408–417.

5. Reed, K. E. & Rice, C. M. (2000). Overview of hepatitisC virus genome structure, polyprotein processing,and protein properties. Curr. Top. Microbiol. Immunol.242, 55–84.

6. Ago, H., Adachi, T., Yoshida, A., Yamamoto, M.,Habuka, N., Yatsunami, K. & Miyano, M. (1999).Crystal structure of the RNA-dependent RNA poly-merase of hepatitis C virus. Structure, 7, 1417–1426.

7. Bressanelli, S., Tomei, L., Roussel, A., Incitti, I., Vitale,R. L., Mathieu, M. et al. (1999). Crystal structure of theRNA-dependent RNA polymerase of hepatitis Cvirus. Proc. Natl Acad. Sci. USA, 96, 13034–13039.

8. Bressanelli, S., Tomei, L., Rey, F. A. & De Francesco, R.(2002). Structural analysis of the hepatitis C virusRNA polymerase in complex with ribonucleotides.J. Virol. 76, 3482–3492.

9. Lesburg, C. A., Cable, M. B., Ferrari, E., Hong, Z.,Mannarino, A. F. & Weber, P. C. (1999). Crystalstructure of the RNA-dependent RNA polymerasefrom hepatitis C virus reveals a fully encircled activesite. Nat. Struct. Biol. 6, 937–943.

10. Wang, M., Ng, K. K., Cherney, M. M., Chan, L.,Yannopoulos, C. G., Bedard, J. et al. (2003). Non-nucleoside analogue inhibitors bind to an allostericsite on HCV NS5B polymerase. Crystal structuresand mechanism of inhibition. J. Biol. Chem. 278,9489–9495.

11. O'Farrell, D., Trowbridge, R., Rowlands, D. & Jager, J.(2003). Substrate complexes of hepatitis C virus RNApolymerase (HC-J4): structural evidence for nucleo-tide import and de-novo initiation. J. Mol. Biol. 326,1025–1035.

12. Biswal, B. K., Cherney, M. M., Wang, M., Chan, L.,Yannopoulos, C. G., Bilimoria, D. et al. (2005). Crystalstructures of the RNA dependent RNA polymerasegenotype 2a of hepatitis C virus reveal two conforma-tions and suggest mechanisms of inhibition by non-nucleoside inhibitors. J. Biol. Chem. 280, 18202–18210.

13. Butcher, S. J., Grimes, J. M., Makeyev, E. V., Bamford,D. H. & Stuart, D. I. (2001). A mechanism for initiatingRNA-dependent RNA polymerization. Nature, 410,235–240.

14. Choi, K. H., Groarke, J. M., Young, D. C., Kuhn, R. J.,Smith, J. L., Pevear, D. C. & Rossmann, M. G. (2004).The structure of the RNA-dependent RNA polymer-ase from bovine viral diarrhea virus establishes therole of GTP in de novo initiation. Proc. Natl Acad. Sci.USA, 101, 4425–4430.

15. Ferrer-Orta, C., Arias, A., Perez-Luque, R., Escarmis,C., Domingo, E. & Verdaguer, N. (2004). Structure offoot-and-mouth disease virus RNA-dependent RNApolymerase and its complex with a template–primerRNA. J. Biol. Chem. 279, 47212–47221.

16. De Francesco, R. & Carfi, A. (2007). Advances in thedevelopment of new therapeutic agents targeting theNS3-4A serine protease or the NS5B RNA-dependentRNA polymerase of the hepatitis C virus. Adv. DrugDelivery Rev. 59, 1242–1262.

17. Kukolj, G., McGibbon, G. A., McKercher, G., Marquis,M., Lefebvre, S., Thauvette, L. et al. (2005). Binding sitecharacterization and resistance to a class of non-nucleoside inhibitors of the hepatitis C virus NS5Bpolymerase. J. Biol. Chem. 280, 39260–39267.

18. Di Marco, S., Volpari, C., Tomei, L., Altamura, S.,Harper, S., Narjes, F. et al. (2005). Interdomaincommunication in hepatitis C virus polymeraseabolished by small molecule inhibitors bound to anovel allosteric site. J. Biol. Chem. 280, 29765–29770.

19. Biswal, B. K., Wang, M., Cherney, M. M., Chan, L.,Yannopoulos, C. G., Bilimoria, D. et al. (2006). Non-nucleoside inhibitors binding to hepatitis C virusNS5B polymerase reveal a novel mechanism ofinhibition. J. Mol. Biol. 361, 33–45.

20. Love, R. A., Parge, H. E., Yu, X., Hickey, M. J.,Diehl, W., Gao, J. et al. (2003). Crystallographicidentification of a noncompetitive inhibitor bindingsite on the hepatitis C virus NS5B RNA polymeraseenzyme. J. Virol. 77, 7575–7581.

21. Pfefferkorn, J. A., Greene, M. L., Nugent, R. A., Gross,R. J., Mitchell, M. A., Finzel, B. C. et al. (2005).Inhibitors of HCV NS5B polymerase: Part 1. Evalua-tion of the southern region of (2Z)-2-(benzoylamino)-3-(5-phenyl-2-furyl)acrylic acid. Bioorg. Med. Chem.Lett. 15, 2481–2486.

22. Tedesco, R., Shaw, A. N., Bambal, R., Chai, D.,Concha, N. O., Darcy, M. G. et al. (2006). 3-(1,1-dioxo-2H-(1,2,4)-benzothiadiazin-3-yl)-4-hydroxy-2(1H)-quinolinones, potent inhibitors of hepatitis Cvirus RNA-dependent RNA polymerase. J. Med.Chem. 49, 971–983.

23. Harper, S., Pacini, B., Avolio, S., Di Filippo, M.,Migliaccio, G., Laufer, R. et al. (2005). Developmentand preliminary optimization of indole-N-acetamideinhibitors of hepatitis C virus NS5B polymerase. J. Med.Chem. 48, 1314–1317.

24. Tomei, L., Altamura, S., Bartholomew, L., Biroccio,A., Ceccacci, A., Pacini, L. et al. (2003). Mechanismof action and antiviral activity of benzimidazole-based allosteric inhibitors of the hepatitis C virusRNA-dependent RNA polymerase. J. Virol. 77,13225–13231.

25. Tomei, L., Altamura, S., Bartholomew, L., Bisbocci,M., Bailey, C., Bosserman, M. et al. (2004). Char-acterization of the inhibition of hepatitis C virusRNA replication by nonnucleosides. J. Virol. 78,938–946.

26. Beaulieu, P. L. & Tsantrizos, Y. S. (2004). Inhibitors ofthe HCV NS5B polymerase: new hope for thetreatment of hepatitis C infections. Curr. Opin. Invest.Drugs, 5, 838–850.

27. Li, H., Tatlock, J., Linton, A., Gonzalez, J., Borchardt,A., Dragovich, P. et al. (2006). Identification andstructure-based optimization of novel dihydropyr-ones as potent HCV RNA polymerase inhibitors.Bioorg. Med. Chem. Lett. 16, 4834–4838.

28. Ma, H., Leveque, V., De Witte, A., Li, W., Hendricks,T., Clausen, S. M. et al. (2005). Inhibition of nativehepatitis C virus replicase by nucleotide and non-nucleoside inhibitors. Virology, 332, 8–15.

29. Graham, D. J., Stahlhut, M., Flores, O., Olsen, D. B.,Hazuda, D. J., Lafemina, R. L. & Ludmerer, S. W.(2006). A genotype 2b NS5B polymerase with novel


substitutions supports replication of a chimeric HCV1b:2b replicon containing a genotype 1b NS3-5Abackground. Antiviral Res. 69, 24–30.

30. Adachi, T., Ago, H., Habuka, N., Okuda, K., Komatsu,M., Ikeda, S. & Yatsunami, K. (2002). The essential roleof C-terminal residues in regulating the activity ofhepatitis C virus RNA-dependent RNA polymerase.Biochim. Biophys. Acta, 1601, 38–48.

31. Pfefferkorn, J. A., Nugent, R., Gross, R. J., Greene, M.,Mitchell, M. A., Reding, M. T. et al. (2005). Inhibitors ofHCV NS5B polymerase: Part 2. Evaluation of thenorthern region of (2Z)-2-benzoylamino-3-(4-phe-noxy-phenyl)-acrylic acid. Bioorg. Med. Chem. Lett.15, 2812–2818.

32. Powers, J. P., Piper, D. E., Li, Y., Mayorga, V., Anzola,J., Chen, J. M. et al. (2006). SAR and mode of action ofnovel non-nucleoside inhibitors of hepatitis C NS5bRNA polymerase. J. Med. Chem. 49, 1034–1046.

33. Lee, G., Piper, D. E., Wang, Z., Anzola, J., Powers, J.,Walker, N. & Li, Y. (2006). Novel inhibitors of hepatitisC virus RNA-dependent RNA polymerases. J. Mol.Biol. 357, 1051–1057.

34. Nguyen, T. T., Gates, A. T., Gutshall, L. L., Johnston,V. K., Gu, B., Duffy, K. J. et al. (2003). Resistanceprofile of a hepatitis C virus RNA-dependent RNApolymerase benzothiadiazine inhibitor. Antimicrob.Agents Chemother. 47, 3525–3530.

35. Ludmerer, S.W.,Graham,D. J., Boots, E.,Murray, E.M.,Simcoe, A., Markel, E. J. et al. (2005). Replication fitnessand NS5B drug sensitivity of diverse hepatitis C virusisolates characterized by using a transient replicationassay. Antimicrob. Agents Chemother. 49, 2059–2069.

36. Tomei, L., Vitale, R. L., Incitti, I., Serafini, S., Altamura,S., Vitelli, A. & De Francesco, R. (2000). Biochemicalcharacterization of a hepatitis C virus RNA-depen-dent RNA polymerase mutant lacking the C-terminalhydrophobic sequence. J. Gen. Virol. 81, 759–767.

37. Harper, S., Avolio, S., Pacini, B., Di Filippo, M.,Altamura, S., Tomei, L. et al. (2005). Potent inhibitorsof subgenomic hepatitis C virus RNA replication

through optimization of indole-N-acetamide allostericinhibitors of the viral NS5B polymerase. J. Med. Chem.48, 4547–4557.

38. Otwinowski, Z. & Minor, W. (1997). Processing of X-ray diffraction data collected in oscillation mode.Methods Enzymol. 276, 307–326.

39. Collaborative Computational Project No. 4. (1994).The CCP4 suite: programs for protein crystallogra-phy. Acta Crystallogr., Sect. D: Biol. Crystallogr. 50,760–763.

40. Vagin, A. & Teplyakov, A. (1997). MOLREP: anautomated program for molecular replacement. J. Appl.Crystallogr. 30, 1022–1025.

41. Jones, T. A., Zou, J. Y., Cowan, S. W. & Kjeldgaard, M.(1991). Improved methods for building proteinmodels in electron density maps and the location oferrors in these models. Acta Crystallogr., Sect. A: Found.Crystallogr. 47, 110–119.

42. Perrakis, A., Morris, R. & Lamzin, V. S. (1999).Automated protein model building combined withiterative structure refinement. Nat. Struct. Biol. 6,458–463.

43. Brunger, A. T., Adams, P. D., Clore, G. M., DeLano,W.L., Gros, P., Grosse-Kunstleve, R. W. et al. (1998).Crystallography &NMR System: a new software suitefor macromolecular structure determination. ActaCrystallogr., Sect. D: Biol. Crystallogr. 54, 905–921.

44. Brünger, A. T., Adams, P. D. & Rice, L. M. (1998).Recent developments for the efficient crystallographicrefinement of macromolecular structures. Curr. Opin.Struct. Biol. 8, 606–611.

45. Laskowski, R. A., MacArthur, M. W., Moss, D. S. &Thornton, J. M. (1993). PROCHECK: a program tocheck the stereochemical quality of protein structures.J. Appl. Crystallogr. 26, 283–291.

46. Trozzi, C., Bartholomew, L., Ceccacci, A., Biasiol, G.,Pacini, L., Altamura, S. et al. (2003). In vitro selectionand characterization of hepatitis C virus serineprotease variants resistant to an active-site peptideinhibitor. J. Virol. 77, 3669–3679.

Structural Basis for Resistance of the Genotype 2b Hepatitis C Virus NS5B Polymerase to Site A...

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