General Catalytic Deficiency of Hepatitis C Virus RNA Polymerase with an S282T Mutation and Mutually Exclusive Resistance towards 2'-Modified Nucleotide Analogues

The hepatitis C virus (HCV) RNA-dependent RNA polymerase NS5Bis an important target for antiviral therapies. NS5B is ableto initiate viral RNA synthesis de novo and then switch to afast and processive RNA elongation synthesis mode. The nucleotideanalogue 2'-C-methyl CTP (2'-C-Me-CTP) is the active metaboliteof NM283, a drug currently in clinical phase II trials. Theresistance mutation S282T can be selected in HCV replicon studies.Likewise, 2'-O-Me nucleotides are active both against the purifiedpolymerase and in replicon studies. We have determined the molecularmechanism by which the S282T mutation confers resistance to2'-modified nucleotide analogues. 2'-C-Me-CTP is no longer incorporatedduring the initiation step of RNA synthesis and is discriminated21-fold during RNA elongation by the NS5B S282T mutant. Strikingly,2'-O-methyl CTP sensitivity does not change during initiation,but the analogue is no longer incorporated during elongation.This mutually exclusive resistance mechanism suggests not onlythat "2'-conformer" analogues target distinct steps in RNA synthesisbut also that these analogues have interesting potential incombination therapies. In addition, the presence of the S282Tmutation induces a general cost in terms of polymerase efficiencythat may translate to decreased viral fitness: natural nucleotidesbecome 5- to 20-fold less efficiently incorporated into RNAby the NS5B S282T mutant. As in the case for human immunodeficiencyvirus, our results might provide a mechanistic basis for therational combination of drugs for low-fitness viruses.

INTRODUCTION

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Introduction

Hepatitis C virus (HCV) infection is the most common blood-borneinfection and a major cause of chronic liver disease in developedcountries. More than 170 million individuals in the world areinfected with HCV, making these individuals at risk of developingliver cirrhosis and hepatocellular carcinoma. Persistent HCVinfection can be controlled by antiviral therapy (30, 40) oreven eradicated from infected patients (43).

Current antiviral therapies rely on the combination of pegylatedalpha interferon and the nucleoside analogue ribavirin (16,18, 29). Less than 50% of treated patients respond when theyare infected with the most prevalent HCV genotype, i.e., genotype1 (23). The HCV RNA-dependent RNA polymerase (RdRp), the NS5Bprotein, plays an essential role in the replication of the viralgenome and is thus an attractive target for the developmentof new antiviral drugs (36). Intensive drug-screening programsled to the discovery of two classes of HCV NS5B inhibitors,namely, nonnucleoside inhibitors (NNIs) and nucleoside inhibitors(NIs). NNIs have been described to bind to one of the threeallosteric sites present at the NS5B surface (for a review,see reference 11). All of the NNIs are noncompetitive inhibitorsrelative to nucleoside triphosphate (NTP) incorporation andtarget the alloenzyme free of substrate. They are inactive whenthe enzyme has entered into the processive elongation phase(3, 17, 31, 45, 46) of in vitro RdRp reactions or when NS5Bis complexed with other nonstructural replicative proteins (28)in replicon-expressing cells. NIs, however, target the activesite and are active on both the purified polymerase and thereplicative complex (7, 28, 39). NM283 is such a compound. Itis an oral prodrug of 2'-C-methyl cytidine (2'-C-Me-cytidine)which is currently under clinical evaluation. 2'-Modified nucleosides,modified with either 2'-C-Me or 2'-O-Me, inhibit RNA replicationin the replicon system and in RdRp assays in vitro (7, 33, 39).Both types of 2'-modified nucleosides promote chain terminationof the neosynthesized RNA when incorporated into nascent RNAby NS5B (7, 33).

Unfortunately, all NNI or NI compounds tested so far in thereplicon system lose efficacy after selection of a single pointmutation in the viral polymerase gene (22, 26, 33-35, 45, 46).Paralleling findings in the human immunodeficiency virus type1 (HIV-1) antiretroviral field, this suggests that viral resistanceto polymerase inhibitors may also occur in patients. To date,no resistant HCVs have been selected during the clinical testingof NM283 (2), although the increase of viral loads observedduring monotherapy suggests the appearance of such resistantmutants. 2'-C-Me-purine nucleoside-resistant replicons can beisolated readily in vitro, and resistance is the result of theS282T substitution in the NS5B gene (S282T NS5B) (33). It hasbeen shown that the S282T substitution increases both 50% inhibitoryconcentrations and K_i values for 2'-C-methyl-modified nucleotidesas well as the ability to extend an analogue-terminated primerrelative to that of the wild type (WT) (33). It was suggestedthat the S282T mutation induces resistance by increased discriminationof the 2'-C-methyl nucleotide analogue at the NS5B active siterelative to the natural substrate NTP. However, the moleculardetails of S282T mutant-mediated resistance are still unclear.Discrimination can result from a selective decrease of eitherthe binding of the analogue (reflected by an increased K_m) orthe catalytic step of analogue incorporation into RNA (reflectedby the decrease of the maximum velocity value [V_max]). Resistancecan also be achieved by the excision of the incorporated drug(32). This particular mechanism is described in vitro for thebovine viral diarrhea virus RdRp, in the case of incorporationof 3'-dNMP (10). No reparation or excision of an NTP analoguehas been reported yet for the HCV NS5B polymerase, and the mechanismleading to discrimination (increased K_m or decreased V_max) ofthe 2'-C-methyl analogue in the context of S282T mutant resistanceis not known.

The Flaviviridae NS5b protein is able to initiate RNA synthesiswithout an RNA primer in a so-called de novo RNA synthesis processas well as to elongate an existing RNA primer (27, 38, 50).The RNA synthesis initiation phase is defined as the formationof the first phosphodiester bridge between two nucleotides toform a diribonucleotide primer. So far, primer-independent RNAsynthesis is unique to viral RNA polymerases. Through evolution,these polymerases have selected unique structural features essentialto the synthesis of their own short RNA primers (4, 6, 9). Thecrystal structure of the HCV polymerase and elegant enzymaticassays have identified some of these structural determinants(4, 19, 24). A peculiar ß-strand-turn-ß-strand(flap) subdomain might belong to the latter. The flap actuallyobstructs the polymerase active site when the site is comparedto those of related primer-dependent polymerases. It has beenproposed that the flap gives physical support to initiatingnucleotides, up to the point where a conformational change occursat the polymerase active site (4, 19, 49). Subsequently, thepolymerase has to adopt an alternate conformation in which theprimer is elongated in a processive fashion, giving rise todouble-stranded RNA (dsRNA). Recently, the flap was shown toplay a role in the repression of primer-directed RNA synthesisin favor of the initiation of RNA synthesis (41).

Using different template systems discriminating initiation fromelongation, we have previously shown that these two steps ofRNA synthesis are structurally and kinetically distinct andmay thus have different nucleotide analogue susceptibilities(15). For example, 2'-O-methyl GTP is incorporated during theRNA elongation step only by WT NS5B. In view of optimal futureHCV drug combination therapies, it is of prime importance tounderstand, at the molecular level, which viral RNA synthesissteps are targeted by a given drug as well as which resistancemechanisms are at play at the NS5B active site.

In this work, we have analyzed the use of 2'-C-methyl and 2'-O-methylCTP (2'-C-Me-CTP and 2'-O-Me-CTP) and the natural CTP substrateby WT and S282T NS5B during initiation and elongation of RNAsynthesis. We show that they have opposite resistance profilesduring initiation and elongation of RNA synthesis and that theS282T mutant induces a general decrease in the efficiency ofincorporation of natural nucleotides.

MATERIALS AND METHODS

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Materials and Methods

HCV 1b polymerase, plasmid constructs, enzyme preparation, and reagents. WT and mutant NS5B-D55 genes were tagged with six C-terminalhistidines and expressed from the pDest 14 vector (Invitrogen)in Escherichia coli BL21(DE3) cells (Novagen). Site-directedmutagenesis was performed using a QuikChange site-directed mutagenesiskit according to the manufacturer's instructions (Stratagene).All constructs were verified by DNA sequencing. WT and S282TNS5B proteins were purified as described previously (15). RNAmolecular weight markers were synthesized using T7 RNA polymeraseand the appropriate template RNAs as previously described (15).RNA oligonucleotides were obtained from MWG-Biotech. DNA oligonucleotideswere obtained from Life Technologies. For RNA elongation experiments,RNA oligonucleotides were 5' ${alpha}$ -³²P labeled, using T4 polynucleotidekinase (New England Biolabs). ${gamma}$ -³²P-labeled ATP (3,000 Ci/mmol), ${alpha}$ -³²P-labeled GTP (3,000 Ci/mmol), and ${alpha}$ -³²P-labeled CTP (3,000Ci/mmol) were purchased from Amersham. 2'-O-Me-CTP was purchasedfrom Trilink, Inc. 2'-C-Me-CTP was kindly provided by Idenix,Inc.

Steady-state kinetics of RNA synthesis using homopolymeric templates. Polymerase activity was assayed by monitoring the incorporationof radiolabeled guanosine into 15-mer cytidine RNA oligonucleotidetemplates, as described previously (15).

Determination of V_max and K_m. An RNA oligonucleotide corresponding to the 3' end of the negativestrand of the HCV genome [RNA H (–) (5'-UCGGGGGCUGGC-3')]or corresponding to a modified 3' end of the positive strandof the HCV genome [RNA H (+2) (5'-CAGAUCAGGU-3')] was used toanalyze the synthesis of the first GC or AC phosphodiester bond.Reactions were performed as described previously (15), using100 µM [ ${alpha}$ -³²P]CTP (1 µCi) and GTP or ATP (1, 5, 10,50, 100, and 500 µM). For V_max and K_m values for CTP orCTP analogue incorporation upon initiation, reactions were assayedwith 100 µM [ ${alpha}$ -³²P]GTP (1 µCi), using increasingconcentrations of either CTP, 2'-C-Me-CTP, or 2'-O-Me-CTP (1,5, 10, 50, 100, and 500 µM). RNA elongation was measuredusing a 5' ${alpha}$ -³²P-labeled hairpin RNA template (HP4 [5'-UGACGGCCCGGAAAACCGGGCC-3']for GTP incorporation, HP3 [5'-ACUGGGCCCGGAAAACCCGGGCC-3'] forCTP incorporation, and HP2 [5'-GACUGGCCCGGAAAACCGGGCC-3'] forATP incorporation). Products were separated using sequencinggel electrophoresis and quantitated using photostimulated platesand a Fuji imager. Product formation during the initial reactionwas assayed as described previously (15) and fitted to a linearcurve as follows: product = V_it, where t is the time in seconds.The dependence of V_i on the NTP concentration is described bythe hyperbolic equation V_i = V_max(NTP)/(K_m + NTP), where V_maxand K_m are the maximal velocity and the apparent affinity constantof NTP incorporation by NS5B, respectively. V_max and K_m weredetermined by curve fitting using Kaleidagraph (Synergy Software).

Molecular modeling. An initiation complex model was built from the superpositionof NS5B structures in complex with UTP (Protein Data Bank no.1GX6) (4) or ssRNA (Protein Data Bank no. 1NB7) (37) onto theternary complex of HIV-1 reverse transcriptase (RT; ProteinData Bank no. 1RTD) (20). The final model consists of WT NS5B,ssRNA, GTP in the +1 position, and CTP in the +2 position. CTPwas then replaced by 2'-C-Me-CTP or 2'-O-Me-CTP. The replacementof residue 282 by a threonine led to the mutant model of theinitiation complex. All models were constructed with the TURBOprogram (42).

RESULTS

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Results

2'-C-Me-CTP and 2'-O-Me-CTP give opposite S282T mutant-mediated resistance profiles during initiation. Figure 1 shows RNA synthesis using the HCV (–) 3'-endRNA as a template. Table 1 shows the corresponding enzymaticconstants determined after product quantitation as describedpreviously (15). WT NS5B incorporates 2'-C-Me-CMP and CMP tothe same extent. Comparatively, although still incorporated,2'-O-Me-CTP yields a pppGC_2'-_O_-Me product with a much lowerefficiency (Table 1 and Fig. 1B). Indeed, the K_m and V_max valuesare similar for 2'-C-Me-CTP and CTP, whereas there is a 14-foldincrease in K_m for 2'-O-Me-CTP (K_m _2'-_O_-methyl-CTP = 189 µMand K_m _CTP = 13 µM). As previously reported, 2'-C-Me-cytidineand 2'-O-Me-cytidine are effective chain terminators when incorporatedinto nascent RNA. No elongation of the pppGC product was observedwhen products were terminated with these analogues, whereasthe pppGCC product was formed in the presence of the naturalCTP (Fig. 1A).

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FIG. 1. Incorporation of CTP, 2'-O-Me-CTP, and 2'-C-Me-CTP into RNA during initiation. (A) Time course of CTP, 2'-O-Me-CTP, and 2'-C-Me-CTP incorporation into RNA during initiation by WT NS5B polymerase, analyzed by 20% denaturing gel electrophoresis. The RNA template (shown at the top) allows the synthesis of pppGC and ppGCC products as described in Materials and Methods. The migration positions of radiolabeled GTP, pppGC, and pppGCC are shown on the left. (B) The experiment shown in panel A was repeated for various CTP and CTP analogue concentrations, the pppGC product was quantitated, and the initial velocity was calculated for each CTP or CTP analogue concentration. Results are represented as the initial velocity for each CTP ( ${circ}$ ), 2'-C-Me-CTP ( ${square}$ ), or 2'-O-Me-CTP ( ${diamond}$ ) concentration as a function of the NTP concentration. Hyperbolic fitting of the data was used to determine values of the enzymatic constants V_max and K_m. (C) The experiment shown in panel A was performed using the S282T NS5B mutant under the same conditions. (D) Product formation by the S282T NS5B mutant obtained with different CTP and CTP analogue concentrations was quantitated, and results were fitted to a hyperbolic equation to determine V_max and K_m values.

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TABLE 1. Steady-state constants of wild-type and S282T NS5B polymerases for RNA templates during initiation^a

S282T NS5B is able to incorporate the natural CTP substrateleading to the formation of the pppGC and pppGCC products (Fig.1C). However, there is a threefold decrease in the catalyticefficiency in the incorporation of CTP (V_max/ K_m _CTP = 0.35x 10^–3) relative to that of the WT (V_max/ K_m _CTP = 0.92x 10^–3) (Table 1) due to a fivefold decrease of V_max.We then measured the incorporation of CTP analogues but couldnot detect any use of 2'-C-Me-CTP, reflecting a drastic effectof the S282T mutation. However, 2'-O-Me-CTP is still a substratefor the S282T mutant (Fig. 1C), but there is a dramatic effecton V_max, which is sixfold lower (V_max = 0.8 x 10^–3 s^–1)than that of the WT enzyme (V_max = 4.7 x 10^–3 s^–1)(Table 1 and Fig. 1D). This V_max decrease is nevertheless balancedby a fourfold increase in affinity (K_m _2'-_O_-Me-CTP = 48 µMfor the S282T mutant and 189 µM for the WT) (Table 1 andFig. 1D). This leads to a catalytic efficiency similar to thatof the WT enzyme and, therefore, to no resistance (Table 1).

Modeling of CTP and CTP analogues in the NS5B initiation complex. In order to gain further insight into the mechanism leadingto the discrimination of 2'-O-Me-CTP, we generated a model basedon the NS5B crystal structure (5, 24) and various structuralpolymerase-substrate complexes (4, 37). Available structuraldata are believed to be representative of the initiation complex.Thus, our model should only be relevant to results obtainedfrom the analysis of the initiation steps of RNA synthesis.As previously described (33), 2'-C-Me-CTP is found in a positionsimilar to that observed for the natural CTP (not shown), andno steric hindrance is observed (Fig. 2A). In contrast, theaddition of the methyl group at the 2'-O position of the sugarchanges the natural 3'-endo conformation of the sugar to a 2'-endoconformation (44). Therefore, the 2'-O-methyl group is foundin close proximity to threonine 287 (Fig. 2B). This leads toan apparent steric hindrance between the methyl group of themodified CTP and both the methyl group of threonine 287 andthe backbone of serine 282.

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FIG. 2. Modeling of 2'-C-Me-CTP and 2'-O-Me-CTP in the active site of the WT NS5B and S282T mutant polymerases. The GTP and ssRNA of initiation complex models are not shown. The active site of WT (A and B) or S282T (C and D) NS5B appears as a blue surface, with catalytic residues represented by sticks with a transparent surface and Mg²⁺ ions represented as green spheres. Residues 282 and 287 appear as sticks, with part of the surface shown as a green grid. Nucleotide analogues (2'-C-Me-CTP [A and C] and 2'-O-Me-CTP [B and D]) appear as sticks, with the surface of the methyl group shown as an orange grid. The figure was created with VMD (21).

To understand the effect of the resistance mutation S282T, therelative position of the 2'-modified nucleotide was also analyzedin the context of this substitution (Fig. 2C and D). For thesake of clarity, the view in Fig. 2C and D is rotated 180°relative to that in Fig. 2A and B. Strikingly, in the S282TNS5B active site, the 2'-C-methyl group is found in a positionleading to a drastic steric clash with both the methyl groupand the hydroxyl group of the substituted threonine at position282 (Fig. 2C). This steric clash might not be compatible withbinding of this analogue by the S282T NS5B and is consistentwith the lack of incorporation observed in vitro at the initiationstep of RNA synthesis (Fig. 1 and Table 1). In contrast, theaccommodation of the 2'-O-Me-CTP is similar to that for theWT. The steric hindrance involving T287 is still present, andno additional hindrance is induced by the mutation of serine282 to threonine (Fig. 2D). This observation is consistent withthe 21-fold discrimination of this analogue by S282T NS5B, asmeasured in polymerase assays (Fig. 1 and Table 1).

Both 2'-C-Me-CTP and 2'-O-Me-CTP are discriminated by S282T NS5B during elongation. We made use of a hairpin RNA primer template mimicking the elongationstep of RNA synthesis (7, 33) (Fig. 3). In contrast to the casefor initiation, both 2'-O-Me-CTP and 2'-C-Me-CTP are incorporatedinto the hairpin RNA to a similar extent as that for CTP (threefolddiscrimination) (Table 2 and Fig. 3B). This result reflectsa marked relaxation of 2'-O-methyl group discrimination at thepolymerase active site, leading to a 10-fold increase in inhibitorsensitivity once the enzyme has entered the elongation phase.Surprisingly, in the case of S282T NS5B, this scenario is exactlythe opposite of what is observed during the initiation step:no incorporation of 2'-O-Me-CTP can be detected, while 2'-C-Me-CTPis incorporated into the hairpin RNA (Fig. 3C and D). However,the S282T substitution promotes a decrease in the apparent affinityfor 2'-C-Me-CTP, which is reflected by an increased K_m (170µM) (Table 2). The 2'-C-Me-CTP analogue is discriminated21-fold by the S282T enzyme relative to CTP, resulting in aninefold resistance to 2'-C-Me-CTP (Table 2). In the case of2'-O-Me-CTP, no incorporation can be detected, leading to completeresistance.

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FIG. 3. Incorporation of CTP, 2'-C-Me-CTP, and 2'-O-Me-CTP into RNA during elongation. (A) Time course of incorporation of CTP, 2'-C-Me-CTP, and 2'-O-Me-CTP into RNA hairpin template (1 mM, shown on the top) by WT NS5B, analyzed by 14% acrylamide denaturing gel electrophoresis. Migration positions of the hairpin and hairpin +1 template are shown on the left. (B) The experiment shown in panel A was repeated for various CTP and CTP analogue concentrations, the +1 product was quantitated, and the initial velocity was calculated for each CTP or CTP analogue concentration. Results are represented as the initial velocity for each CTP ( ${circ}$ ), 2'-C-Me-CTP ( ${square}$ ), or 2'-O-Me-CTP ( ${diamond}$ ) concentration as a function of the NTP concentration. Hyperbolic fitting of the data was used to determine values of the enzymatic constants V_max and K_m. (C) The experiment shown in panel A was performed using the S282T NS5B mutant under the same conditions. (D) Product formation by the S282T NS5B mutant with different CTP and CTP analogue concentrations was quantitated, and the results were fitted to a hyperbolic equation to determine V_max and K_m values.

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TABLE 2. Steady-state constants of wild-type and S282T NS5B polymerases for RNA templates during elongation^a

General loss of efficiency toward natural nucleotides by S282T NS5B. We noticed that the CTP incorporation efficiency is decreasedthreefold for S282T NS5B relative to that of the WT. If sucha loss of efficiency is true for other NTPs, then this singleNS5B substitution might be one of the factors responsible fora loss of fitness at the viral level. In order to gain insightinto the molecular mechanism of this reduced substrate efficiency,we measured the efficiencies of incorporation of natural nucleotidesinto RNA. We first looked at the kinetics of incorporation ofGTP, using an oligo(C)₁₅ RNA template (Fig. 4). The synthesisof the G2 product, reflecting the initiation step of RNA synthesis(15), is barely affected by the S282T mutation, nor are theG3 and G4 products (Fig. 4). However, the amounts of G5 andlonger products clearly decrease (30% of the amount of productsynthesized by the WT enzyme) (Fig. 4B), although they are stilldetectable, suggesting a more drastic effect of the S282T mutationon the elongation step of RNA synthesis. It is also apparentfrom Tables 1 and 2 that the S282T mutation impedes incorporationof the natural CTP substrate (see above). GTP or ATP and CTPare the nucleotides incorporated during initiation in vivo atthe +1 and +2 positions, respectively. Thus, since UTP is neverused during this step, it was not investigated. For both initiationand elongation steps, GTP and ATP incorporation was affectedby the S282T substitution, with a general decrease in catalyticefficiency (Fig. 5 and Tables 3 and 4). However, a more pronouncedeffect was observed during elongation, where the catalytic efficienciesfor all substrates, either natural or 2' modified, were below20% of that for WT NS5B (Fig. 5 and Tables 2 and 4). UTP incorporationby WT NS5B was barely detected and was undetectable when assayedwith S282T NS5B, confirming the same trend (not shown). Thereduced catalytic efficiency for the incorporation of CTP orCTP analogues (Tables 1 and 2) and ATP or GTP (Tables 3 and4) is the consequence of a general decrease of V_max during bothinitiation and elongation (Fig. 5, insets). However, duringinitiation only, this V_max decrease is balanced by a two- tofourfold increase of the apparent affinity for CTP or ATP (Fig.5, insets). In summary, the S282T NS5B catalytic efficiencyduring initiation is 2-fold lower than that of the WT enzyme(Table 3), while it is reduced 5- to 20-fold during elongation(Fig. 5 and Table 4).

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FIG. 4. Kinetics of RNA synthesis by WT and S282T NS5B polymerases, using oligo(C) as a template. (A) RNA products of reactions by either WT or S282T NS5B were resolved in a 14% polyacrylamide-7 M urea gel. RNA markers were synthesized using T7 RNA polymerase as described in Materials and Methods. Each product band is indicated on the right. (B) Comparative quantitation of reaction products from the experiment shown in panel A. Each individual P_i product band from WT or S282T NS5B was quantitated, and the percentage of the ratio of S282T(P_i)/WT(P_i) products was plotted for each i-mer product.

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FIG. 5. Comparative incorporation efficiencies for nucleotide utilization by WT and S282T mutant NS5B polymerases during initiation and elongation. Calculations were based on the values taken from Tables 1 and 3 for comparative incorporation during initiation and from Tables 2 and 4 for comparative incorporation during elongation. The catalytic efficiency (V_max/K_m) for each nucleotide is expressed as a percentage of the catalytic efficiency determined for WT NS5B polymerase. The percent binding affinity (1/K_m) and maximal velocity (V_max) were calculated in the same manner and are shown in the insets.

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TABLE 3. Steady-state constants of wild-type and S282T NS5B polymerases for RNA templates during initiation^a

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TABLE 4. Steady-state constants of wild-type and S282T NS5B polymerases for RNA templates during elongation^a

We conclude that the molecular determinants of S282T NS5B-mediated2'-C-Me analogue resistance are completely discriminated duringinitiation and discriminated 21-fold during the elongation stepof RNA synthesis. Strikingly, for 2'-O-Me-CTP incorporation,the picture is opposite, leading to a similar incorporationduring initiation but to apparently complete discriminationduring elongation. This S282T mutation-mediated resistance,however, drastically reduces the polymerization efficiency ofnatural nucleotides, raising interesting perspectives aboutviral fitness and its use in optimized anti-HCV therapeuticprotocols.

DISCUSSION

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Discussion

In this study, we have characterized the molecular mode of actionof triphosphate derivatives of 2'-O-Me- and 2'-C-Me-cytidine,with the latter currently being evaluated in clinical trials.2'-C-Me-CTP is incorporated as efficiently as CTP by the WTenzyme during either initiation or elongation, consistent withthe antiviral activities observed in patients after 2 weeksof treatment (1). 2'-O-Me-CTP has also been described as anHCV polymerase inhibitor which is active as a chain terminator(7), with a 50% inhibitory concentration measured by RdRp assaysimilar to that of 2'-C-Me-ATP. In contrast to 2'-C-Me-CTP,we show that 2'-O-Me-CTP is preferentially incorporated duringelongation, as is 2'-O-Me-GTP (15). The 36-fold discriminationof 2'-O-Me-CTP at initiation is probably due to steric hindranceby the methyl group of threonine 287 during this step. Duringelongation, no discrimination is observed, and the 2'-O-Me-CTPis incorporated equally as well as the natural CTP or 2'-C-Me-CTP.This suggests that important modifications of the active siteoccur during the switch from initiation to elongation, leadingto the displacement of threonine 287 and the relaxation of thesteric hindrance induced by this residue during initiation.These results are consistent with our previous observation thatthe conformation of the active site during elongation is morepermissive to sugar modifications than that during initiation(15).

Conformational changes of the active site are also illustratedby the analysis of the S282T mutant enzyme. This mutant wasselected and isolated after treatment of replicon-expressingcells with a suboptimal concentration of 2'-C-Me adenosine orguanosine (33). It was reported that the S282T substitutionconfers resistance to both 2'-C-Me nucleosides but does notaffect 2'-O-Me nucleoside susceptibility. The molecular basisof this differential resistance profile for these closely related2'-modified analogues is interesting. We show in this studythat the S282T mutation-mediated 2'-C-Me-CTP resistance is theresult of a steric hindrance translated at the molecular level,with an increased K_m and an unchanged V_max during elongation.During initiation, this steric hindrance is drastic, and noincorporation is detected, probably due to a steric clash betweenthe methyl group of the modified analogue and the methyl groupof the substituting threonine (Fig. 2). It was recently reportedthat HCV and bovine viral diarrhea virus polymerase resistanceto nucleotide analogues could also be the result of pyrophosphorolysisleading to the excision of the analogue after its incorporationinto the nascent RNA (10, 14). No pyrophosphorolysis could beobserved in our experimental system with either WT or S282TNS5B, probably because of specific template requirements forthis particular mechanism (14). Nevertheless, it was shown thatS282T NS5B is able to excise chain-terminator analogues to thesame extent as WT NS5B (14). Therefore, S282T mutant-mediatedresistance to 2'-C-Me-NTP is only due to K_m-based discrimination(Table 2).

Surprisingly, we noticed that the S282T substitution also impairsthe incorporation of 2'-O-Me-CTP (Tables 1 and 2). This mutation,however, does not lead to resistance toward this analogue inthe replicon system (33). We have no explanation for this discrepancy.However, RNA synthesis in vivo occurs in a replication complexinvolving other nonstructural proteins, which might influencethe three-dimensional conformation of NS5B. Thus, the structureof the NS5B active site might be slightly different in the replicationcomplex than the one determined with the isolated enzyme. Alternatively,and perhaps more likely, 2'-O-Me-CTP may inhibit another targetbesides NS5B when RNA synthesis is measured either from a purifiedreplicative complex or in the replicon system. Nevertheless,in our in vitro RdRp assay, S282T NS5B was fully resistant to2'-O-Me-CTP during elongation (Table 2) but was as competentas WT NS5B for incorporation during initiation. Again, our resultsillustrate an important conformational change occurring duringthe switch from initiation to elongation.

Mutations conferring resistance to HCV polymerase or proteaseinhibitors are often associated with a decrease in replicativefitness (25, 26, 34), although the molecular mechanisms arenot fully understood. The S282T mutation is associated withsuch replicative defects (26). Our study suggests that reducedfitness may be due to a decrease in the catalytic rate (V_max)of incorporation for all natural nucleotides, with the affinity(K_m) being unaffected. Surprisingly, the S282T mutation alsoaffects the incorporation of GTP and ATP during initiation,although this mutation is located 10 Å away from the +1position, where GTP or ATP is positioned during this step (4).This suggests that the S282T mutation may have a long-rangeeffect, e.g., an effect transmitted through the stacking ofnucleotides +2 and +1. We did not observe a burst of productformation under any circumstances, using either a preformedNS5B-template or NS5B-nucleotide complex with either WT or S282TNS5B, ruling out a low dissociation rate as a rate-limitingstep (not shown). Thus, variation of the dissociation rate ofS282T NS5B does not explain the differences in V_max observedwith different nucleotides.

For the poliovirus 3D polymerase, it has been suggested thatthe correct positioning of the NTP is dependent on its correctbinding at both the sugar binding site and the triphosphatebinding site (8). The HCV NS5B sugar binding site is made upof serine 282, asparagine 291, and glutamic acid 225 (4). Asin the poliovirus model, the ribose in the NS5B active siteis thought to be held firmly by an interaction between its 3'-OHand the backbone of D225, in addition to its 2'-OH and bothN291 and S282. Indeed, any alteration in this hydrogen bondnetwork, by either sugar modification or amino acid substitutions,would induce a reduced stabilization of the ribose moiety anda probable loosening of binding at both the sugar and triphosphatebinding sites (8). Based on this selectivity model, the S282Tsubstitution in the HCV polymerase is indeed able to alter thesugar binding site, and hence triphosphate positioning, afterpromoting local rearrangement up to the catalytic center (V_maxeffect).

Our results might have clinical relevance. Although no resistantviruses have been reported yet for NM283 clinical trials, theincrease in viral load during monotherapy suggests the appearanceof such resistant mutants (1). If this mutation appears in theclinic and in the absence of compensatory mutations (34), theresulting mutant viruses will have an impaired replication capacitythat may translate into better control by the immune systemor by other drugs. It is important to draw from the HIV-1 experience.The nucleoside analogue lamivudine is an RT inhibitor elicitingresistance through the emergence of the M184V substitution inRT (48). The M184V mutation alters viral fitness through a decreasedincorporation efficiency of natural nucleotides by RT (13).It has been suggested, however, that there is a clinical benefitto maintaining the presence of the M184V substitution by uninterruptedlamivudine treatment (47). Indeed, the loss of viral fitnesspotentiates the efficacy of additional HIV-1 inhibitors (12).If the S282T mutation arises in patients, then the S282T mutationand 2'-C-methyl cytosine may well be the first example of sucha rational approach of drug-resistant HCV therapy through thecontrol of the fitness/resistance balance.

ACKNOWLEDGMENTS

We thank B. Selisko, K. Alvarez, J. C. Guillemot, E. Decroly,and D. Storer for critical readings of the manuscript and helpfuldiscussions. We thank J. L. Romette for handling intellectualproperty rights.

This work was supported by the European Community (FlavitherapeuticsEuropean contract no. QLK3-CT-2001-00506), the Association pourla Recherche contre le Cancer (ARC), and the Centre Nationalde Recherche Scientifique (CNRS). H.D. is an INSERM appointee.

FOOTNOTES

* Corresponding author. Mailing address: AFMB-CNRS-ESIL, Case 925, 163 avenue de Luminy, 13288 Marseille Cedex 9, France. Phone: 33-491-828644. Fax: 33-491-828646. E-mail: Bruno.Canard{at}afmb.univ-mrs.fr.

Published ahead of print on 25 September 2006.

REFERENCES

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