Determinants of RNA-Dependent RNA Polymerase (In)fidelity Revealed by Kinetic Analysis of the Polymerase Encoded by a Foot-and-Mouth Disease Virus Mutant with Reduced Sensitivity to Ribavirin

A mutant poliovirus (PV) encoding a change in its polymerase(3Dpol) at a site remote from the catalytic center (G64S) confersreduced sensitivity to ribavirin and forms a restricted quasispecies,because G64S 3Dpol is a high-fidelity enzyme. A foot-and-mouthdisease virus (FMDV) mutant that encodes a change in the polymerasecatalytic site (M296I) exhibits reduced sensitivity to ribavirinwithout restricting the viral quasispecies. In order to resolvethis apparent paradox, we have established a minimal kineticmechanism for nucleotide addition by wild-type (WT) FMDV 3Dpolthat permits a direct comparison to PV 3Dpol as well as to FMDV3Dpol derivatives. Rate constants for correct nucleotide additionwere on par with those of PV 3Dpol, but apparent binding constantsfor correct nucleotides were higher than those observed forPV 3Dpol. The A-to-G transition frequency was calculated tobe 1/20,000, which is quite similar to that calculated for PV3Dpol. The analysis of FMDV M296I 3Dpol revealed a decreasein the calculated ribavirin incorporation frequency (1/8,000)relative to that (1/4,000) observed for the WT enzyme. Unexpectedly,the A-to-G transition frequency was higher (1/8,000) than thatobserved for the WT enzyme. Therefore, FMDV selected a polymerasethat increases the frequency of the misincorporation of naturalnucleotides while specifically decreasing the frequency of theincorporation of ribavirin nucleotide. These studies providea mechanistic framework for understanding FMDV 3Dpol structure-functionrelationships, provide the first direct analysis of the fidelityof FMDV 3Dpol in vitro, identify the β9- ${alpha}$ 11 loop as a (in)fidelitydeterminant, and demonstrate that not all ribavirin-resistantmutants will encode high-fidelity polymerases.

INTRODUCTION

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INTRODUCTION

Viral polymerases are important targets for the therapeuticintervention of viral infections (3, 10, 15, 30, 33, 43). Thebest example is the reverse transcriptase from human immunodeficiencyvirus (5, 22, 23, 28, 47, 49). In most cases, (deoxy)nucleosideanalogs that enter the clinic exploit the relaxed fidelity ofviral polymerases relative to those of related cellular polymerases(3, 10, 15, 23, 28, 30, 33, 43, 47). The efficacy of an antiviralnucleoside is compromised by the development of resistance.In most cases, virus mutants that exhibit decreased sensitivityto antiviral nucleosides encode polymerases that exhibit theincreased fidelity of substrate selection and/or incorporation(11, 24, 49). An understanding of the structural and kineticbases for polymerase fidelity should reveal mechanisms thatviruses will employ to escape the antiviral activity of (deoxy)nucleosidepolymerase inhibitors. Importantly, this information may facilitatethe development of antiviral nucleosides to which the emergenceof viral resistance is less facile.

The purine analogue ribavirin (1-β-D-ribofuranosyl-1,2,4-triazole-carboxamide)is a synthetic antiviral ribonucleoside with activity againsta broad spectrum of RNA viruses (26). Unlike most antiviralnucleosides, ribavirin does not function as a chain terminator.Ribavirin monophosphate (RMP) is a known inhibitor of IMP dehydrogenase,causing a twofold reduction in guanine nucleotide pools in cellculture (26). The inhibition of this cellular target may accountfor ribavirin's activity against some viruses (26). Ribavirintriphosphate (RTP) is the most abundant ribavirin metabolitein cells. RTP has been shown to act as an inhibitor and substrateof the viral RNA-dependent RNA polymerase (RdRp) (26). The utilizationof RTP as a GTP analogue during the initiation of RNA synthesiscan lead to uncapped transcripts that cannot be translated efficiently(26). The ambiguous utilization of RTP by the RdRp during genomereplication leads to the lethal mutagenesis of the virus population(1, 13, 14, 48). Given the unique, and somewhat controversial,mechanisms of action proposed for this clinically importantnucleoside, viral mutants capable of evading the antiviral activityof ribavirin should shed additional light on its mechanism ofaction and might suggest strategies for the design of more efficaciousanalogues of ribavirin (25, 26). Indeed, mutations in genescoding for functions other than that of the RdRp have been associatedwith ribavirin resistance (37).

The serial passage of poliovirus (PV) in cell culture in thepresence of sublethal concentrations of ribavirin selected viruspopulations with decreased sensitivity to ribavirin, and theresistance was mapped to a Gly-to-Ser substitution at position64 (G64S) of PV RdRp (3Dpol) (9, 38). PV G64S 3Dpol exhibitsa threefold increase in nucleotide incorporation fidelity relativeto that of wild-type (WT) 3Dpol, causing a corresponding reductionin viral quasispecies diversity (9, 36, 38, 45). Interestingly,the restricted quasispecies renders the resulting PV populationsless adaptable to a complex biological environment and documentsthe behavior of the entire quasispecies as a unit of selectionin vivo (36, 45, 46). The fact that the same, unstable PV mutantconferring reduced sensitivity to ribavirin was isolated bytwo laboratories (9, 38) suggests that few mechanisms existto escape the antiviral activity of this compound. Whether ornot this observation relates to the structure of the compoundor its mechanism of action remains unclear.

Recently, we identified M296I as a change in 3Dpol of foot-and-mouthdisease virus (FMDV) that confers decreased sensitivity to ribavirin(39). FMDV M296I 3Dpol became dominant in the population oftwo parallel lineages of FMDV passaged in the presence of ribavirin(but not in control populations passaged in the absence of ribavirin).Importantly, FMDV M296I 3Dpol incorporates RMP less efficientlythan WT 3Dpol (39).

In contrast to Gly-64, which is remote from the catalytic site,Met-296 lies within the β9- ${alpha}$ 11 loop (residues 294 to 302)of the FMDV 3Dpol catalytic site (19) (Fig. 1). This loop couldcontribute to polymerase fidelity, given its interactions withthe base of the incoming nucleotide as well as the templatemolecule (18). In order to elucidate the mechanistic basis forthe decreased sensitivity of FMDV M296I 3Dpol, we have performeda detailed kinetic analysis of FMDV 3Dpol, yielding the firstdirect comparison of the minimal kinetic mechanism and the fidelityof nucleotide addition for two picornaviral polymerases fromevolutionarily distant genera. Importantly, this comparativeanalysis revealed numerous differences in the biochemical propertiesof these enzymes that may have significance for the biologyof these viruses. Consistently with our expectations, we findthat FMDV M296I 3Dpol incorporated RMP less efficiently thanWT FMDV 3Dpol. Surprisingly, FMDV M296I 3Dpol misincorporatedGMP more efficiently than WT FMDV 3Dpol. Therefore, in contrastto the high-fidelity variant created by PV when faced with theantiviral activity of ribavirin, FMDV created a low-fidelityvariant with a specific defect for ribavirin utilization. Thisobservation suggests that counterintuitive solutions exist tothe problem of the ribavirin antagonism of virus production.In addition, this study highlights the ability of the β9- ${alpha}$ 11loop and/or elements with which it interacts to contribute tothe (in)fidelity of nucleotide selection depending upon thenature of the nascent base pair.

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FIG. 1. Structure of FMDV 3Dpol-RNA-RTP catalytic complex. (A) FMDV 3Dpol is shown in gray, the template-primer molecule is shown in light green, and the RTP molecule is represented by dark green sticks. Loop β9- ${alpha}$ 11 of FMDV 3Dpol is shown in blue, and the residue Met-296 is represented by red sticks. (B) A closer view of the β9- ${alpha}$ 11 loop (blue), with the 3Dpol residue Met-296 (red) and the RTP molecule (green). Blue sticks are residues Ser-298 and Gly-299, which establish interactions with the incoming nucleotide base (18). Asp-245 and Asn-307, which interact with the ribose of the incoming nucleotide, are represented by gray sticks. (C) Superimposition of structures of different FMDV 3Dpol complexes. Shown are the binary complex with template-primer RNA (Protein Database accession no. [pdb] 1WNE; gray); ternary complex with UTP/ATP (pdb 2EC0; magenta); and ternary complex with ribavirin (pdb 2E9R; cyan). The loop of residues 294 to 301 is shown in yellow. The distance between C ${alpha}$ atoms of Gly299 in the different complexes shown (taken as a measure of the movement of the loop of residues 297 to 301) is about 0.9 and 1.4 Å in the complexes with UTP/ATP and ribavirin, respectively.

MATERIALS AND METHODS

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MATERIALS AND METHODS

Molecular cloning, expression, and purification of FMDV 3Dpol. The FMDV polymerase 3D coding region (cloned into pET-28a [pET28a3D])was expressed in Escherichia coli BL21 and purified as previouslydescribed (4, 19). The polymerase was fused at its C terminusto a His tag sequence that allowed purification by Ni-nitrilotriaceticacid affinity chromatography (ProBond Resin; Invitrogen). FMDVpolymerase encoding M296I (M296I 3Dpol) was obtained by thesite-directed mutagenesis of pET-28a3D using the QuikChangesite-directed mutagenesis kit (Stratagene) as previously described(17). Briefly, pET-28a3D was amplified by using mutagenic primersthat include the substitution G7497A (sense primer) or C7497U(antisense primer). PCR amplification was carried out usingPfu Ultra (Roche); the amplified DNA was treated with DpnI at37°C for 1 h and transformed into E. coli XL1-Blue-competentcells, which were provided by the manufacturer. Plasmids fromkanamycin-resistant colonies were sequenced to ascertain thatthe whole 3D coding region was properly cloned and that thedesired mutation was present. The M296I mutant was expressedand purified as described for WT 3Dpol. Proteins were dialyzedthree times against 50 mM Tris-HCl, pH 7.5, 500 mM NaCl, 1 mMEDTA, 1 mM dithiothreitol, 10% glycerol. Both proteins obtainedwere >95% pure by sodium dodecyl sulfate-polyacrylamide gelelectrophoresis analysis.

Poly(U) synthesis activity assay. The specific activity of purified WT FMDV 3Dpol was 160 pmolUMP incorporated/min/µg of protein in the standard incorporationassay. Reaction mixtures contained 30 mM morpholinepropanesulfonicacid, pH 7.0, 33 mM NaCl, 5 mM Mg(CH₃COO)₂, 40 ng/µl polyriboadenylate(average length of 300 residues) (Amersham), 2.4 µM oligo(dT)₁₅(Invitrogen), 500 µM [ ${alpha}$ -³²P]UTP (200 mCi/mmol) (Amersham).Purity and specific activity were indistinguishable from thoseof previous preparations of the enzyme (4, 19, 39). M296I 3Dpolshowed a specific activity similar to that of WT 3Dpol (>75%specific activity relative to that of WT 3Dpol).

Sym/sub incorporation assays. Reaction mixtures contained 50 mM HEPES, pH 7.5, 10 mM 2-mercaptoethanol,5 mM MgCl₂, 500 µM nucleoside triphosphate (NTP), 5'-³²Pend-labeled sym/sub (s/s; a symmetrical substrate primer-template),and FMDV 3Dpol, as previously described for PV 3Dpol (6, 9).5'-³²P labeling and the annealing of s/s were performed as previouslydescribed (6, 9). Reactions were quenched by the addition ofEDTA to a final concentration of 50 mM. Product was resolvedfrom the substrate by electrophoresis through a denaturing 23%polyacrylamide gel, visualized by phosphorimaging, and quantifiedby using ImageQuant software (Molecular Dynamics) as previouslydescribed (6, 9). Specific concentrations of s/s, 3Dpol, and/ornucleotide are indicated in the figure legends. FMDV 3Dpol wasdiluted immediately before use in 50 mM HEPES, pH 7.5, 10 mM2-mercaptoethanol, and 20% glycerol. The volume of enzyme addedto the reaction mixture was, at most, 1/10 of the total reactionvolume. Reactions were performed at 30°C.

Rapid chemical quenched-flow experiments. Rapid mixing/quenching experiments were performed by using amodel RQF-3 chemical quench-flow apparatus (KinTek Corp., Austin,TX). Experiments were performed at 30°C by using a circulatingwater bath. FMDV 3Dpol (2 µM) was incubated with eithers/s-AU or s/s-AC (2 µM; 1 µM duplex) and UTP (10µM) for 900 to 1,200 s to allow the formation of 3Dpol-s/sproduct complexes. The 3Dpol-s/s product complex then was rapidlymixed with various concentrations of nucleotide substrate (apparentdissociation constant [K_d,app] of 0.2 to 5). After being mixed,reactant concentrations were reduced by 50%. At various timesafter mixing, reactions were quenched by the addition of EDTAto a final concentration of 0.3 M.

Determination of the kinetic parameters (k_pol and K_d,app) for FMDV 3Dpol-catalyzed nucleotide incorporation. Data were fit by nonlinear regression using the program KaleidaGraph(Synergy Software, Reading, PA). Time courses at fixed nucleotideconcentrations were fit to the following equation:

Formula 1 (1)
where A is the amplitude of theburst, k_obs is the observed first-order rate constant describingthe burst, t is the time, and C is a constant. The k_obs valueswere plotted as a function of nucleotide concentration, andthe data were fitted to the following equation:

Formula 2 (2)
where k_pol is the maximal ratefor nucleotide incorporation.

RESULTS

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RESULTS

PV 3Dpol is the only picornavirus 3Dpol for which detailed kineticand thermodynamic data exist (12, 40). This information hasbeen invaluable to our understanding of the molecular mechanismsthat govern the fidelity of RNA synthesis by RdRps (7-9, 27),the establishment of structure-function relationships for RdRps(8, 9, 20, 21, 27, 41, 42), the mechanism of action of antiviralcompounds (13, 14), and the mechanisms of resistance to antiviralcompounds (9). However, the extent to which the paradigms establishedfor PV 3Dpol extend to other RdRps, especially other picornaviral3Dpols, has yet to be evaluated empirically. The interpretationof recent structures for FMDV 3Dpol and its complexes with primedtemplate in the absence and presence of natural and antiviralnucleotides has relied heavily on the mechanistic data availablefor PV 3Dpol (18, 19). Therefore, the initial motivation forthis study was to create a mechanistic framework for the nucleotideaddition cycle of FMDV 3Dpol that would illuminate both theunique and universal features of the picornaviral 3Dpol nucleotideaddition cycle and permit the elucidation of FMDV structure-functionrelationships.

A minimal kinetic mechanism for single-nucleotide addition byPV 3Dpol is shown in Fig. 2. The binding of 3Dpol (^oE) to primedtemplate RNA (R_n) to form a 3Dpol-RNA complex (^oER_n) is in arapid equilibrium. The partitioning of the 3Dpol-RNA complexinto an activated complex (ER_n) is slow and governed by therate constant k_assembly. The quantity of ER_n formed at any givenconcentration of 3Dpol is attenuated by the inactivation of3Dpol, which is governed by the temperature-dependent rate constantk_inact. The activated 3Dpol-RNA complex binds nucleotides withan apparent binding affinity given by K_d,app to form the 3Dpol-RNA-nucleotidecomplex (ER_nNTP). This complex undergoes catalysis at a rategoverned by the rate constant k_pol, yielding the product 3Dpol-extendedRNA complex (ER_{n + 1}) and pyrophosphate. 3Dpol is present inactivated complexes; ER_n and ER_{n + 1} dissociate from RNA ata rate governed by the rate constants k_off,ERn and k_{off,ERn+ 1}, respectively. This mechanism is sufficient to explain thekinetics and mechanism of PV 3Dpol RNA synthesis in vitro andexplains biological phenotypes observed for PV 3Dpol and itsderivatives (6, 21). This framework guided our studies of FMDV3Dpol.

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FIG. 2. Mechanism of 3Dpol-RNA primer-template complex assembly and nucleotide incorporation. A minimal kinetic mechanism for single-nucleotide addition by PV 3Dpol is shown. X, inactivated 3Dpol.

Assembly of active FMDV 3Dpol-RNA complexes is slow and attenuated by thermal inactivation of 3Dpol. We have employed a symmetrical primer-template (s/s) that permitsnucleotide addition to be monitored by evaluating the extensionof end-labeled primer (Fig. 3A) (6). We will maintain a conventionin which the templating nucleotide(s) employed for an s/s ineach experiment is indicated explicitly. For example, s/s-Urefers to a substrate with uridine as the templating nucleotide.

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FIG. 3. Kinetics of assembly of WT FMDV 3Dpol-s/s-U complexes. (A) Sequence of s/s-U. (B) AMP incorporation into s/s-U by WT FMDV 3Dpol. Products were resolved by electrophoresis on a denaturing 23% polyacrylamide gel. (C) Concentration dependence of WT FMDV 3Dpol. Reaction mixtures contained 1 µM s/s-U (0.5 µM duplex), 500 µM ATP, 5 mM MgCl₂, and either 0.25 ( ${circ}$ ), 0.5 (•), 1 ( ${square}$ ), or 5 ( ${blacksquare}$ ) µM 3Dpol. Reactions were initiated by the addition of 3Dpol, incubated at 30°C, and quenched at the indicated times by the addition of EDTA (50 mM). The solid lines represent the fit of the data to a single exponential. The observed rate of assembly was 0.003 s^–1 for all concentrations of 3Dpol. (D) Concentration dependence of s/s-U. Reactions were performed as described above, but the mixtures contained 1 µM 3Dpol and either 1 (•), 2 ( ${circ}$ ), 4 ( ${blacksquare}$ ), 10 ( ${square}$ ), or 20 ( ${blacktriangleup}$ ) µM s/s-U. The solid lines represent the fit of the data to a single exponential. The observed rates of assembly were 0.004, 0.004, 0.002, 0.002, and 0.004 s^–1 for 20, 10, 4, 2, and 1 µM s/s-U, respectively (average, 0.003 s^–1).

In order to determine if the FMDV 3Dpol utilization of s/s waslimited by a slow assembly step, reaction mixtures containings/s-U (0.5 µM duplex) and ATP (500 µM) were initiatedby the addition of FMDV 3Dpol (0.25 to 5 µM). Reactionswere quenched at various times by the addition of EDTA (50 mM).Products were resolved by denaturing polyacrylamide gel electrophoresisand visualized by phosphorimaging (Fig. 3B). The kinetics ofAMP incorporation at the various concentrations of enzyme areshown in Fig. 3C. In no case did the end point reach the expectedvalue of 0.5 µM. At 5 µM enzyme, 0.44 µM s/s-Uwas extended. Moreover, at all enzyme concentrations employed,the observed rate constant was the same (0.003/s). A concentration-independentrate constant suggests that a first-order process occurs afterthe formation of the bimolecular collision complex that limitsthe rate of 3Dpol activation.

The inability to reach the expected end point could have beencaused by inactive enzyme present at the start of the experimentor inactivated during the course of the experiment. To distinguishbetween these two possibilities, the reaction was repeated underconditions in which the concentration of 3Dpol (1 µM)was held constant while varying the concentration of s/s-U (1to 20 µM). In this experiment also, a concentration-independentrate constant of 0.003/s was observed (Fig. 3D). However, theexpected end point of 1 µM was achieved by using highconcentrations of s/s-U (Fig. 3D). The finding that both 10and 20 µM s/s-U yielded essentially overlapping curves(Fig. 3D) can be interpreted only to mean that the observedrate constant reflects a step after s/s-U binding to 3Dpol.The ability to observe the stoichiometric utilization of enzyme(Fig. 3D) confirms that all of the enzyme in the preparationis active. Therefore, when lower concentrations of s/s-U areemployed, the enzyme must be inactivating during the reactionto produce the lower-than-expected end points.

In order to examine enzyme inactivation directly, we performedthe experiment shown in Fig. 4A. FMDV 3Dpol was incubated inbuffer at different temperatures for various amounts of time,followed by the addition of s/s-U (0.5 µM) and ATP (500µM). Primer extension was permitted for 10 min. In thisexperiment, FMDV 3Dpol was compared directly to PV 3Dpol asa control. The preincubation of both enzymes at 30°C priorto initiating the reaction caused a reduction in product formation(Fig. 4B). FMDV 3Dpol was clearly more stable than PV 3Dpol(Fig. 4B). Product formed was plotted as a function of preincubationtime. Data were fit to a single exponential decay, yieldingobserved rate constants for inactivation (k_inact) of 0.002 and0.02/s for FMDV 3Dpol and PV 3Dpol, respectively (Fig. 4C).At temperatures of 22 and 4°C, FMDV 3Dpol remained one totwo orders of magnitude more stable than PV 3Dpol (Table 1).

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FIG. 4. Inactivation of WT FMDV 3Dpol and PV 3Dpol. (A) Experimental design. WT FMDV or PV 3D^pol (2 µM) was incubated at 30°C in 1x reaction buffer for various amounts of time. The reaction was initiated by the addition of s/s-U (1 µM) and ATP (500 µM) and was allowed to proceed for an additional 10 min at 30°C, at which time the reaction was quenched by the addition of EDTA (50 mM). (B) Products from reactions described in panel A resolved by electrophoresis on a denaturing 23% polyacrylamide gel. (C) Kinetics of inactivation of WT FMDV (•) and PV ( ${circ}$ ) 3Dpol from the reactions described in panel A. The amount of 11-mer product was plotted as a function of the time of 3Dpol incubation. The solid lines represent the fit of the data to a single exponential and gives a k_inact of 3 x 10^–3 s^–1 for FMDV 3Dpol and 20 x 10^–3 s^–1 for PV 3Dpol.

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TABLE 1. Kinetic parameters for the mechanism of FMDV and PV 3Dpol stability and 3Dpol primer-template complex assembly/stability^a

FMDV 3D polymerase forms a very stable complex with RNA. An important determinant of polymerase processivity is the rateconstant for the dissociation of the enzyme from nascent RNAand template. In the system employed here, two FMDV 3Dpol-nucleicacid complexes form: 3Dpol-s/s-U substrate complex (ER_n) and3Dpol-s/s-U product complex (ER_{n + 1}). The dissociation of 3Dpolfrom the latter complex will set the upper limit for primerextension under steady-state conditions (i.e., excess s/s-U).

We measured the rate constant for the dissociation of FMDV 3Dpolfrom s/s-U by mixing 3Dpol and 5'-³²P-labeled s/s-U and incubatingit for 1,200 s to permit complexes to form (Fig. 5A). We nextadded a 1,000-fold molar excess of unlabeled s/s-U as a trapfor free and dissociating enzyme (Fig. 5A). At various timesafter the addition of trap, ATP was added for 30 s (Fig. 5A).The extended primer provided an indication of the amount ofcomplex remaining (Fig. 5B). The percentage of primer extendedwas plotted as a function of time after the addition of trap,and the data were fit to a single exponential decay (Fig. 5C).This analysis revealed a dissociation rate constant of 0.00009/s(half-life of 2 h). Dissociation from the s/s-U-product complexwas measured by using the same approach; however, during theinitial incubation of 3Dpol with s/s-U, a concentration of ATPstoichiometric with enzyme was added (Fig. 5D). After the additionof trap, complex stability was monitored by evaluating the formationof the two-nucleotide-extended product after the addition ofUTP (Fig. 5E). Quantitative analysis yielded a dissociationrate constant of 0.000007/s (half-life of 27.5 h) (Fig. 5F).

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FIG. 5. Kinetics of dissociation of FMDV 3Dpol-s/s-U substrate and 3Dpol-s/s-U product complexes. (A) Experimental design. WT FMDV 3Dpol (1 µM) was incubated with stoichiometrically ³²P-labeled s/s-U (0.1 µM; 0.05 µM duplex) for 1,200 s at 30°C, at which point trap (100 µM unlabeled s/s-U) was added to the reaction mixture. At fixed times after the addition of trap, ATP (500 µM) was added, and the reaction was allowed to proceed for 30 s and then quenched by the addition of EDTA (50 mM). (B) Products from reactions described in panel A were resolved by electrophoresis on a denaturing 23% polyacrylamide gel. (C) Kinetics of dissociation of FMDV 3Dpol-s/s-U complexes from the reactions described in panel A. The solid lines represent the fit of the data to a single exponential and give a k_off,ERn of 9 x 10^–5 s^–1. (D) Experimental design. Reactions were performed as described for panel A but contained ATP (1 µM) during the assembly reaction, and at fixed times after the addition of trap, UTP (500 µM) was added. (E) Products from reactions described in panel D resolved by electrophoresis on a denaturing 23% polyacrylamide gel. (F) Kinetics of dissociation of FMDV 3Dpol-s/s-U product complexes from the reactions described in panel D. The solid lines represent the fit of the data to a single exponential and give a k_{off,ERn + 1} of 0.7 x 10^–5 s^–1.

These data reveal a very stable association of FMDV 3Dpol withs/s-U. Similarly to observations made for PV 3Dpol (6), theFMDV 3Dpol-s/s-U product complex is more stable than the initialcomplex. In the case of FMDV 3Dpol, a 13-fold difference wasobserved, whereas only a 6-fold difference was observed forPV 3Dpol (6). Also, the FMDV 3Dpol-s/s-U complexes ER_n and ER_{n+ 1} are 7- and 14-fold more stable than the corresponding PV3Dpol-s/s-U complexes.

Nucleotide addition by FMDV 3Dpol is fast and faithful. Given the substantial differences in the stability of 3Dpol-s/s-Usubstrate and product complexes (Fig. 5), we decided to evaluatethe kinetics and fidelity of nucleotide incorporation afterthe first cycle of nucleotide addition. Additional justificationfor the evaluation of the ER_{n + 1} complex is provided by structuraldata that reveal more contacts in the 3Dpol-ER_{n + 1} complexthan in the 3Dpol-ER_n complex (18). We developed two new substratesfor these experiments, s/s-AU and s/s-AC (Fig. 6A). These substratespermit the assembly of product complexes by incubating 3Dpol,s/s-AU, and UTP prior to the addition of ATP (correct for s/s-AU)or GTP (correct for s/s-AC).

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FIG. 6. Pre-steady-state kinetic analysis of nucleotide incorporation into s/s-AU and s/s-AC by WT FMDV 3Dpol. (A) Sequences of s/s-AU and s/s-AC. (B) Experimental design. WT FMDV 3Dpol (2 µM) was incubated with either s/s-AU or s/s-AC (2 µM; 1 µM duplex) and UTP (10 µM) for 900 s to allow the formation of 3Dpol-s/s product complexes. The 3Dpol-s/s product complex then was rapidly mixed using a rapid chemical quench-flow apparatus with various concentrations of nucleotide substrate (either ATP or GTP, with K_d,apps of 0.2 to 5). At various times after being mixed, reactions were quenched by the addition of EDTA (0.3 M). Time courses at fixed nucleotide concentrations were fit to a single exponential to obtain the observed rate constant for nucleotide incorporation, k_obs (see equation 1). The observed rate constants then were plotted as a function of nucleotide concentration, and the data were fit to a hyperbola (see equation 2) to obtain the maximal observed rate constant for nucleotide incorporation, k_pol, and the apparent dissociation constant, K_d,app. (C) AMP incorporation into s/s-AU. Shown is a replot of the observed rate constants as a function of nucleotide concentration to yield a k_pol of 80 s^–1 and a K_d,app of 500 µM. (D) GMP incorporation into s/s-AC. Shown is a replot of the observed rate constants as a function of nucleotide concentration to yield a k_pol of 200 s^–1 and a K_d,app of 50 µM. (E) GMP incorporation into s/s-AU. Shown is a replot of the observed rate constants as a function of nucleotide concentration to yield a k_pol of 0.01 s^–1 and a K_d,app of 800 µM.

The general design of these experiments is presented in Fig.6B. 3Dpol was assembled with the appropriate primed templatein the presence of UTP, followed by the addition of the indicatednucleotide at different concentrations for various amounts oftime. Reactions were quenched, and product analysis performedas described above. When correct nucleotide incorporation experimentswere performed, the reactions occurred too fast for manual quenching,so a chemical quench-flow apparatus was employed as describedin Materials and Methods. Product formed as a function of timewas plotted, and data were fitted to a single exponential rise,yielding an observed rate constant (k_obs). The observed rateconstants were plotted as a function of nucleotide concentrationand data were fitted to a hyperbola, yielding an apparent dissociationconstant (K_d,app) and a maximal rate of nucleotide incorporation(k_pol).

We evaluated the incorporation of AMP (Fig. 6C) and GMP (Fig.6D) as the correct nucleotide and the incorporation of GMP asthe incorrect nucleotide (Fig. 6E). The values for K_d,app, k_pol,and k_pol/K_d,app are reported in Table 2. The K_d,app values fornucleotide binding ranged from 50 to 500 µM. As the correctnucleotide, GTP bound 10-fold more efficiently than ATP; a similarobservation was made for PV 3Dpol (7). As the incorrect nucleotide,GTP bound to 3Dpol as efficiently as ATP. These data suggestthat GTP binding as the correct nucleotide is a special case,and that the initial binding of both correct and incorrect nucleotideslikely is driven by using the triphosphate moiety of incomingnucleotide instead of by base pairing. The k_pol values for correctnucleotide incorporation ranged from 80 to 200/s, which is onpar with values measured for PV 3Dpol (7). The k_pol value forGMP misincorporation was 0.01/s. Thus, FMDV 3Dpol fidelity isdetermined by kinetic events reported by k_pol. By using (k_pol/K_d,app)_incorr/(k_pol/K_d,app)_corr,where incorr is the incorrect nucleotide and corr is the correctnucleotide, to calculate the frequency of A $->$ G transition mutations,a mutation frequency of 1/20,000 was obtained (Table 2). Thisobservation predicts less than one A $->$ G transition mutation perround of genome replication for FMDV. Clearly, this mutationfrequency will contribute substantial sequence diversity atthe population level. We conclude that FMDV 3Dpol is fast andquite faithful, even in the absence of a proofreading nuclease.

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TABLE 2. Kinetic constants for nucleotide incorporation by FMDV WT and M296I 3Dpol^a

Nucleotide addition by FMDV M296I 3Dpol is slower and less faithful than that of WT 3Dpol. To study the biochemical properties of FMDV M296I 3Dpol, theM296I-encoding mutation was engineered into the E. coli expressionvector employed for WT FMDV 3Dpol as described in Materialsand Methods. FMDV M296I 3Dpol was expressed and purified aspreviously reported for WT FMDV 3Dpol (4).

We evaluated the kinetics of nucleotide (mis)incorporation asdescribed above for WT FMDV 3Dpol (Fig. 7 and Table 2). A two-to threefold reduction in the overall efficiency (k_pol/K_d,app)of correct nucleotide incorporation was observed relative tothat of WT enzyme (Table 2). This reduction was caused primarilyby a decrease in the k_pol value. Surprisingly, the overall efficiencyof GMP misincorporation for FMDV M296I 3Dpol was unchanged relativeto that for WT enzyme. However, the values for both K_d,app andk_pol decreased somewhat (Table 2). We conclude that the fidelityof FMDV M296I is reduced relative to that of the WT enzyme,with a mutation frequency of 1/8,000, which is 2.5-fold higherthan that for the WT (Table 3). These data suggest that FMDVencoding the M296I change in 3Dpol should exhibit a mutatorphenotype. The reevaluation of the biological data (39) leadsto a similar conclusion (Table 3).

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FIG. 7. Pre-steady-state kinetic analysis of nucleotide incorporation into s/s-AU and s/s-AC by FMDV M296I 3Dpol. Reactions were performed as described in the legend to Fig. 6 but contained FMDV M296I 3Dpol. (A) AMP incorporation into s/s-AU. Shown is a replot of the observed rate constants as a function of nucleotide concentration to yield a k_pol of 30 s^–1 and a K_d,app of 400 µM. (B) GMP incorporation into s/s-AC. Shown is a replot of the observed rate constants as a function of nucleotide concentration to yield a k_pol of 100 s^–1 and a K_d,app of 70 µM. (C) GMP incorporation into s/s-AU. Shown is a replot of the observed rate constants as a function of nucleotide concentration to yield a k_pol of 0.006 s^–1 and a K_d,app of 500 µM.

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TABLE 3. Mutation frequency of FMDV WT and M296I 3Dpol

FMDV M296I 3Dpol incorporates ribavirin less efficiently than WT 3Dpol. The observation that FMDV M296I 3Dpol is a low-fidelity enzymewas inconsistent with our expectations. FMDV encoding this changeconfers reduced sensitivity to ribavirin. The resistance ofPV to ribavirin also was conferred by a mutation in its 3Dpol(G64S) (9, 38); however, PV G64S 3Dpol was a high-fidelity enzyme(9, 38).

In order to resolve this apparent paradox, we evaluated thekinetics and specificity of ribavirin incorporation by bothFMDV WT and M296I 3Dpol enzymes (Fig. 8 and Table 2). For bothenzymes, ribavirin utilization was templated by uridine andcytidine (Fig. 8 and Table 2). The overall efficiency of ribavirinincorporation by WT FMDV 3Dpol was four- to fivefold higherthan that for GMP misincorporation (Table 2). In contrast, theoverall efficiency of ribavirin incorporation by FMDV M296I3Dpol was equal to that of GMP misincorporation (Table 2). Therefore,despite the low-fidelity behavior of the enzyme for GMP misincorporation,FMDV M296I 3Dpol is four- to fivefold less capable of incorporatingribavirin relative to the ability of WT FMDV 3Dpol. These dataare consistent with the biological observation that FMDV encodingM296I 3Dpol is less sensitive to ribavirin (39), but they showthat ribavirin resistance in this case comes with the penaltyof higher frequencies of GMP misincorporation. We conclude thatthe molecular mechanism for FMDV resistance to ribavirin isunique. Therefore, at least two mechanisms exist for picornavirusesto evade the antiviral activity of ribavirin; the productionof a high-fidelity polymerase is only one. Perhaps many othermechanisms remain to be discovered.

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FIG. 8. Ribavirin incorporation into s/s-AU and s/s-AC by FMDV WT and M296I 3Dpol. (A) RMP incorporation into s/s-AU by FMDV WT (•) and M296I ( ${circ}$ ) 3Dpol. Shown is a replot of the observed rate constants as a function of nucleotide concentration to yield a k_pol of 0.03 s^–1 and a K_d,app of 600 µM for WT FMDV 3Dpol and a k_pol of 0.01 s^–1 and a K_d,app of 1,000 µM for FMDV M296I 3Dpol. (B) RMP incorporation into s/s-AC by FMDV WT (•) and M296I ( ${circ}$ ) 3Dpol. Shown is a replot of the observed rate constants as a function of nucleotide concentration to yield a k_pol of 0.02 s^–1 and a K_d,app of 500 µM for WT FMDV 3Dpol and a k_pol of 0.009 s^–1 and a K_d,app of 800 µM for FMDV M296I 3Dpol.

DISCUSSION

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DISCUSSION

The elaboration of a kinetic and thermodynamic description ofthe nucleotide addition cycle for a polymerase permits the establishmentof structure-function relationships controlling catalysis andthe fidelity of nucleotide selection and incorporation, as wellas the elucidation of molecular mechanisms for antiviral compoundsand corresponding resistance mechanisms (11, 12, 22, 24, 31,40, 49). A wealth of biochemical and structural informationexists for a large number of RdRps (32); however, PV 3Dpol isthe only RdRp for which a complete kinetic and thermodynamicdescription of the nucleotide addition cycle exists (6-8, 12,20, 40). The availability of mechanistic information for a secondRdRp permits direct comparisons of RdRps in vitro that shouldilluminate similarities and differences. Significant differencesobserved in vitro may have implications for virus biology.

In this study, we have elucidated the minimal mechanism fornucleotide addition catalyzed by FMDV 3Dpol. We find that thekinetic pathway employed by FMDV 3Dpol is identical to thatemployed by PV 3Dpol (Fig. 2) (6). This observation is not surprising,given the conservation of kinetic pathways across diverse classesof nucleic acid polymerases (40). Importantly, numerous differencesin the kinetics and/or thermodynamics of the steps comprisingthe pathway are revealed by comparing FMDV 3Dpol to PV 3Dpol.

During the first step of the reaction, 3Dpol must associatewith primed template. We have employed the artificial primer-templates/s, which was developed for PV 3Dpol (6). This primer-templatehas been shown to recapitulate the biological elongation complex(6, 21). However, the in vitro assembly of 3Dpol onto duplexRNA does not have a known biological equivalent. During assembly,there is a race between 3Dpol binding to nucleic acid and temperature-dependentinactivation. The assembly of FMDV 3Dpol-s/s complexes was 30-foldslower than the rate observed for PV 3Dpol (Fig. 3; Table 1).Unexpectedly, FMDV 3Dpol produced more complexes than PV 3Dpolunder identical conditions (Fig. 3; Table 1) (6). The reasonfor this apparent paradox was that the rate of the thermal inactivationof FMDV 3Dpol was at least 7-fold but as much as 500-fold slowerthan that of the thermal inactivation of PV 3Dpol dependingupon the temperature employed (Table 1). These results suggestthat a correlation exists between the rate of assembly and therate of thermal inactivation. Note that we do not believe thatthe thermal inactivation of 3Dpol for s/s utilization equatesto irreversible enzyme inactivation due to an unfolding event,because 3Dpol-catalyzed viral protein genome-linked uridylylationreactions can proceed linearly for hours (34, 35). The enhancedstability of FMDV 3Dpol on nucleic acid coupled with the enhancedthermal stability may provide explanations for the facile crystallizationof this protein with so many ligands (17-19).

Peersen and colleagues originally suggested that PV 3Dpol assemblywas limited by a cis-trans isomerization of Pro-119 (42). Thispossibility was ruled out by the finding that the structurallyequivalent residue (Pro-120) in FMDV 3Dpol remains in the cisconformation when bound to s/s (19). Structures of FMDV 3Dpoland PV 3Dpol are essentially superimposable (19, 42). However,one exception has been noted recently. The β9- ${alpha}$ 11 (FMDV3Dpol nomenclature [19]) loop of PV 3Dpol (residues 288 to 292)exists in two conformations: in and out (Olve Peersen, personalcommunication). The out conformation occludes the s/s bindingsite described for FMDV 3Dpol (19). Only the in conformationis observed for FMDV 3Dpol in the absence of nucleic acid andnucleotide (19). As discussed below, this loop exhibits changesin position in response to different template-incoming nucleotidepairs (18). Changes in the dynamic behavior and/or equilibriumposition of this element in the absence of nucleic acid for3Dpols from different picornaviruses may contribute to the rateof assembly and/or inactivation. Consistently with this hypothesisis the finding that some substitutions in this element, forexample, Cys-290 to Val in PV 3Dpol, decrease the rate of assembly(J. J. Arnold and O. Peersen, unpublished observations).

The final complex formed after s/s binding to 3Dpol is verystable and becomes even more stable after the incorporationof the first nucleotide (Fig. 5; Table 1). The half-lives ofthe FMDV 3Dpol-s/s complexes before and after nucleotide incorporationare 2 and 27 h, respectively (Table 1). Complexes formed byFMDV 3Dpol are at least sixfold more stable than the correspondingPV complexes (Table 1). The half-lives for other polymerase-primedtemplate complexes are on the order of seconds to minutes (2,31). The structure of the FMDV 3Dpol-s/s complex does not revealmore interactions with s/s than those observed for other classesof nucleic acid polymerase (19). What is different, however,is the absence of large domain movements after binding (19).Therefore, the increased binding affinity may derive from therigidity of the RNA binding site and the absence of large domainmovements that could induce dissociation. We hypothesize thatthe restriction of interdomain movements is caused by the interactionof the 3Dpol fingertip subdomain with the thumb subdomain (41,42).

After the formation of the 3Dpol-s/s complex, nucleotide bindsto form the 3Dpol-s/s-NTP complex. The apparent dissociationconstant, K_d,app, is derived from kinetic data. Our studiesof PV 3Dpol have shown that nucleotide binding is a two-stepprocess (7). The first step is driven by interactions of thetriphosphate with motif F of 3Dpol (residues 155 to 176 forPV and 160 to 181 for FMDV). The data obtained for FMDV 3Dpolare consistent with this mechanism (Fig. 6 and 8 and Table 2).The K_d,app values measured for correct nucleotides, incorrectnucleotides, and nucleotide analogues are essentially the same.GTP appears to be a special case, as the K_d,app value of thisnucleotide for FMDV 3Dpol was 10-fold lower than those of othernucleotides (Table 2); similar observations were made for PV3Dpol (7). Interestingly, the K_d,app values measured for FMDV3Dpol are 5- to 10-fold higher than those measured for PV 3Dpol(Table 2) (7). This observation suggests that FMDV multiplicationshould be more sensitive to changes in intracellular nucleotideconcentration than PV. In addition, FMDV should be less susceptibleto the antiviral action of nucleotide analogues than PV in directcomparisons. This difference may have contributed to a low-fidelityvariant instead of a high-fidelity variant.

Finally, the formation of the 3Dpol-s/s-NTP complex leads tocatalysis and the formation of one-nucleotide-extended RNA.The kinetics of nucleotide addition are governed by the macroscopicrate constant, k_pol (7). This rate constant contains terms describingnucleotidyl transfer but also contains terms describing thesecond step of nucleotide binding (7). The second step of nucleotidebinding is a conformational change of the complex that leadsto the orientation of the triphosphate into a position suitablefor catalysis (7, 8, 20). Importantly, this second step is usedas a fidelity checkpoint (8, 9, 20). The k_pol values measuredfor correct nucleotide incorporation by FMDV 3Dpol were fast(Fig. 6; Table 2) and on par with those measured for PV 3Dpol(7). The fidelity of nucleotide incorporation for FMDV 3Dpolis governed by steps reflected in k_pol, as the k_pol values forincorrect nucleotides and nucleotide analogues were decreasedby a factor of $~$ 10,000 relative to those for correct nucleotides(Fig. 6, Table 2). Therefore, FMDV replication is as faithfulas that of any DNA virus would be in the absence of proofreadingand repair mechanisms. Nevertheless, over multiple rounds ofreplication, the calculated mutation frequency of 1/20,000 (Table3) is still sufficient to create substantial genetic diversityin the virus population.

With this minimal description of the WT FMDV 3Dpol in hand,we were in a position to probe the molecular mechanism for thedecreased ribavirin sensitivity of an FMDV variant encodingM296I 3Dpol (39). Our expectation was that FMDV M296I 3Dpolwould be a higher-fidelity polymerase than the WT based on PVG64S 3Dpol, which also confers decreased sensitivity to ribavirin(9). Surprisingly, FMDV M296I 3Dpol exhibited a lower fidelitythan that of WT FMDV 3Dpol. The overall efficiency (k_pol/K_d,app)of correct nucleotide incorporation for FMDV M296I 3Dpol wasdecreased 2.5-fold relative to that of the WT enzyme withoutexhibiting any change in the overall efficiency of incorrectnucleotide incorporation (Fig. 7; Table 2). This change in fidelitydecreases the calculated transition mutation frequency (Table3). Importantly, evidence for low-fidelity replication by thisvariant is apparent in the transition mutation frequency observedin cell culture (Table 3) (39). The mutation frequency calculatedfrom biochemical data is higher than that observed in cell culturefor both FMDV WT and M296I 3Dpols (Table 3). The differenceis due, in part, to the fact that all four possible mispairedcombinations have not been considered. In addition, intracellularconditions, for example, nucleotide pools, may be conduciveto more faithful RNA synthesis. Finally, selection occurs duringvirus multiplication in cell culture, which will reduce theobserved mutation frequency. These data provide another correlationbetween polymerase error frequency in vitro and virus populationdiversity in cell culture. However, future experiments willbe required to prove that the increased error frequency of M296I3Dpol observed in vitro is the cause of the reduced fitnessof M296I FMDV observed in cell culture.

This is the first FMDV polymerase variant with a mutator phenotype.A PV 3Dpol variant that changes Asn-297 in motif B to Glu alsoexhibits a mutator phenotype (27). The corresponding residuein FMDV 3Dpol is Asn-307. As shown in Fig. 1B, Asn-307 alsointeracts with the nucleotide. This illustration also conveysthe ability of Asn-307 to influence the β9- ${alpha}$ 11 loop andvice versa. The kinetic data show that substitutions in bothmotif B and the β9- ${alpha}$ 11 loop cause the mutator phenotypeby decreasing the efficiency of correct nucleotide incorporation,not by increasing the efficiency of incorrect nucleotide incorporation.This observation suggests that the RdRp active site employstwo distinct conformations for correct versus incorrect nucleotideincorporation.

Why is M296I FMDV less sensitive to ribavirin than the WT virus?Although FMDV M296I 3Dpol is more efficient at GMP misincorporationthan the WT enzyme, FMDV M296I 3Dpol is less efficient at ribavirinincorporation (Fig. 8). In fact, the overall efficiency of ribavirinincorporation by FMDV M296I 3Dpol is four- to fivefold lowerthan that observed for WT FMDV 3Dpol (Table 2). The abilityto decrease the efficiency of ribavirin incorporation withoutchanging the efficiency of GMP misincorporation suggests thatthe active-site conformations supporting these incorporationevents are different. We now have to conclude that there areat least three distinct conformations assumed by the RdRp thatare competent for nucleotide incorporation. Therefore, substantialflexibility exists in the active-site conformations employedfor nucleotide incorporation, and these active-site conformationsare induced by the nascent base pair. This interpretation isconsistent with recent reports on 2'-deoxynucleotide selectionby DNA polymerases (29, 44).

Residue 296 is located on the β9- ${alpha}$ 11 loop (Fig. 1). Thisloop moves as much as 1.5 Å in response to nucleotidebinding and the nature of the nascent base pair (Fig. 1C) (18).It is likely that changes in the position of this loop contributeto the induced-fit mechanism revealed by the biochemical studiesreported here. In the context of our other studies of RdRp structure-functionrelationships (9, 20, 21, 27), we conclude that cross-talk betweenresidues in the ribose binding pocket, including motifs A andB, and the β9- ${alpha}$ 11 loop determines RdRp (in)fidelity.

The M296I substitution was identified as a common change encodedby a variety of FMDV 3Dpol genes present in populations seriallypassaged in the presence of ribavirin that exhibited reducedsensitivity to the drug (39). The distribution of mutationsobserved in the presence of ribavirin for the population wasbiased heavily toward G $->$ A and C $->$ U transition mutations, suggestingthat ribavirin was incorporated preferentially opposite C inthe template (13). In contrast, FMDV encoding the M296I substitutionexhibited a bias for A $->$ G and U $->$ C transition mutations, suggestingthat ribavirin was incorporated preferentially opposite U inthe template (39). Our biochemical data explain that the biasfor FMDV M296I 3Dpol as the frequency of ribavirin incorporationopposite C is 1/100,000, a value 12.5-fold lower than that observedfor incorporation opposite U (Table 3). Therefore, even moreinsight into RdRp fidelity may be obtained from the furtheranalysis of mutations in the 3Dpol coding sequence present inthe original populations of FMDV selected in the presence ofribavirin.

The viral RdRp is responsible for generating genomic diversityduring replication, leading to the phenotypic flexibility ofRNA virus quasispecies. Viral quasispecies dynamics dependsnot only on the genomic diversity of the population but alsoon selection imposed by the environment and changes therein(recently reviewed in reference 16). Although not proven, itis likely that viral quasispecies dynamics are optimized forviral pathogenesis. Clearly, even in the absence of extinctionor even detectable mutagenesis, nucleotide analogues like ribavirincould alter viral quasispecies dynamics, leading to an antiviraleffect, especially in the complex environment of a mammalianhost. Consistently with this possibility is the finding thatthe ribavirin-resistant PV variant encoding the G64S 3Dpol exhibitsa restricted quasispecies (36, 45) and is attenuated in a mousemodel for PV infection (36, 45). G64S PV grows as well as theWT virus in cell culture, and the population diversity is decreasedby only threefold (9, 38, 45). G64S PV is capable of protectingmice against a lethal challenge with WT PV (46). Therefore,processes selecting for virus variants displaying a restrictedquasispecies may represent processes for viral attenuation andvaccine development.

Is the selection of viral mutants conferring resistance to ribavirina general approach for viral attenuation by quasispecies restriction?Clearly, multiple mechanisms for resistance to ribavirin exist.FMDV has found a counterintuitive solution: a low-fidelity polymerasewith a specific defect in ribavirin utilization. In the caseof hepatitis C virus, mutations in both the RdRp (NS5b) (50)and another replicase component (NS5a) (37) have been implicatedin ribavirin resistance, but the molecular mechanism(s) is notknown. The study presented here underscores the impact of mechanisticstudies of the RdRp in vitro on defining the mechanisms of resistancein cell culture and the elucidation of structural elements controllingRdRp fidelity/genomic diversity, which are potential targetsfor viral attenuation and vaccine development.

ACKNOWLEDGMENTS

We thank Ibrahim Moustafa for preparing Fig. 1C. We are indebtedto C. Ferrer-Orta and N. Verdaguer for many valuable discussionsof FMDV polymerase structure. We thank R. Agudo and C. Escarmísfor their many helpful suggestions.

This study was supported by the following grants: AI45818 fromNIAID/NIH (to C.E.C.), BFU 2005-00863 from MEC, Proyecto Intramuralde Frontera 2005-20F-0221 from CSIC, and Fundación RamónAreces. CIBERehd is funded by the Instituto de Salud CarlosIII. A.A. is a recipient of an I3P contract from CSIC, and M.S.was supported by a predoctoral fellowship from the Ministeriode Educación y Ciencia.

FOOTNOTES

* Corresponding author. Mailing address for C. E. Cameron: Department of Biochemistry and Molecular Biology, Pennsylvania State University, University Park, PA 16802. Phone: (814) 863-8705. Fax: (814) 865-7927. E-mail: cec9{at}psu.edu. Mailing address for E. Domingo: Centro de Biología Molecular Severo Ochoa (CSIC-UAM), Cantoblanco, E-28049 Madrid, Spain. Phone: 34 91 1964540. Fax: 34 91 1964420. E-mail: edomingo{at}cbm.uam.es

Published ahead of print on 1 October 2008.

REFERENCES

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