Effect of Metal Ion Binding on the Structural Stability of the Hepatitis C Virus RNA Polymerase*

The RNA polymerase activity of the hepatitis C virus, a major human pathogen, has previously been shown to be supported by metal ions. In the present study, we report a systematic analysis of the effect of metal ion binding on the structural stability of the hepatitis C virus RNA polymerase. Chemical and thermal denaturation assays revealed that the stability of the protein is increased significantly in the presence of metal ions. Structural analyses clearly established that metal ion binding increases hydrophobic exposure on the RNA polymerase surface. Furthermore, our denaturation studies, coupled with polymerization assays, demonstrate that the active site region of the polymerase is more sensitive to chemical denaturant than other structural scaffolds. We also report the first detailed study of the thermodynamic parameters involved in the interaction between the hepatitis C virus RNA polymerase and metal ions. Finally, a mutational analysis was also performed to investigate the importance of Asp220, Asp318, and Asp319 for metal ion binding. This mutational study underscores a strict requirement for each of the residues for metal binding, indicating that the active center of the HCV RNA polymerase is intolerant to virtually any perturbations of the metal coordination sphere, thereby highlighting the critical role of the enzyme-bound metal ions. Overall, our results indicate that metal ions play a dual modulatory role in the RNA polymerase reaction by promoting both a favorable geometry of the active site for catalysis and by increasing the structural stability of the enzyme.

The RNA polymerase activity of the hepatitis C virus, a major human pathogen, has previously been shown to be supported by metal ions. In the present study, we report a systematic analysis of the effect of metal ion binding on the structural stability of the hepatitis C virus RNA polymerase. Chemical and thermal denaturation assays revealed that the stability of the protein is increased significantly in the presence of metal ions. Structural analyses clearly established that metal ion binding increases hydrophobic exposure on the RNA polymerase surface. Furthermore, our denaturation studies, coupled with polymerization assays, demonstrate that the active site region of the polymerase is more sensitive to chemical denaturant than other structural scaffolds. We also report the first detailed study of the thermodynamic parameters involved in the interaction between the hepatitis C virus RNA polymerase and metal ions. Finally, a mutational analysis was also performed to investigate the importance of Asp 220 , Asp 318 , and Asp 319 for metal ion binding. This mutational study underscores a strict requirement for each of the residues for metal binding, indicating that the active center of the HCV RNA polymerase is intolerant to virtually any perturbations of the metal coordination sphere, thereby highlighting the critical role of the enzymebound metal ions. Overall, our results indicate that metal ions play a dual modulatory role in the RNA polymerase reaction by promoting both a favorable geometry of the active site for catalysis and by increasing the structural stability of the enzyme.
Recent estimates indicate that more than 170 million people worldwide are infected with the hepatitis C virus (HCV) 1 (1). It is estimated that about 80% of patients with acute HCV infection will progress to chronic hepatitis. Of these, 20% will develop cirrhosis, and 1-5% will develop hepatocellular carcinoma (2)(3)(4)(5). There is thus an urgent need for the development of antiviral drugs aimed at inhibiting this pathogen.
The HCV nonstructural 5B protein (NS5B) has been shown to be an RNA-dependent RNA polymerase (6 -11). The protein contains characteristic motifs, such as the GDD motif, shared by RNA-dependent RNA polymerases, and is believed to be responsible for the genome replication of HCV (12). The NS5B protein has been studied extensively during the past few years because it is one of the major targets for the development of antiviral drugs (7,(13)(14)(15)(16)(17)(18)(19)(20)(21)(22)(23)(24)(25). The enzyme can utilize a wide range of RNA molecules as template, although it appears to prefer certain homopolyribonucleotides (26). By itself, NS5B appears to lack specificity for HCV RNA and displays activity on heterologous nonviral RNA (3). This lack of specificity for HCV RNA supports the notion that additional viral or cellular factors are required for specific recognition of the viral replication signal.
The HCV RNA polymerase activity has been shown to be supported by both magnesium and manganese ions (7,13,(21)(22)(23)(24)(25). However, the recent characterization of the affinity of the enzyme for metal ions suggests that magnesium is the cation that is used in vivo during polymerization (27). Analysis of the crystal structure of NS5B revealed that the protein is folded into characteristic fingers, palm, and thumb subdomains (28,29). The particular fold adopted by the palm subdomain is shared by many proteins that bind nucleotides and/or nucleic acids (30). The crystal structure of the HCV RNA polymerase showed that it contains two absolutely conserved aspartic acid residues that coordinate two metal ions in the active site of the protein (31). These two metal ions are in contact with both the phosphate of the nucleotide and several acidic amino acids residues (31). A catalytic mechanism has been proposed for polymerases in which one metal ion is involved in both positioning the substrate and in the activation of an incoming nucleophile (32). Nucleophilic attack would then generate a trigonal bipyramidal transition state that would be stabilized by both metal ions. The second metal ion also stabilizes the negative charge that appears on the leaving 3Ј-oxygen, thus facilitating its departure from the phosphate. Analysis of the NS5B protein crystal structure indicated that the two metal ions are about 3.6 Å apart in the active site of the protein (31). Crystallographic and fluorescence spectroscopy data indicate that metal ion binding seems to be limited to the active site region and does not involve other subdomains of the protein (27,31). Finally, we and others recently demonstrated that the enzyme undergoes conformational changes upon binding of metal ions (27,33). However, this process does not significantly stimulate the binding of the enzyme to the RNA or NTP substrates (27).
In the present study, we report a systematic analysis of the effect of metal ion binding on the structural stability of the HCV RNA polymerase. Using fluorescence spectroscopy, circular dichroism (CD), and denaturation assays, we demonstrate that the binding of metal ions to the enzyme is critical for both structural stabilization and catalysis. Mutational analysis also * This work was supported by grants from the Canadian Institutes for Health Research and Fonds de la Recherche en Santé du Québec. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18  revealed the importance of specific aspartate residues for metal binding. Our data provide insights on the precise role of metal ions in the NS5B-mediated RNA polymerase reaction.

MATERIALS AND METHODS
HCV NS5B Expression and Purification-The expression and purification of a truncated form of HCV NS5B protein (NS5B⌬21) lacking the last 21 amino acids of the protein were performed as described previously (27). Alanine substitutions were introduced into the NS5B gene by using the two-stage overlap extension method (34).
Fluorescence Measurements-Fluorescence was measured using a Hitachi F-2500 fluorescence spectrophotometer. Background emission was eliminated by subtracting the signal from either buffer alone or buffer containing the appropriate quantity of substrate.
The extent to which ligands bind to the NS5B protein was determined by monitoring the fluorescence emission of a fixed concentration of proteins and titrating with a given ligand. The binding can be described by Equation 1, The K d values were determined from a nonlinear least squares regression analysis of titration data using Equation 3.
Thermodynamics of Binding-The temperature dependence of the association constants (K a ) for ligand binding was analyzed according to the van't Hoff isobaric equation, assuming that the entropy change (⌬S o ) and the enthalpy change (⌬H o ) remained constant over the whole range of temperatures (Equation 4).
Equilibrium Unfolding Experiments-A 100 nM solution of NS5B was adjusted to the desired final concentration of guanidinium hydrochloride (GdmHCl) and incubated for 60 min at 22°C. The parameters ⌬G u°( free energy of unfolding in the absence of denaturant), m (cooperativity of unfolding), and C m (midpoint concentration of denaturant required to unfold half of the protein) were obtained as previously outlined using Equation 5 ⌬G u ϭ Ϫ RT ln K u (Eq. 5) and Equation 6.
CD Spectroscopy Measurements-CD measurements were performed with a Jasco J-810 spectropolarimeter. The samples were analyzed in quartz cells with path lengths of 1 mm. Thermal transitions were monitored by following the change in ellipticity at 222 nm. The samples were heated from 20 to 95°C, at a heating rate of 1°C/min. The ellipticity results were expressed as mean residue ellipticity, [], in degrees⅐cm 2 ⅐dmol Ϫ1 . The fraction of unfolded protein at each temperature was determined by calculating the ratio [ 222 ]/[ 222 ] d , where [ 222 ] d is the molar ellipticity for the completely unfolded enzyme.
ANS Binding Measurements-Binding of ANS (1-anilino-8-naphthalenesulfonate) was evaluated by measuring the fluorescence enhancement of ANS (50 M) upon excitation at a wavelength of 380 nm. The emission spectra were integrated from 400 to 600 nm.
Quenching of HCV RNA Polymerase by Acrylamide-Quenching experiments were performed at 22°C by adding increasing concentrations of acrylamide. The excitation wavelength was set at 290 nm, and the fluorescence emission spectra were scanned from 300 to 400 nm. The integration area between 330 and 360 nm was used for data analysis. The fluorescence quenching data in the presence of acrylamide were analyzed according to the Stern-Volmer equation (35), where F o and F are the fluorescence intensities in the absence or presence of the quencher, respectively. K sv is the dynamic Stern-Volmer quenching constant, and [Q] is the quencher concentration.
When the Stern-Volmer displayed an upward curvature, the static quenching concept was used, and the experimental data were fitted to a revised Stern-Volmer equation, where V is the static quenching constant measuring the complex formation between acrylamide and the enzyme.

Expression, Purification, and Intrinsic Fluorescence
Properties of the HCV RNA Polymerase-To evaluate the role of metal ions in the NS5B-mediated RNA polymerase activity, the enzyme was expressed as described previously (27). SDS-PAGE analysis demonstrated that the 65-kDa NS5B protein was the predominant polypeptide in the purified fraction (Fig. 1A). The identity of the protein was also confirmed by immunoblotting analysis, using a monospecific antibody (data not shown). The fluorescence properties of the purified NS5B protein in standard buffer at 22°C are shown in Fig. 1B. Analysis of the background corrected fluorescence emission spectrum of the NS5B protein revealed an emission maximum ( max ϭ 335 nm) that is blue shifted relative to that of free L-tryptophan, which under the same conditions is observed to be at 350 nm. Blue shifts of protein emission spectra have been ascribed to shielding of the tryptophan residues from the aqueous phase, a result of the three-dimensional structure of the protein. Accordingly, denaturation of NS5B with 8 M urea results in a red shift of max toward 350 nm (Fig. 1B). Analysis of the molar intensity of the fluorescence emission spectrum of NS5B revealed a linear change of 0.16 fluorescence intensity units/nM protein concentration (Fig. 1B). This relatively small change can probably be attributed to small losses of proteins from solution through adhesion. All subsequent binding experiments were therefore performed at a protein concentration of 100 nM, with the assumption that the binding equilibrium was not complicated by the presence of an aggregation equilibrium.
Denaturation Studies-In a recent study, we demonstrated that the HCV RNA polymerase undergoes subtle conformational changes upon the binding of metal ions, but this process does not significantly stimulate the binding of RNA or NTP substrates (27). Because metal ions have the potential to play both catalytic and structural roles in protein chemistry, we analyzed the effect of magnesium ions on the structural stability of the HCV RNA polymerase. The effect of metal ion binding on the NS5B structural stability was initially assessed by GdmHCl denaturation assays performed at 22°C. Upon an increase of the GdmHCl concentration, the fluorescence emission maximum of the unliganded NS5B protein shifted to 350 nm (data not shown), reflecting the transfer of tryptophan residues to a more polar environment. The unliganded protein structure reacted very sensitively to the slightest concentration changes in the lower concentration range between 0.5 and 3.0 M where the strongest effects on emission changes are observed ( Fig. 2A). No changes could be observed at GdmHCl concentrations higher than 3.5 M. The C m value (midpoint concentration of denaturant required to unfold half of the protein) was reached at 1.05 M GdmHCl. Protein denaturation assays were then performed in the presence of saturating concentrations of Mg 2ϩ ions. The complete thermodynamic unfolding parameters were determined, and the values are presented in Table I. A significant stabilization of the NS5B structure was observed in the presence of magnesium ions, as reflected by the shift in the C m value that was now reached at 1.91 M GdmHCl ( Fig. 2A). The informative finding is that the stability of the enzyme is increased by 2.22 kJ⅐mol Ϫ1 in the presence of Mg 2ϩ ions.
The chemical denaturation assays were also analyzed in terms of catalytic activity. Two distinct approaches were undertaken to study the effect of denaturation on the catalytic activity of the protein. In the first approach, the HCV RNA polymerase was initially denatured with increasing concentrations of GdmHCl. The RNA polymerase activity of the protein was then monitored by incubating the denatured protein with magnesium and a proper combination of template/primer/nucleotide. The data indicated that the RNA polymerase activity of the enzyme was reduced by 50% at a concentration of 0.81 M GdmHCl (Fig. 2B). In the second approach, the NS5B protein was initially incubated with magnesium. The protein-ion complex was then denatured with GdmHCl and subsequently assayed for RNA polymerase activity. Using this second approach, a concentration of 1.32 M GdmHCl was necessary to reduce the NS5B-mediated RNA polymerase activity by half (Fig. 2B). Overall these data indicate that the binding of metal ions to the HCV RNA polymerase protects the enzyme against chemical denaturation. By comparison with the structural stability assay ( Fig. 2A), these data suggest that the active site region of the enzyme is more sensitive to chemical denaturant than other structural scaffolds.
The effects of metal binding on the structural stability of the HCV RNA polymerase were also assessed by CD spectroscopy. Thermal denaturation assays were performed in both the presence and absence of Mg 2ϩ ions, and unfolding of the enzyme was evaluated by monitoring the changes in the ␣-helix content of the protein (222 nm). Thermal denaturation of the unliganded NS5B protein initially revealed a midpoint of thermal transition (T m ) of 40.2°C (Fig. 3A). The addition of saturating concentrations of Mg 2ϩ ions resulted in a shift of the T m value to 45.7°C. These results demonstrate that the binding of Mg 2ϩ ions to the NS5B protein significantly increases the structural stability of the enzyme.
Thermal stability was also monitored in terms of catalytic activity. This was done by preincubating either the unliganded NS5B protein or the protein saturated with magnesium ions for 15 min at various temperatures, followed by quenching on ice. The protein samples were then assayed for RNA polymerase activity at 18°C in the standard RNA polymerase buffer. The thermal inactivation curves are shown in Fig. 3B. The results indicate that preincubation of the HCV RNA polymerase with Mg 2ϩ ions protects the enzyme against thermal denaturation. The inactivation curve of the protein preincubated with magnesium ions is shifted 15°C to the right relative to the enzyme that had not been preincubated with metal ions (Fig. 3B). Note that all of the denaturation assays, performed either with increasing GdmHCl concentrations or by thermal denaturation, revealed monophasic unfolding curves, suggestive of a two-state unfolding model. No intermediate form could be detected during the unfolding process.
Thermodynamic Analysis-Using a combination of crystallography, fluorescence, and CD, the HCV RNA polymerase has previously been shown to interact with metal ions (27,31,33). In the present study, a thermodynamic investigation of the metal ion binding process was initiated to provide additional insight into the energetic and entropic characteristics of the binding reaction. Using fluorescence spectroscopy, the thermodynamic parameters of the binding reaction were evaluated by determining the temperature dependence of the association constant (K as ) for Mg 2ϩ ions. Analysis of the van't Hoff plot for the initial interaction between Mg 2ϩ and the HCV RNA polymerase reveals that the interaction is connected with a high enthalpy of association, ⌬H o ϭ Ϫ33.4 kJ/mol (Fig. 4). Furthermore, the binding reaction is clearly enthalpy-driven with the resultant ⌬S o ϭ Ϫ88.7 J/mol⅐K. These data indicate that the reaction is spontaneous at low temperatures but tends to reverse at higher temperatures. Accordingly, the binding of metal ions could not be observed repeatedly at temperatures higher than 50°C (data not shown). This can most probably be attributed to the denaturation of the protein that occurs at higher temperatures and the concomitant destabilization of the active site.
Metal Ion Binding Increases Hydrophobic Exposure at Low Urea Concentrations-To gain additional insights into the structural modifications that occur upon metal ion binding, we investigated the binding of structural fluorescent reporters to the enzyme. The exposure of the hydrophobic area of the HCV RNA polymerase was evaluated by measuring the binding of ANS to the protein. ANS is a reporter of exposed hydrophobic surfaces on proteins which binds with high affinity to hydrophobic patches, which results in an enhancement of ANS intrinsic fluorescence (36). Our data revealed that the unliganded NS5B protein binds very weakly to ANS, probably reflecting limited hydrophobic regions at the surface of the protein (Fig.  5). However, a significant increase of the ANS fluorescence is observed when the protein is incubated in the presence of saturating concentrations of magnesium ions (Fig. 5). As expected, increasing the urea concentrations resulted in a drastic decrease of the emission intensity, as the protein with bound Mg 2ϩ became unfolded by the denaturant. Overall, these data indicate that magnesium ion binding increases hydrophobic exposure on the HCV RNA polymerase surface.
To understand better the conformational changes that occur after the binding of magnesium ions to the HCV RNA polymerase, quenching experiments were performed in the presence of acrylamide. Acrylamide is a nonselective quencher of proteins which can penetrate into the interior of the protein matrix (37). In the absence of denaturant, acrylamide displayed greater accessibility for the NS5B protein bound to magnesium ions, reflecting the structural difference between the unliganded NS5B protein and the protein bound to Mg 2ϩ ions (Fig. 6). As observed in our ANS binding assays, acrylamide has increased access to the interior of the NS5B protein when the protein is bound to magnesium ions (Fig. 6). The various quenching constants are summarized in Table II.
The acrylamide quenching experiments were then performed in the presence of increasing urea concentrations. As seen in Fig. 6, metal ion binding provided significant protection against acrylamide quenching when the protein was denatured with high concentrations of urea. This protection was particularly evident in the presence of 4.5 M urea, where the binding of Mg 2ϩ ions clearly protected the NS5B protein from acrylamide quenching. Clearly, the acrylamide quenching assays performed in the presence of increasing urea concentrations indicate that magnesium ion binding to the HCV RNA polymerase significantly increases the structural stability of the protein.
Mutational Analysis-Analysis of the crystal structure of the HCV RNA polymerase has revealed the presence of two metal ions that lie 3.6 Å apart in the active center of the enzyme (31). The side chains of Asp 220 and Asp 318 have been shown to bridge the two metal ions, whereas the side chain of Asp 319 interacts with only one of the two metal ions (31). The respective importance of each of these side chains for catalysis has been confirmed previously by mutational analysis (9). To investigate whether the metal ion binding activity detected in our assays is exclusively dependent on the active center of the HCV RNA polymerase, we synthesized a series of enzymatic mutants. Asp 220 , Asp 318 , and Asp 319 were substituted for alanine, and the mutant polypeptides were expressed and purified in parallel with the wild-type enzyme. The effects of single alanine mutations on the metal ion binding activity were investigated by fluorescence spectroscopy. No significant changes in fluorescence intensity could be observed upon the addition of Mg 2ϩ ions (up to 100 mM) to these alanine mutants (Fig. 7A). We conclude that each of the three aspartate residues (Asp at 220, 318, and 319) is critical for metal ion binding. The thermodynamic stability of these mutants was then analyzed by Gdm-HCl denaturation (Fig. 7B). In contrast to the wild-type enzyme, no significant stabilization was observed when the D220A mutant was incubated with a high concentration of Mg 2ϩ ions (50 mM). Similar results were obtained for both the D318A and D319A mutants. Finally, no significant increase of the ANS fluorescence was observed when the D220A mutant proteins were incubated with Mg 2ϩ (Fig. 7C). Again, similar results were obtained for the two other alanine mutants.  Binding of Other Metal Ions-Previous studies have shown that certain divalent ions, such as zinc, copper, and nickel, do not support the catalytic activity of the HCV RNA polymerase but can very efficiently inhibit the activity of the enzyme (13,25). Using fluorescence spectroscopy, we have been unable to detect efficiently the binding of these ions to the HCV RNA polymerase. Because no modifications in the fluorescence intensity are observed upon the addition of zinc, copper, and nickel, we conclude that these ions do not bind in close proximity to the indole side chain of a tryptophan and/or do not induce a conformational change that results in alterations in the microenvironments of tryptophan residues of the enzyme upon binding. Nonetheless, we incubated the HCV RNA polymerase with these metal ions, and we intended to monitor the binding of ANS to the enzyme. Unfortunately, high concentrations of Zn 2ϩ , Cu 2ϩ , and Ni 2ϩ ions (2-20 mM) led to the precipitation of the ANS structural reporter thereby hindering the fluorescence analysis. However, no significant changes in the ANS fluorescence intensity were observed when the enzyme was incubated with lower concentrations (100 -1,000 M) of copper, nickel, and zinc ions (data not shown).

DISCUSSION
Studies performed with purified proteins in solution add crucial information to crystallographic data. In this regard, the multidimensional properties of both fluorescence and CD spectroscopy provide accurate sensitivity to monitor numerous aspects of protein-ligand interactions. In the present study, the effect of metal ion binding on the stability of the HCV RNA polymerase was investigated.
The RNA polymerase activity of the HCV NS5B protein has been characterized extensively (7,(13)(14)(15)(16)(17)(18)(19)(20)(21)(22)(23)(24)(25). The protein is believed to be responsible for the genome replication of HCV and is thus a critical protein of the virus. Magnesium or manganese ions have been shown to be involved in the catalytic activity of the enzyme (7,13,(21)(22)(23)(24)(25). The novel aspect of the current study is that we demonstrate the structural role of metal ions in the HCV RNA polymerase. Both CD and fluorescence spectroscopy studies clearly demonstrated that the protein has an increased structural stability in the presence of metal ions, compared with the unliganded state. The results are supported by previous observations that demonstrated that the HCV RNA polymerase undergoes conformational changes upon metal ion binding (27,33). The use of both the ANS structural reporter and acrylamide quencher clearly established structural differences between the unliganded enzyme, and the enzyme bound to magnesium ions. A dual role for metal ions in both structural stabilization and catalysis has also been observed in other proteins (38 -41). For instance, the presence of magnesium in the active site of the Escherichia coli alkaline phosphatase has been shown to be important for both catalytic activity and stability of the enzyme (38).
What is the biological relevance of the present findings? Mounting evidence suggests that a conformational change is required for the HCV RNA polymerase to provide an adequate platform for initiation of transcription/replication. For instance, it has been suggested that a modest movement of a ␤-hairpin that hangs over the active site appears to be neces- sary to position the 3Ј-end of the RNA substrate and/or accommodate the elongation of the nascent double-stranded RNA molecules (42,43). This is reminiscent of the situation that is found in the rabbit hemorrhagic disease virus, where it was shown that the conformation of the active site can vary dramatically depending on the ions present during crystallization (44). Comparisons among various RNA polymerases strongly suggest that conformational changes similar to those seen in DNA polymerases may be important for the catalytic activity of RNA polymerases (45,46). Our study clearly showed that the binding of metal ions induces a conformational change that is characterized by an increased exposure of hydrophobic regions. This conformational modification may reflect movement of flexible structures to accommodate the substrate, or alternatively, it may reflect structural rearrangements that are required for additional functions. For instance, structural modifications may be required to promote interactions with other proteins (47) or to facilitate the formation of the membrane-associated HCV replication complex (48). Alternatively, it has been suggested previously that the ⌬1 loop that connects the fingers and the thumb subdomains of the HCV RNA polymerase could contribute to modulate the conformational state of the enzyme (28). It should be noted that inhibition of such a conformational change may provide a target for the development of novel antiviral drugs. In fact, a recent study suggested that the binding of the HCV NS5A protein to the HCV RNA polymerase may inhibit conformational changes that are required for the RNA polymerase activity (49). Finally, modulation of the HCV RNA polymerase activity has also been noted upon interaction with the HCV NS3 and NS4B proteins. However, the involvement of precise conformational changes in these processes remains to be addressed.
The present work has demonstrated major changes in the structural stability of the HCV RNA polymerase when it binds to magnesium ions. Our denaturation studies, coupled with polymerization assays, demonstrated that the active site region of the polymerase is more sensitive to chemical denaturant than other structural scaffolds. Indeed, mutational studies have shown that the active site of HCV RNA polymerase is composed of an intricate network of hydrogen bonds and electrostatic interactions, of which a surprisingly high proportion is required for reaction chemistry (9). The amino acids that are coordinating the two metal ions (metals A and B) found in the NS5B active site have also been identified by crystallographic studies (31). The side chains of Asp 220 and Asp 318 bridge the two metal ions that lie 3.6 Å apart in the crystal structure, whereas the side chain of Asp 319 interacts with only one of the two metal ions (metal A) (31). The respective importance of these side chains for catalysis has been confirmed by mutational analysis. Although changes to either the Asp 220 or Asp 318 residues completely abolished the enzymatic activity, substitution of the Asp 319 residue by asparagine or glutamate is tolerated (9). In addition to their contacts with aspartate residues, the two metal ions are engaged in a network of interactions with the phosphates of nucleotides (31). For instance, metal B is coordinated by all three phosphates of the nucleotide, whereas metal A interacts with the ␣-phosphate, likely in position to contact the 3Ј-OH of the ultimate nucleotide of the nascent RNA chain during polymerization. The present mutational analysis revealed the importance of each of the aspartate residues (Asp 220 , Asp 318 , and Asp 319 ) that directly contact the metal ions in the previously determined crystal structure of the HCV RNA polymerase. The mutational study underscores a strict requirement for the bidentate interactions made by both Asp 220 and Asp 318 , but the informative finding is that substitution of the Asp 319 residue by alanine completely abolished the Mg 2ϩ binding activity. The absence of the Asp 319 side chain clearly modifies the geometry of the enzyme active center and completely abrogates the Mg 2ϩ binding activity. This indicates that the active center of the HCV RNA polymerase is intolerant to virtually any perturbations of the metal coordination sphere, highlighting the critical role of the enzyme-bound metal ions. Based on the results of our fluorescence, CD, and denaturation experiments, we demonstrated that the NS5B protein undergoes a conformational change upon the binding of metal ions which is characterized by an increased stability of the enzyme. Furthermore, previous reports indicated that the binding of metal ions does not significantly stimulate the binding of the HCV RNA polymerase to its RNA or NTP substrates (27). We thus envisage that the ion-induced conformational change is a prerequisite for catalytic activity by correctly positioning the side chains of residues located in the active site of  the enzyme, while at the same time contributing to the stabilization of the enzyme architecture. Therefore, we propose that magnesium ions play a dual modulatory role in the HCV RNA polymerase reaction by promoting both a favorable geometry of the active site for catalysis and by increasing the structural stability of the enzyme. Although the complete understanding of the mechanisms underlying HCV replication and the cellular and viral factors required for these processes is still incomplete, characterization of the biochemical properties of NS5B should provide the basis for further studies in this direction. Structural and enzymatic studies are clearly beginning to reveal the essential features of the polymerase reaction. These new insights should eventually lead to the design of effective antiviral drugs to inhibit viral replication and, ultimately, cure infected patients.