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INTRODUCTION |
Currently, hepatitis C virus
(HCV)1 is the leading
etiological agent of non-A non-B hepatitis, with more than 170 million
people worldwide being infected with HCV (1). 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 (2-5). HCV is a positive, single-stranded RNA virus of the
Flaviviridae family. The genome is ~10,000 nucleotides long and encodes a single polyprotein of about 3,010 amino acids (6).
The polyprotein is processed by both host cell and viral proteases into
three major structural proteins and several nonstructural proteins
necessary for viral replication (6).
One key enzyme encoded by HCV is the nonstructural 5B protein (NS5B),
which has been shown to be an RNA-dependent RNA polymerase (7-12). The HCV NS5B protein contains characteristic motifs, such as
the GDD motif, shared by RNA-dependent RNA polymerases
(13). The NS5B protein is thus believed to be responsible for the
genome replication of HCV. Indeed, polymerase activity has been
demonstrated with recombinant NS5B expressed in both insect cells and
Escherichia coli (8, 14-26). The activity has been
extensively studied because it is one of the major targets for the
development of antiviral drugs. The NS5B protein can utilize a wide
range of RNA molecules as template, although it appears to prefer
certain homopolyribonucleotides (27). By itself, NS5B appears to lack specificity for HCV RNA and displays activity on heterologous nonviral
RNA (8). 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.
Previous reports showed that both magnesium and manganese ions can
support the polymerase activity (8, 14, 22-26), although manganese
ions appear to be more effective in promoting optimal catalytic action
(14). No information is currently available on the precise affinity of
the enzyme for metal ions nor on their possible roles in catalysis, but
manganese has recently been found in the crystal structure of NS5B
(28). Metal ions have the potential to fulfill multiple functional
roles in catalysis such as: (i) increasing the affinity of the enzyme
for RNA; (ii) increasing the affinity of the enzyme for NTPs; (iii)
inducing the proper folding of the protein; and (iv) being directly
involved in catalysis by promoting the activation of nucleophiles.
As a first step toward elucidating the nature and the role(s) of metal
ions in the reaction chemistry, we have utilized endogenous tryptophan
fluorescence to evaluate the interactions of metal ions with the NS5B
protein. Quenching of the fluorescence signals by titration of the
protein with metal ions provides a straightforward and powerful
technique for evaluating the binding of metal ions to proteins. Our
data provide insights on the precise role of metal ions in the
NS5B-mediated RNA polymerase reaction.
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EXPERIMENTAL PROCEDURES |
HCV NS5B Expression and Purification--
A plasmid for
expression of a truncated form of HCV NS5B protein (NS5B
21) lacking
the last 21 amino acids of the protein was generated by inserting the
truncated NS5B gene between the BamHI and XhoI
cloning sites of the pET21b expression plasmid (Novagen). In this
context, the NS5B protein is fused in frame with a C-terminal peptide
containing six tandem histidine residues, and expression of the
His-tagged protein is driven by a T7 RNA polymerase promoter. The
resulting recombinant plasmid, pET-NS5B, was transformed into E. coli BL21(DE3). A 100-ml culture of E. coli
BL21(DE3)/pET-NS5B was grown at 37 °C in Luria-Bertani medium containing 0.1-mg/ml ampicillin until the A600
reached 0.5. The culture was adjusted to 0.4 mM
isopropyl-
-D-thiogalactopyranoside, and the incubation
was continued at 18 °C for 20 h. The cells were then harvested
by centrifugation, and the pellet was stored at
80 °C. All
subsequent procedures were performed at 4 °C. Thawed bacteria
pellets were resuspended in 5 ml of lysis buffer A (50 mM
Tris-HCl, pH 7.5, 150 mM NaCl, 10% sucrose), and cell
lysis was achieved by the addition of lysozyme and Triton X-100 to
final concentrations of 50 µg/ml and 0.1%, respectively. The lysates were sonicated to reduce viscosity, and any insoluble material was
removed by centrifugation at 13,000 rpm for 45 min. The soluble extract
was applied to a 2-ml column of nickel-nitrilotriacetic acid-agarose
(Qiagen) that had been equilibrated with buffer A containing 0.1%
Triton X-100. The column was washed with the same buffer and then
eluted stepwise with buffer B (50 mM Tris-HCl, pH 8.0, 0.1 M NaCl, 10% glycerol) containing 50, 100, 200, 500, and
1000 mM imidazole. The polypeptide composition of the
column fractions was monitored by SDS-PAGE. The recombinant NS5B
protein was retained on the column and recovered in the 200 mM imidazole eluate. This fraction was dialyzed against
buffer C (50 mM Tris-HCl, pH 8.0, 50 mM NaCl, 2 mM dithiothreitol, 10% glycerol, 0.05% Triton X-100), and
the dialysate was applied to a 2-ml column of phosphocellulose that had
been equilibrated in buffer C. The column was washed with the same
buffer and then eluted stepwise with buffer C containing 0.1, 0.2, 0.3, 0.4, 0.5, and 1.0 M NaCl. The recombinant protein was
retained on the column and was recovered predominantly in the 1.0 M NaCl fraction. Following dialysis against buffer C, the
phosphocellulose preparation was stored at
80 °C. The protein concentration was determined by the Bio-Rad dye binding method with
bovine serum albumin as the standard.
Fluorescence Measurements--
Fluorescence was measured using
an 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 HCV 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.
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(Eq. 1)
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where Kd is the apparent dissociation
constant, [NS5B] is the concentration of the protein,
[NS5B·ligand] is the concentration of complexed protein, and
[ligand] is the concentration of unbound ligand.
The proportion of ligand-bound protein as described by Equation 1 is
related to measured fluorescence emission intensity by Equation 2.
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(Eq. 2)
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where
F is the magnitude of the difference between
the observed fluorescence intensity at a given concentration of ligand and the fluorescence intensity in the absence of ligand,
Fmax is the difference at infinite
[ligand], and [NS5B]tot is the total protein concentration.
If the total ligand concentration, [ligand]tot, is in
large molar excess relative to [NS5B]tot, then it can be
assumed that [ligand] is approximately equal to
[ligand]tot. Equations 1 and 2 can then be combined to
give Equation 3.
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(Eq. 3)
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The Kd values were determined from a
nonlinear least square regression analysis of titration data by using
Equation 3. The stoichiometry of binding was established from a linear version of the Hill equation.
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(Eq. 4)
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where n is the order of the binding reaction with
respect to ligand concentration, and K' is the concentration
of ion that yields 50% of
Fmax.
Circular Dichroism Spectroscopy Measurements--
Circular
dichroism measurements were performed with a Jasco J-810
spectropolarimeter. The samples were analyzed in quartz cells with
pathlengths of 1 mm. Far-UV and near-UV wavelength scans were recorded
from 200 to 250 nm and from 250 to 340 nm, respectively. All of the
dichroic spectra were corrected by subtraction of the background for
the spectrum obtained with either buffer alone or buffer containing
metal ions. An average of six wavelength scans is presented. The
ellipticity results were expressed as mean residue ellipticity,
[
], in degrees·cm2·dmol
1.
Analysis of Competitive Binding--
Analysis of the effect of a
fixed concentration of one metal ion ligand (iona) on the
binding of a second ion ligand (ionb) was performed in a
manner analogous to that previously reported for analyzing the kinetics
of a system in which two alternative substrates compete for the same
enzyme binding site (29). The change in fluorescence (
F)
observed upon titration of NS5B with iona in the presence
of a fixed concentration of competing substrate (ionb) can
be described by Equation 5.
|
(Eq. 5)
|
where
Fmax(a) and
Fmax(b) are the changes in fluorescence
produced at infinite concentrations of iona and
ionb, respectively. Ka and
Kb are the apparent dissociation constants for
iona and ionb, respectively. Equation 5 was fit
to the simple ligand saturation isotherms for both iona and
ionb.
Electrophoretic Mobility Shift Assays--
Electrophoretic
mobility shift assays were used to measure the binding of HCV NS5B
protein to RNA. The standard binding reactions were performed in buffer
containing 10 mM Tris-HCl, pH 7.5, 10 mM NaCl,
10 mM MgCl2 and incubated for 30 min at
25 °C. The concentrations of divalent ions were modified in some
experiments as indicated in the text. Radiolabeled RNA was synthesized
from a plasmid containing the NS5B gene flanked by the 5'- and
3'-terminal sequences found in the HCV genome. This RNA contains
the N-terminal region of the genome (nucleotides 1-571, including the
5'-untranslated region) fused to the C-terminal portion of the genome
(starting at nucleotide 9068 and including the 3'-untranslated region).
The plasmid was linearized with XbaI (located at the 3' end
of the construct) and gel-purified. The RNA transcript (1442 nucleotides) was synthesized with the MAXIscript kit (Ambion) using T7
RNA polymerase. Radiolabeled [
-32P]UTP was added to
the reaction, and the RNA fragment was purified with the QIAquick
nucleotide removal kit (Qiagen). The binding reaction mixtures were
directly subjected to electrophoresis on 1.5% agarose gels, and the
bands were visualized by autoradiography. The bands were then excised
from the dried agarose gels, and the amount of radioactivity was
counted with a liquid scintillation counter. The apparent dissociation
constant (Kd) for each probe was determined
according to Equation 6.
|
(Eq. 6)
|
Where fD represents the fraction of the
shifted nucleic acids, [NS5B] is the total protein concentration, and
Kd is the dissociation constant for the binding reaction.
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RESULTS |
Expression, Purification, and Intrinsic Fluorescence Properties of
HCV NS5B Protein--
The HCV NS5B protein contains motifs shared by
RNA polymerases, and its activity has been shown to be dependent on the
presence of either magnesium or manganese ions (8, 14, 22-26). To
further characterize the metal binding activity of the enzyme, the NS5B protein was expressed in E. coli as described under
"Experimental Procedures." A truncated form of NS5B (NS5B
21)
lacking the previously identified 21-amino acid hydrophobic domain (14,
22, 24, 25) was expressed and purified. SDS-PAGE analysis showed that the 65-kDa NS5B
21 protein was the predominant polypeptide in the
purified fraction (Fig. 1A).
The amount of NS5B
21 protein in this fraction was estimated to be 60 µg/ml. Immunoblotting analysis, using a monospecific antibody, also
confirmed the identity of this protein (data not shown).

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Fig. 1.
Expression, purification, and fluorescence
properties of NS5B. A, an aliquot (2 µg) of the
purified preparation of NS5B 21 was analyzed by electrophoresis
through a 12.5% polyacrylamide gel containing 0.1% SDS and visualized
with Coomassie Blue Dye. The positions and sizes (in kDa) of the size
markers are indicated on the left. B, background
corrected fluorescence emission spectra of NS5B. A, purified
protein in 50 mM Tris-HCl, 50 mM KOAc, pH 7.5. B, purified protein after a 2-h exposure to an 8 M solution of urea at 25 °C. Fluorescence spectra were
recorded at an excitation wavelength of 290 nm. C, molar
fluorescence of NS5B. Various concentrations of the purified NS5B
protein were assayed in 50 mM Tris-HCl, 50 mM
KOAc, pH 7.5. Emission was monitored at 335 nm, and excitation was
performed at 290 nm.
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The fluorescence emission spectrum of purified NS5B in standard buffer
at 22 °C is shown in Fig. 1B. To obtain the maximal emission peak at the low concentrations of protein required to accurately determine Kd values, excitation was
carried out at 290 nm. Both tyrosine and tryptophan absorb at this
wavelength (29). However, varying the excitation wavelength from 254 nm, where the contribution of tyrosine fluorescence to the emission spectrum would be the greatest, to 295 nm, where the emission spectrum
would arise almost exclusively from tryptophan, produced no change in
the position of
max (335 nm) or in the spectral bandwidth (55 nm at half-height) (data not shown). Thus, despite the
fact that NS5B contains 22 tyrosines in addition to the 9 tryptophans,
the emission spectrum is dominated by the indole fluorophores. This
dominance is due, in part, to the higher extinction coefficient of
tryptophan and to resonance energy transfer from tyrosine to tryptophan.
The emission maximum of the enzyme (335 nm) is blue-shifted relative to
that of free L-tryptophan, which under the same conditions is observed to be at 350 nm. The
max of tryptophan is
highly sensitive to the polarity of the microenvironment in which its indole side chain is localized. Blue shifts of protein emission spectra
have been ascribed to shielding of the tryptophan residues from the
aqueous phase (30). This shielding is the 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).
The molar intensity of the fluorescence emission spectrum of NS5B was
also determined. This spectrum was determined to see whether
significant protein aggregation or whether the loss of protein from
solution through adhesion could influence the data. As can be seen in
Fig. 1C, a decrease in fluorescence is observed with
decreasing concentrations of NS5B. A linear change of 0.16 fluorescence
intensity units/nM of protein was observed over the range
examined. This relatively small change can probably be attributed to
small losses of proteins from solution through adhesion. Alternatively, some photobleaching may occur over the period of time in which the
experiment is carried out. All of the binding experiments were thus
performed at a protein concentration of 250 nM, with the
assumption that the binding equilibrium was not complicated by the
presence of an aggregation equilibrium.
Binding of Mg2+ and Mn2+ Metal Ions to the
HCV NS5B Protein--
The binding of metal ions to free enzymes has
been shown to result in a significant decrease in emission fluorescence
intensities (31-33). We observed that the binding of both
Mg2+ and Mn2+ ions to the NS5B protein resulted
in a modification of the intensity of the intrinsic fluorescence of
this protein. As a consequence we were able to evaluate
Kd values for both Mg2+ and
Mn2+ ions, the cofactors necessary for the NS5B-mediated
polymerase activity, by titrating the binding of increasing amounts of
metal ion to a fixed concentration of the NS5B protein. Typical
emission spectra obtained from the titration of MgCl2 are
shown in Fig. 2A. The addition
of increasing amounts of Mg2+ produced a decrease in the
fluorescence intensity, but the emission maximum (335 nm) and spectral
bandwidth were unaffected. The corresponding saturation isotherm
generated by plotting the change in fluorescence intensity at 335 nm as
a function of added MgCl2 is shown in Fig. 2B.
Quenching saturated at millimolar Mg2+ concentrations, and
a 3.1 mM Kd value could be estimated for
Mg2+ from a fit of Equation 3 to the generated saturation
isotherm. About 50% of the intrinsic protein fluorescence was
accessible to the quencher Mg2+ ion (Fig. 2B).
Analysis of a Hill plot generated from the Mg2+ ion binding
data yielded a Hill coefficient of 1.04, indicating a lack of
cooperativity (Table I). Furthermore, a
Scatchard plot of Mg2+ binding data is linear, providing no
evidence for multiple classes of independent magnesium ion binding
sites or of cooperative binding sites (data not shown). Note that
binding of the Mg2+ ions could not be detected after
heating the enzyme at 60 °C in the presence of 0.1% SDS prior to
the titration (data not shown). The addition of EDTA to the reaction
reversed the effects on fluorescence and showed that the change in
fluorescence observed by the addition of the metal ion is not solely
due to a change in the ionic strength of the solution (data not shown).
Accordingly, electrostatic interactions appear to make only minor
contributions to the overall binding energy, as illustrated by the
minimal effect of ionic strength on the apparent Kd
value of NS5B for Mg2+ ions (Fig. 2C).

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Fig. 2.
Titration of NS5B with Mg2+
ions. A, increasing amounts of MgCl2 were
added to a 250 nM solution of the enzyme in binding buffer
(50 mM Tris-HCl, 50 mM KOAc, pH 7.5), and the
emission spectrum was scanned from 310 to 440 nm. B, a
saturation isotherm can be generated from these data by plotting the
change in fluorescence intensity at 335 nm as a function of added
MgCl2. C, the effect of increasing ionic
strength on the apparent dissociation constant of NS5B for
MgCl2 was investigated. Increasing concentrations of KCl
were added to the reactions to generate the desired ionic strength.
D, kinetic analysis of real time binding of
MgCl2 to the NS5B protein. A 300 µM solution
of the enzyme was incubated with 150 mM MgCl2.
Emission was monitored for 30 s at 335 nm, and excitation was
performed at 290 nm.
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Table I
Dissociation constants (Kd), maximal decrease of fluorescence
( F/Fo/max), Hill coefficients (n), and
association rates for the interaction of NS5B with various metal ions
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The kinetics of real time Mg2+ ion binding to the NS5B
protein were investigated by monitoring the intrinsic protein
fluorescence of NS5B following the addition of Mg2+ ions
(Fig. 2D). The progress of the binding reaction was followed and showed that there was a rapid exponential decrease in fluorescence following the addition of the Mg2+ ions. An apparent
association rate of 0.2 µM
s
1
was estimated from the data. Half-maximal quenching was observed at
~2 s, whereas maximal quenching was achieved after 5 s of
incubation with Mg2+ ions and remained constant thereafter.
The exponential decrease in fluorescence observed following the
addition of metal ions was not due to photobleaching, because similar
results were obtained when the NS5B protein was incubated away from the
light source.
The binding of Mn2+ ions to the NS5B protein was
investigated in an analogous manner by monitoring the decrease in the
intrinsic protein fluorescence following binding to the ion. Again
quenching saturated at millimolar Mn2+ concentrations, and
a 0.3 mM Kd value was estimated from a
fit of Equation 3 to the generated saturation isotherm (Table I). About
25% of the intrinsic protein fluorescence was accessible to the
quencher Mn2+ ion. A Hill plot was generated from the
Mg2+ ion binding data and yielded a Hill coefficient of
0.98 (Table I). Finally, the addition of Mn2+ ions resulted
in a rapid exponential decrease in fluorescence yielding an apparent
association rate of 0.3 µM
1
s
1 (Table I).
The presence of multiple tryptophan residues in the NS5B protein
allowed binding assays to be performed with a high degree of
sensitivity. Interpretation of the quenching data in terms of spatial
relationships is complicated because the tryptophan residues are
distributed rather uniformly throughout the protein. To further
characterize the interaction between the metal ions and NS5B, far- and
near-UV CD spectra were recorded both in the presence and the absence
of metal ions. Analysis of the far-UV CD spectra (Fig.
3A) revealed that the binding
of Mg2+ ions to the NS5B protein does not induce a
significant modification of the secondary structure component of the
protein. The far-UV CD spectra thus suggest that the NS5B protein
maintains a comparable ordered secondary structure following the
binding of the metal ions. Although the far-UV CD data indicate that no
significant changes in secondary structure are occurring, analysis of
the near-UV CD spectra was performed to verify that the decrease of fluorescence intensity observed upon binding of metal ions is indeed
reflecting conformational changes. Analysis of the near-UV CD spectra
of the NS5B protein in both the absence and presence of
Mg2+ ions was performed from 250 to 340 nm. As can be seen
in Fig. 3B, a significant reduction of the amplitude of the
signal is observed over the 280-300-nm region when the protein is
incubated with Mg2+ ions. Overall, the CD spectra suggest
that the protein undergoes a subtle conformational change upon the
binding of metal ions rather than a radical modification of the overall
protein architecture.

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Fig. 3.
Far-UV and near-UV circular dichroism spectra
of the NS5B protein. Far-UV (A) and near-UV
(B) CD spectra were recorded for the NS5B protein in both
the absence (spectrum a) and presence (spectrum
b) of 20 mM MgCl2. In each case the enzyme
concentration was 20 µM, and the spectra were recorded
from 200 to 250 nm. The averages of six wavelength scans are
presented.
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Binding of Other Divalent Cations and Competitive
Binding--
Previous studies showed that other divalent cations, such
as zinc, cobalt, copper, and calcium, do not support the NS5B-mediated RNA polymerase activity (14, 25, 26). However, it has been noted that
these ions can efficiently inhibit both the Mn2+- and the
Mg2+-dependent RNA polymerase activity (14, 25,
26). Inhibition by these ions presents several interesting scenarios:
(i) Do these ions bind to the same active site as Mn2+ and
Mg2+? or (ii) Do they bind at a different site on the
protein, thereby somehow allosterically hindering the polymerase
reaction? To answer these questions, we attempted to evaluate the
binding of other divalent cations to the NS5B protein using
fluorescence spectroscopy. However, interaction of the protein with
Zn2+, Ca2+, and Cu2+ could not be
efficiently and repeatedly evaluated. Significant quenching of the
intrinsic protein fluorescence was only observed in the presence of
Co2+ ions. Analysis of the binding data yielded a
Kd value of 35 mM (Table I).
Competitive alternative ligand binding experiments were carried out to
determine whether the different metal ions compete for a common binding
site. In the first experiment, the combined
F produced at
335 nm by the addition of various manganese concentrations was plotted
against the concentration of Mg2+ ions. Three
concentrations of Mn2+ were used in this competition
experiment. The saturation isotherm resulting from the competitive
ligand experiment conducted with Mg2+ as iona
and Mn2+ as ionb is shown in Fig.
4. The pattern of lines is in accordance with a model in which the binding of Mg2+ and
Mn2+ is mutually exclusive (29). A quantitative analysis of
the compliance of the experiment with the competitive binding model was
made by comparing the Kd and
Fmax values derived from the fit of Equation 5 to the dual titration data with the Kd and
Fmax values determined in single ligand
titrations. In each case, the values were statistically
indistinguishable. The reverse experiment, using Mn2+ as
iona and Mg2+ as ionb, yielded
similar conclusions (data not shown). Similar competitive experiments
also demonstrated that Co2+ ions are competing for the same
site as Mn2+ and Mg2+ ions (data not
shown).

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Fig. 4.
Dual ligand titration using Mg2+
as iona and Mn2+ as ionb.
Standard titration assays were performed using Mg2+ ions in
the presence of increasing amounts of Mn2+ ions. The
concentrations of Mn2+ ions used in these experiments were
0 mM ( ), 0.05 mM ( ), 0.2 mM
( ), and 20 mM ( ).
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Importance of the Metal Ions in the Nucleic Acid Binding Activity
of NS5B--
Magnesium and manganese ions have previously been shown
to be essential for the NS5B-mediated polymerase activity (8, 14, 22-26). We were thus interested in determining whether the ion binding
activity is necessary for the binding of NS5B to the HCV RNA template,
an essential step in the initiation of polymerization. An
electrophoretic mobility shift assay was thus performed to evaluate the
binding to RNA in both the presence and the absence of metal ions.
The binding of the HCV NS5B protein to a single-stranded RNA transcript
containing the NS5B gene flanked by the 5'- and 3'-terminal sequences
found in the HCV genome was initially evaluated in the presence of 10 mM MgCl2 (Fig.
5A). The amount of the shifted
species reached a maximum around 15 nM. Higher
concentrations of the protein did not further enhance the amount of
shifted RNA, suggesting that the reaction had come to an equilibrium.
An apparent Kd of 10 nM could be
estimated for this RNA substrate (Fig. 5B). Note that the
binding to an HCV-unrelated single-stranded RNA sequence was also
evaluated and yielded a similar apparent Kd value (8 nM), indicating that NS5B does not interact with RNA substrates in a sequence-specific manner (data not shown).

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Fig. 5.
Affinity of the NS5B protein for RNA.
A, electrophoretic mobility shift assay using HCV-specific
RNA as substrate. The samples were run on a 1.5% agarose gel, and the
amounts of protein used in the assay are indicated at the
top of the gel. B, evaluation of the dissociation
constant for RNA. The percentages of free probes are presented
graphically to be able to estimate the dissociation constant.
C and D, the effects of magnesium (C)
and manganese (D) on the binding NS5B to RNA were analyzed.
E, real time kinetic analysis of the binding of RNA to NS5B.
A 250 nM solution of the enzyme was incubated with 750 nM of RNA in 50 mM Tris-HCl, 10 mM
NaCl, pH 7.5. Emission was monitored for 30 min at 335 nm, and
excitation was performed at 290 nm. F, determination of the
apparent binding site size (napp) for the
interaction between RNA and NS5B. The effect of increasing molar ratios
of RNA to NS5B protein was investigated by fluorescence spectroscopy.
Excitation was performed at 290 nm, and emission was monitored at 335 nm. Linear approximations were fitted to the initial and final slopes
of the curves, and the intersection of the two lines corresponds to the
apparent binding site size.
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The divalent ion requirement for the binding of NS5B protein to nucleic
acids was then investigated using 5 nM of NS5B protein, an
amount near the Kd value and thus in the linear
range of the binding reaction. The results showed that RNA binding can occur in the absence of ions and that binding is only modestly stimulated by lower concentrations of magnesium (Fig. 5C).
The data indicated that the binding activity reached a maximum at ~5
mM of MgCl2 and that a decrease was noted at
higher concentrations. On the other hand, Mn2+ ions were
able to support the binding activity over a wide range of
concentrations ranging from 0 to 25 mM (Fig.
5D). Overall, these binding studies indicated that NS5B can
bind to RNA substrates in the absence of Mg2+ or
Mn2+ ions and that the presence of these ions only modestly
affects the binding reaction.
Preliminary studies showed that the binding of RNA to the NS5B protein
results in a significant decrease in emission fluorescence intensities
(data not shown). The kinetics of RNA binding to the NS5B protein were
thus investigated in both the presence and the absence of
Mg2+ or Mn2+ ions by monitoring the intrinsic
protein fluorescence of NS5B following the addition of RNA. The
progress of the binding reaction was analyzed and showed that there was
an exponential decrease in fluorescence upon the addition of RNA,
followed by a slower linear decrease (Fig. 5E). Half-maximal
quenching was observed at ~2 min, whereas maximal quenching was
achieved after 6 min of incubation with RNA and remained constant
thereafter (t1/2 = 2 min). The presence of either
Mg2+ or Mn2+ ions did not significantly
influence the rate of binding to the RNA substrate (data not shown).
Again, the exponential decrease in fluorescence observed following the
addition of RNA was not due to photobleaching because similar results
were obtained when the NS5B protein was incubated away from the light source.
To evaluate the stoichiometry of NS5B binding to nucleic acids, the
extent of binding was evaluated by monitoring the intrinsic fluorescence of NS5B upon interaction with RNA. The binding site size
(napp) was determined from fluorescence
titration curves in which increasing amounts of RNA were added to a
fixed amount of protein (Fig. 5F). A plot of the absolute
fluorescence change, normalized by the initial fluorescence in the
absence of any RNA, versus the ratio of total RNA
(nucleotides) to total protein was generated. Linear approximations
were fitted to the initial and final slopes of the curves, and the
intersection of the two lines corresponds to the apparent binding site
size (34). The apparent binding site size of NS5B on the RNA substrate
was estimated to be 8 nucleotides. The addition of various
concentrations of metal ions did not significantly alter this value
(data not shown). Taken together, the results indicate that metal ions
do not influence the RNA binding activity of the NS5B protein.
Importance of the Metal Ions in the Nucleotide Binding Activity of
NS5B--
The effect of magnesium and manganese ions on the binding of
free nucleotides was also investigated by fluorescence spectroscopy. Initial experiments, performed in the absence of added metal ions, indicated that binding of NTP ligands to the NS5B protein results in a
significant decrease in the fluorescence emission intensity. A typical
titration experiment, using GTP as the ligand, is shown in Fig.
6. An apparent Kd
value of 65 µM could be estimated from the generated
saturation isotherm. To determine the role of metal ions in NTP
binding, the enzyme was titrated with GTP in the presence of increasing
concentrations of either MgCl2 or MnCl2. The
addition of increasing concentrations of either ion, up to 10 mM, did not significantly modify the apparent
Kd value for GTP (data not shown). Similar
conclusions were drawn when titration experiments were performed with
other NTPs (data not shown). These results clearly indicate that both
Mg2+ and Mn2+ do not influence the RNA binding
activity of the NS5B protein.

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Fig. 6.
Titration of NS5B with GTP. Increasing
amounts of GTP were added to a 250 nM solution of the
enzyme in binding buffer (50 mM Tris-HCl, 50 mM
KOAc, pH 7.5), and the emission spectrum was scanned from 310 to 440 nm. A double-reciprocal plot of the saturation isotherm, generated from
these data by plotting the change in fluorescence intensity at 335 nm
as a function of added GTP, is shown in the right-hand
corner.
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 |
DISCUSSION |
The enzymatic activity of the HCV NS5B protein has been
extensively characterized (8, 14-26). The protein is believed to be
responsible for the genome replication of HCV and is thus a critical
protein of the virus. Magnesium and manganese ions have been shown to
support the polymerase activity mediated by NS5B (8, 14, 22-26), and
manganese has recently been found in the crystal structure of this
protein (28). However, the precise role of these ions in the reaction
chemistry has not been determined. As a first step toward elucidating
the nature of the metal ion binding to the NS5B protein, we have
utilized the endogenous tryptophan fluorescence to precisely
quantitate the interactions of metal ions with the enzyme.
Quenching of the fluorescence signals by titration of the protein with
metal ions provides a straightforward technique for determining
apparent Kd values. The high intrinsic fluorescence signal of the NS5B protein allowed binding assays to be carried out
with a high degree of sensitivity. The decrease in fluorescence intensity observed upon saturation of the enzyme with these ions can be
produced by contact of the quenching agent with the indole side chain
of a tryptophan and/or by inducing a conformational change in the
enzyme that results in alterations in the microenvironments of
tryptophan residues distal to the ion binding site. The fact that no
tryptophan residues appear to be located in the active site of the NS5B
crystals suggests that the decrease in fluorescence intensity is not
solely the result of the selective quenching of a specific
subpopulation of indole fluorophores but that it also involves a subtle
conformational change. Similar findings have been noted with other
ion-binding proteins (35). Although crystallographic studies have not
yet identified different conformations of NS5B (28, 36, 37), analysis
of the near-UV CD data clearly suggests that a conformational change is
induced upon the binding of metal ions to the NS5B protein. In fact,
comparisons with closely related polymerases strongly suggest that
conformational changes are required for NS5B to efficiently initiate
polymerization (28). However, analysis of the crystal structure the RNA
polymerase of the rabbit hemorrhagic disease virus, a virus closely
related to HCV, revealed the presence of both active and inactive
conformations within the same crystal form (38). The conformations
adopted by the rabbit hemorrhagic disease virus polymerase can vary
dramatically depending on the ions present in the crystallization
solution. It was suggested that these structural changes may be
important for the enzymatic activity of the protein (38). As reported previously for DNA polymerases (39, 40), these conformational changes
are probably required for the catalytic activity of rabbit hemorrhagic
disease virus and HCV polymerases.
Analysis of the crystal structure of NS5B revealed that the protein is
folded into characteristic fingers, palm, and thumb subdomains (36,
37). The particular fold adopted by the palm subdomain is shared by
many proteins that bind nucleotides and/or nucleic acids (41). The
crystal structure of DNA polymerase I from E. coli complexed
with nucleotides showed that it contains two absolutely conserved
aspartic acid residues that coordinate two Mg2+ ions in the
active site of the protein (41). These two metal ions are in contact
with both the phosphate of the nucleotide and several acidic amino
acids residues (41). Two metal ions are also bound in the active site
of the rabbit hemorrhagic disease virus (38) and HCV polymerases (28).
A catalytic mechanism has been proposed in which one metal ion is
involved both in positioning the substrate and in the activation of an
incoming nucleophile (42). Nucleophilic attack then generates a
trigonal bipyramidal transition state that is stabilized by both metal
ions. The second metal ion also stabilizes the negative charge that
appears on the leaving 3' oxygen, thus facilitating its departure from
the phosphate. Analysis of the DNA polymerase I crystal structure indicated that the two metal ions are about 4.0 Å apart in the active
site of the protein (41). Metal ion binding thus seems to be limited to
the active site region and does not involve other subdomains of the
protein. Our titration and competition experiments performed with the
NS5B protein suggest a similar mechanism, as illustrated by the data
indicating that the metal ions bind to a single site on NS5B.
Based on the results of our fluorescence and near-UV CD experiments, we
demonstrated that NS5B undergoes conformational changes upon the
binding of metal ions. This process, however, does not significantly
stimulate NS5B binding to its RNA or NTP substrates. Far-UV circular
dichroism measurements also revealed that the binding of metal ions
does not significantly modify the secondary structure of the protein.
This is in agreement with various studies that showed that NS5B has a
preformed active site (36, 37, 43) in contrast to many other
polymerases, in which major domain rearrangements are needed to form a
catalytically active site (40, 44). 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 intermediate transition state. A number of
acidic amino acid residues located in the active site of the NS5B
protein have the potential to coordinate metal ions through a network
of hydrogen bonds. Following the binding of metal ions, the original
hydrogen bonding interactions would be replaced by interactions with
metal ions and possibly with water molecules, positioning the residues for efficient enzymatic catalysis. Further analysis by site-directed mutagenesis could precisely identify the role of these residues and
provide additional information on the enzyme mechanism.
Fluorescence spectroscopy has tremendous potential for the screening of
antiviral drugs aimed at inhibiting the NS5B-mediated RNA polymerase
activity. The availability and simplicity of data acquisition and
analysis are important practical features behind this possibility. Many
aspects, including characterization of the binding of various ligands,
analysis of the thermodynamics of the interactions, and competition
experiments can easily be performed by fluorescence spectroscopy
analysis. Although the 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. Replication of the HCV genome is a complex event that
probably requires not only the polymerase but also additional viral and cellular factors to form a functional replicative complex. Structural and enzymatic studies are beginning to reveal the essential features of
the polymerase reaction and should ultimately lead to the design of
effective antiviral drugs.