Advertisement
JBC

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Originally published In Press as doi:10.1074/jbc.M308917200 on October 16, 2003

J. Biol. Chem., Vol. 278, Issue 52, 52471-52478, December 26, 2003
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
278/52/52471    most recent
M308917200v1
Right arrow Submit a Letter to Editor
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowRequest Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Bougie, I.
Right arrow Articles by Bisaillon, M.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Bougie, I.
Right arrow Articles by Bisaillon, M.
Social Bookmarking
Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati  
What's this?

Initial Binding of the Broad Spectrum Antiviral Nucleoside Ribavirin to the Hepatitis C Virus RNA Polymerase*

Isabelle Bougie and Martin Bisaillon{ddagger}

From the Département de Biochimie, Faculté de Médecine, Université de Sherbrooke, Sherbrooke, Québec J1H 5N4, Canada

Received for publication, August 12, 2003 , and in revised form, October 16, 2003.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Ribavirin is a broad spectrum antiviral nucleoside that displays activity against a variety of RNA and DNA viruses. Ribavirin is currently used in combination with interferon-{alpha} for the treatment of hepatitis C virus (HCV) infection and was recently shown to be directly incorporated by the HCV RNA polymerase into RNA products. This capacity ultimately leads to increased mutation rates and drastically reduces the viral fitness. As a first step toward elucidating the nature of the specific interaction between ribavirin and the HCV polymerase, we have utilized fluorescence spectroscopy to monitor precisely the binding of ribavirin triphosphate (RTP) to the viral polymerase. This spectroscopic approach allowed us to clearly separate the RTP binding activity from the concomitant catalytic steps. We report here the first detailed study of the binding kinetics and thermodynamic parameters involved in the interaction between RTP and an RNA polymerase. We demonstrate that RTP binds to the same active site as nucleotides. Furthermore, we provide evidence that the HCV polymerase cannot only bind to RTP but also to nonphosphorylated ribavirin, albeit with less affinity. By using various combinations of template-primers, we also demonstrate that base pairing is not involved in the initial binding of RTP to the HCV polymerase. Based on the results of circular dichroism and denaturation studies, we show that the RNA polymerase undergoes subtle conformational changes upon the binding of RTP, although the interaction does not significantly modify the stability of the protein. Finally, although metal ions are required for catalytic activity, they are not required for the initial binding of RTP to the polymerase. Such quantitative analyses are of primary importance for the rational design of new ribavirin analogues of potential therapeutic value and provide crucial insights on the interaction between RTP and the HCV RNA polymerase.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Ribavirin is a broad spectrum antiviral nucleoside that displays activity against a variety of RNA and DNA viruses (1, 2). Once inside the cells, ribavirin is phosphorylated by cellular kinases, with ribavirin triphosphate as the major intracellular metabolite (3, 4). A number of possible mechanisms have been proposed over the years to account for the antiviral activity of ribavirin. Ribavirin has been shown to inhibit the host cell inosine monophosphate dehydrogenase, an enzyme involved in the de novo synthesis of GTP (5, 6). Because GTP is required for the transcription of viral genomes, and replication of RNA viruses, it has been assumed that the decrease in cytosolic concentration of GTP could affect the multiplication of viruses. Ribavirin has also been shown to modulate the host immune system by engendering a bias toward helper T-cells type 1 cytokine response (7, 8). This would ultimately lead to an enhanced immune response against viral infections.

Ribavirin has also been shown to have an inhibitory effect on viral polymerases (9, 10). It has been suggested that ribavirin triphosphate could bind to the nucleotide-binding site of polymerases, thereby competitively inhibiting the binding of the nucleotides (11). More recently, elegant studies demonstrated that ribavirin can actually be used by viral polymerases and incorporated into viral RNA, with the potential to base pair with UMP and CMP, leading to ribavirin-mediated mutagenesis of viral genomes (12–14). This would ultimately drive the viruses beyond a critical mutation rate and lead to an overall reduced fitness of the viral populations.

Ribavirin is currently used in combination with pegylated interferon-{alpha} for the treatment of hepatitis C virus (HCV)1 infection (15, 16). More than 170 million people worldwide are currently infected with HCV (17). Infection with HCV results in persistent infection of the liver, which can eventually lead to the development of cirrhosis and cancer. Recent estimates revealed that at least four million Americans are carriers of the virus, and about 80% of the patients will eventually progress to chronic hepatitis (18–21). Treatment of the HCV infection appears problematic, although significant progress has been made with the use of the ribavirin-interferon-{alpha} combination, which yields a sustained virologic response in more than 40% of patients (22, 23). Ribavirin was shown recently to be directly incorporated by the HCV RNA polymerase into RNA products (13). Incorporation of RTP into RNA transcripts is templated by both cytidine and uridine. This promiscuous base pairing capacity ultimately leads to increased mutation rates and drastically reduces the viral fitness (13, 24). These studies suggest that ribavirin actually binds to the HCV polymerase and can be recognized as a result of substrate mimicry.

As a first step toward elucidating the nature of the specific interaction between ribavirin and the HCV polymerase, we have utilized fluorescence spectroscopy to monitor precisely the binding of RTP to the viral polymerase. This spectroscopic approach allowed us to clearly separate the RTP binding activity from the concomitant catalytic steps. We report here the first detailed study of the binding kinetics and thermodynamic parameters involved in the interaction between RTP and an RNA polymerase. We demonstrate that RTP binds to the same active site as nucleotides. We also provide evidence that the HCV polymerase cannot only bind to RTP but also to nonphosphorylated ribavirin, albeit with less affinity. By using various combinations of template-primers, we also demonstrate that base pairing is not involved in the initial binding of RTP to the HCV polymerase. Based on the results of circular dichroism and denaturation studies, we show that the RNA polymerase undergoes subtle conformational changes upon the binding of RTP, although the interaction does not significantly modify the stability of the protein. Finally, although metal ions are required for catalytic activity, they are not required for the initial binding of RTP to the polymerase. Such quantitative analyses are of primary importance for the rational design of new ribavirin analogues of potential therapeutic value, and provide crucial insights on the interaction between RTP and the HCV RNA polymerase.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
HCV NS5B Expression and Purification—The expression and purification of a truncated form of HCV NS5B protein (NS5B{Delta}21) lacking the last 21 amino acids of the protein were performed as described previously (35).

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,

(Eq. 1)
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,

(Eq. 2)
where {Delta}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, {Delta}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,

(Eq. 3)

The Kd values were determined from a nonlinear least square regression analysis of titration data by using Equation 3.

Thermodynamics of Binding—The temperature dependence of the association constants (Ka) for ligand binding was analyzed according to the van't Hoff isobaric equation, assuming that the entropy change ({Delta}S0) and the enthalpy change ({Delta}H0) remained constant over the whole range of temperatures (Equation 4).

(Eq. 4)

Nucleotide Binding Assay—Nucleotide binding was assessed by analyzing the interaction of [3H]RTP with the NS5B protein. Standard binding reactions containing 10 mM Tris-HCl, pH 7.5, 10 mM NaCl, 1 mM [3H]RTP (Moravek Biochemicals) and 50 nM of enzyme were incubated for 10 min at 25 °C. Protein-nucleotide complexes were precipitated by addition of trichloroacetic acid to a 10% final concentration and 5 µg of bovine serum albumin and washed extensively with 10% trichloroacetic acid. Some experiments were also performed in the presence of the indicated amounts of competitor nucleotides. The radioactivity was quantitated by liquid scintillation counting. The apparent dissociation constant (Kd) for each probe was determined according to Equation 5,

(Eq. 5)
where fD represents the fraction of bound nucleotides, [NS5B] the total protein concentration, and Kd the dissociation constant for the binding reaction.

Circular Dichroism Spectroscopy Measurements—Circular dichroism measurements were performed with a Jasco J-810 spectropolarimeter. The samples were analyzed in quartz cells with path lengths 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 dichroic spectra were corrected by subtraction of the background for the spectrum obtained with either buffer alone or buffer containing RTP. The average of 6 wavelength scans is presented. The ellipticity results were expressed as mean residue ellipticity, [{theta}], in degrees·cm2·dmol-1.

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 30 °C. The parameters (free energy of unfolding in the absence of denaturant), m (cooperativity of unfolding), and Cm (midpoint concentration of denaturant required to unfold half of the protein) were obtained as previously outlined by using Equation 6,

(Eq. 6)
and Equation 7,

(Eq. 7)


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Expression, Purification, and Intrinsic Fluorescence Properties of the NS5B Protein—In order to characterize the interaction between ribavirin and the HCV RNA polymerase, the NS5B protein was expressed as described previously (35). SDS-PAGE analysis showed that the 65-kDa NS5B protein was the predominant polypeptide in the purified fraction (Fig. 1A). Immunoblotting analysis, using a monospecific antibody, also confirmed the identity of the NS5B protein (data not shown).



View larger version (18K):
[in this window]
[in a new window]
  		FIG. 1.
Expression, purification, and fluorescent properties of NS5B. A, an aliquot (2 µg) of the purified preparation of NS5B was analyzed by electrophoresis through a 12.5% polyacrylamide gel containing 0.1% SDS and visualized by staining 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. Curve 1, purified protein in 50 mM Tris-HCl and 50 mM KOAc, pH 7.5; curve 2, 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. The molar fluorescence of NS5B is shown in the inset. Various concentrations of the purified NS5B protein were assayed in 50 mM Tris-HCl and 50 mM KOAc, pH 7.5. Emission was monitored at 339 nm, and excitation was performed at 290 nm.

 
The fluorescence properties of the purified NS5B protein in standard buffer at 22 °C are shown in Fig. 1B. In order to obtain the maximal emission peak at the low concentrations of protein required to accurately monitor the binding of ribavirin to the NS5B protein, excitation was carried out at 290 nm. Analysis of the background corrected fluorescence emission spectrum of the NS5B protein revealed an emission maximum ({lambda}max = 339 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 (25). 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 {lambda}max toward 350 nm (Fig. 1B).

Analysis of the molar intensity of the fluorescence emission spectrum of NS5B was also determined in order to determine whether significant protein aggregation, or if the loss of protein from solution through adhesion, could influence the data. As can be seen in Fig. 1B, 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. All subsequent binding experiments were therefore performed at a protein concentration of 50 nM, with the assumption that the binding equilibrium was not complicated by the presence of an aggregation equilibrium.

Binding of Ribavirin Triphosphate to the NS5B Protein—The binding of nucleotides to enzymes has been shown to result in a significant decrease in emission fluorescence intensities (26–28). In the present study, we intended to characterize the binding of RTP to the HCV NS5B protein by using fluorescence spectroscopy. We observed that the binding of RTP to the enzyme resulted in a significant modification of the intensity of the intrinsic fluorescence of the protein. As a consequence we were able to evaluate the Kd value for RTP by titrating the binding of increasing amounts of RTP to a fixed concentration of the NS5B protein. Typical emission spectra obtained from the titration of RTP are shown in Fig. 2A. The addition of increasing amounts of RTP produced a decrease in the fluorescence intensity, and the emission maximum shifted from 339 nm for the free protein to 345 nm in the presence of higher concentrations of RTP, indicating movement of the tryptophan residues to a relatively more polar environment. The corresponding saturation isotherm generated by plotting the change in fluorescence intensity as a function of added RTP is shown in Fig. 2B. Quenching saturated at millimolar RTP concentrations and a 800 µM Kd value could be estimated for RTP from a fit of Equation 3 to the generated saturation isotherm (Table I). About 15% of the intrinsic protein fluorescence was accessible to the quencher RTP (Fig. 2B). Analysis of the Scatchard plot for the RTP binding data is linear, providing no evidence for multiple classes of independent RTP-binding sites or of cooperative binding sites (data not shown). Finally, the interaction between RTP and NS5B is dependent on the tertiary structure of the protein as indicated by the fact that binding of RTP could not be detected after heating of the enzyme at 60 °C in the presence of 0.1% SDS prior to performing the titration (data not shown).



View larger version (23K):
[in this window]
[in a new window]
  		FIG. 2.
Binding of RTP to the NS5B protein. A, increasing amounts of RTP were added to a 50 nM solution of the enzyme in binding buffer (50 mM Tris-HCl and 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 339 nm as a function of added RTP. A double-reciprocal plot is shown in the inset. C, kinetic analysis of real time binding of RTP to the NS5B protein. A 50 nM solution of the enzyme was incubated with 20 mM RTP. Emission was monitored for 60 s at 339 nm, and excitation was performed at 290 nm. D, the effect of increasing ionic strength on the association constant of NS5B for RTP was investigated. Increasing concentrations of KCl were added to the reactions to generate the desired ionic strength. A graph of the effect of increasing ionic strength on the apparent dissociation constant of NS5B for RTP is shown in inset.

 


View this table:
[in this window]
[in a new window]
  		TABLE I
Kd, Ka, {Delta}F/Fo, and {Delta}G0 values for the interaction of NS5B with ribavirin and ribavirin triphosphate

 
The kinetics of real time RTP binding to the NS5B protein were investigated by monitoring the intrinsic protein fluorescence of NS5B following the addition of RTP (Fig. 2C). The progress of the binding reaction was followed upon addition of saturating amounts of RTP. The results show that there is a rapid exponential decrease in fluorescence intensity following the addition of RTP. An apparent association rate of 0.24 µM-1 s-1 was estimated from the data. Half-maximal quenching was observed at ~1.5 s. The exponential decrease in fluorescence observed following the addition of RTP was not due to photobleaching, as similar results were obtained when the NS5B protein was incubated away from the light source.

In order to evaluate the contribution of the electrostatic interactions to the NS5B-RTP binding activity, binding assays were performed in the presence of increasing ionic strength. The data revealed that the interaction is strongly attenuated at elevated KCl concentrations (Fig. 2D). Electrostatic interactions appear to be making a significant contribution to the overall binding energy as indicated by the effect of ionic strength on the binding of RTP. Extrapolation of the graphic to an ionic strength of 1 M, the standard state where electrostatic interactions are effectively eliminated, yields a Kd of 22.5 mM. Evaluation of the Gibbs energy due to electrostatic interactions ({Delta}{Delta}GES) reveals that 47% of the binding energy is derived from electrostatic interactions.

The effect of temperature on the binding of RTP to the NS5B protein was also evaluated by fluorescence spectroscopy. The temperature dependence of Ka for RTP provides the thermodynamic parameters of the binding. Analysis of the van't Hoff plot for the interaction between RTP and NS5B reveals that the binding is connected with a high enthalpy of association, {Delta}H0 = -9600 J/mol (Fig. 3). Furthermore, the binding reaction is clearly favorable with the resultant {Delta}S0 = 24 J/mol K.



View larger version (10K):
[in this window]
[in a new window]
  		FIG. 3.
Thermodynamic parameters of the interaction between RTP and NS5B. Binding assays were performed at various temperatures. A van't Hoff plot for the interaction between RTP and NS5B is shown. The effect of temperature on the association constant was evaluated at pH 7.0.

 
RTP and Nucleotides Bind to a Common Active Site—We next sought to investigate whether or not RTP binds to the same site as nucleotides. Initial fluorescence spectroscopy experiments suggested that RTP and nucleotides bind to a common site because binding of RTP could not be detected when the NS5B protein had been saturated with nucleotides (data not shown). Therefore, competitive binding experiments were carried out to determine whether RTP and nucleotides compete for a common binding site. Radiolabeled RTP was used as a probe in the binding assay, and nucleotide binding was analyzed by quantitation of bound substrate following trichloroacetic acid precipitation.

The ability of ATP to competitively inhibit the RTP binding activity was initially evaluated. The Lineweaver-Burk plot of the binding data reveals that ATP is a linear competitive inhibitor of the RTP binding activity (Fig. 4B). Consistent with a competitive inhibition, the addition of ATP increased the apparent Kd value of the NS5B protein for RTP (Fig. 4B), whereas no significant changes were made to the maximal binding value. The apparent Kd for RTP increased from 0.8 to 16.7 mM in the presence of 100 µM ATP (20-fold increase). The Ki value for this inhibition was calculated from a Dixon plot to be 65 µM, which is comparable with the Kd value of the protein for ATP (69 µM), as predicted if both RTP and ATP are competing for the same active site (Fig. 4, C and D, respectively). Similar conclusions were reached when other nucleotides (CTP, GTP, and UTP) were used in the competition experiments (data not shown). Taken together these data indicate that RTP and nucleotides bind to a common active.



View larger version (26K):
[in this window]
[in a new window]
  		FIG. 4.
Competitive inhibition of RTP binding by nucleotides. A, competitive inhibition of RTP binding by ATP. Reaction mixtures (50 µl) containing either 0.25, 0.5, 1, and 2 mM [3H]RTP and varying concentrations of ATP, as indicated, were incubated for 10 min at 25 °C. Protein-nucleotide complexes were precipitated by addition of trichloroacetic acid. The radioactivity was quantitated by liquid scintillation counting. B, the effect of increasing ATP concentrations on the apparent dissociation constant of NS5B for RTP was investigated. C, both the mechanism of inhibition and the inhibition constant (Ki) were determined from a Dixon plot of the reciprocal of the RTP-binding reaction versus the concentration of competitor ATP at each fixed concentration of the RTP substrate: 0.5, 1, 2, and 3 mM. D, increasing amounts of ATP were added to a 50 nM solution of the enzyme in binding buffer (50 mM Tris-HCl and 50 mM KOAc, pH 7.5), and the emission spectrum was scanned from 310 to 440 nm. A saturation isotherm can be generated from these data by plotting the change in fluorescence intensity at 339 nm as a function of added ATP. A double-reciprocal plot is shown in the inset.

 
Circular Dichroism Analysis—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 characterize further the interaction between RTP and NS5B, far- and near-UV CD spectra were recorded both in the absence and the presence of RTP. Analysis of the far-UV CD spectra (Fig. 5A) revealed that the binding of RTP 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 RTP. 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 in order to verify that the decrease of fluorescence intensity observed upon binding of RTP is truly reflecting conformational changes. Analysis of the near-UV CD spectra of the NS5B protein in both the absence and presence of RTP was performed from 250 to 340 nm. As can be seen in Fig. 5B, a significant reduction of the amplitude of the signal is observed over the 280–300 nm region when the protein is incubated with RTP. Overall, the CD spectra suggest that the protein undergoes a subtle conformational change upon the binding of RTP, rather than a radical modification of the overall protein architecture.



View larger version (11K):
[in this window]
[in a new window]
  		FIG. 5.
Structural consequences of RTP binding to the NS5B protein. A, far-UV CD spectra were recorded for the free NS5B protein (curve 1) and the protein bound to RTP (curve 2). Formation of the enzyme-RTP complex was induced by incubating the NS5B protein (50 nM) with 5 mM RTP. The spectra were recorded from 200 to 250 nm, and the average of 6 wavelength scans is presented. B, near-UV CD spectra were recorded for the free NS5B protein (curve 1) and the protein bound to RTP (curve 2). The spectra were recorded from 250 to 340 nm, and the average of 6 wavelength scans is presented.

 
Thermodynamic Studies—The previous CD data indicated that the binding of RTP to the NS5B protein results in a subtle conformational change of the protein. We therefore examined the stability of the free protein and the protein bound to RTP. Stability was assessed by GdmHCl denaturation assays performed at 30 °C. The change of the microenvironment of the tryptophan residues as a function of the GdmHCl concentration was investigated by determining the emission maxima and by integration of the fluorescence intensity. Both approaches yielded similar thermodynamic parameters for the free energy of unfolding (), cooperativity of unfolding (m), and midpoint concentration of denaturant required to unfold half of the protein (Cm). Upon an increase of the GdmHCl concentration, the emission maximum of the free NS5B protein shifted to 350 nm (data not shown), reflecting the transfer of tryptophan residues to a more polar environment. The free protein structure reacts 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. 6). No changes could be visualized at GdmHCl concentrations higher than 3.5 M. The change of the integrated fluorescence intensity as a function of GdmHCl concentration for the NS5B protein incubated with RTP is shown in Fig. 6. The sample displays a smooth transition curve, indicative of a cooperative unfolding event. Analysis of the thermodynamic parameters revealed statistically similar values regardless of whether the enzyme is free or bound to RTP (Table II). These results indicate that the binding of RTP to the NS5B protein does not significantly stabilize the protein structure.



View larger version (14K):
[in this window]
[in a new window]
  		FIG. 6.
GdmHCl-induced unfolding equilibrium of NS5B. Transition curves for GdmHCl-induced unfolding of the free NS5B protein ({circ}) and the protein bound to RTP ({square}) were determined. Formation of the enzyme-RTP complex was induced by incubating the NS5B protein (50 nM) with 5 mM RTP. Equilibrium unfolding transitions were monitored by integration of the fluorescence intensity.

 


View this table:
[in this window]
[in a new window]
  		TABLE II
Thermodynamic unfolding parameters measured by equilibrium guanidium chloride denaturation The parameters (free energy of unfolding in the absence of denaturant), m (cooperativity of unfolding), and Cm (midpoint concentration of denaturant required to unfold half of the protein) were determined by guanidium chloride denaturation and from the integration of the fluorescence intensity. The differences in Cm and values in comparison to the free NS5B protein are also shown ({Delta}Cm and , respectively).

 
Contributions of the Phosphates to the Binding Energy— Traditional polymerization assays rely on the transfer of a nucleoside monophosphate to a growing chain of nucleotides. Such assays are frequently used to evaluate the affinity of an enzyme for nucleotides, although they are actually measuring a combination of different steps, i.e. initial binding of the nucleoside triphosphate, hydrolysis to a nucleoside monophosphate, and subsequent transfer to the RNA nascent chain. These assays do not allow the detection of the nucleotide binding activity per se, rather only that of the transfer of the nucleoside monophosphate to a growing chain of nucleotides. The use of fluorescence spectroscopy can allow the identification of the initial nucleotide binding step, thereby separating the binding activity from catalysis. By using fluorescence spectroscopy, we characterized the specific requirements for the binding of RTP to the NS5B protein. The contribution of the triphosphate moiety to the binding activity can be efficiently investigated using this technique. The informative finding is that the protein can actually bind to both ribavirin and to RTP (Fig. 7 and Table I). However, analysis of the thermodynamic parameters of binding reveals the importance of the triphosphate group for the binding reaction. The effect of increasing the number of phosphates, from zero to three, on the total free energy of binding was monitored and revealed that the addition of the triphosphate group had a significant effect on the binding activity. The data revealed that addition of the triphosphate group resulted in a free energy of stabilization of 2805 J/mol. Accordingly, the Ka value for ribavirin increased from 403 to 1250 M-1 in the presence of the triphosphate moiety.



View larger version (13K):
[in this window]
[in a new window]
  		FIG. 7.
Binding of nonphosphorylated ribavirin to the NS5B protein. A, increasing amounts of ribavirin were added to a 50 nM solution of the enzyme in binding buffer (50 mM Tris-HCl and 50 mM KOAc, pH 7.5), and the emission spectrum was scanned from 310 to 440 nm. B, saturation isotherm can be generated from these data by plotting the change in fluorescence intensity at 339 nm as a function of added ribavirin. A double-reciprocal plot is shown in the inset.

 
Binding of RTP in the Presence of a Template-Primer—The effect of the presence of a template-primer on the binding of RTP was analyzed by addition of RTP to the NS5B protein that had been saturated previously with a template-primer substrate. Three combinations of template-primers were used in separate assays. The Kd, Ka, and free energy of binding ({Delta}G0) values were determined for RTP in the presence of NS5B that had been incubated previously with either poly(rA)500· poly(dT)12–18, poly(rC)500·poly(dG)12–18, or an HCV-specific 3' end template (Table III). Overall, the data indicate that the binding of RTP to the NS5B protein is not influenced by the presence of a template-primer, as similar binding parameters are observed in the presence or absence of various templateprimer combinations. Although the presence of template-primers resulted in a significant decrease in the fluorescence intensity, sufficient signal remained to evaluate accurately the binding parameters. Template-primers were used at a concentration of 100 nM in these assays, well above the Kd value of NS5B for RNA as established previously. Saturation of the enzyme with each primer was also monitored by fluorescence spectroscopy in separate binding assays (data not shown). Note that the HCV-specific 3' end template used in this assay terminates with a 3'-C and can serve as a template to generate products that are full-length complements and contain GTP as the initiating nucleotide (data not shown).


View this table:
[in this window]
[in a new window]
  		TABLE III
Kd, Ka, {Delta}F/Fo(max), and {Delta}G0 values for the interaction of NS5B with ribavirin triphosphate in the presence of saturating amounts of various template-primer combinations

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
Several attempts have been made during the past 3 decades to determine the mechanism of action of the antiviral nucleoside ribavirin. Numerous efforts have been made to determine whether RTP can actually serve as a substrate for viral RNA polymerases. Recent studies (12, 13) performed with the picornavirus and hepatitis C virus RNA polymerases clearly demonstrated that RTP can be incorporated by the viral polymerases into nascent chains of RNA. In an effort to characterize the initial binding of RTP to the HCV RNA polymerase (NS5B), fluorescence spectroscopy was utilized to monitor the interaction between the enzyme and RTP. This approach allowed us to separate the binding activity from the hydrolysis of the nucleotide to nucleoside monophosphate and the concomitant transfer to the nascent RNA transcript.

The affinity of the enzyme for RTP is sufficiently high to allow for detailed spectroscopic analyses. Quenching of the fluorescence signals by titration of the protein with RTP provides a straightforward technique for evaluating the kinetic and thermodynamic parameters of the interaction between RTP and NS5B. The high intrinsic fluorescence signal of the HCV RNA polymerase 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 RTP 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 RTP-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. This is also reflected by the change in the fluorescence emission maximum that is detected upon addition of RTP. A conformational change was also detected in our circular dichroism study performed in the presence of RTP. Although crystallographic studies have not yet identified different conformations of NS5B (29–31), our data suggest that a small conformational change is induced upon the binding of RTP to the NS5B protein. In fact, comparisons with closely related polymerases strongly suggest that conformational changes are required in order for NS5B to initiate polymerization efficiently (29). 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 (32). It was suggested that these structural changes may be important for the enzymatic activity of the protein (32). As reported previously (33, 34) for DNA polymerases, these conformational changes are probably required for the catalytic activity of rabbit hemorrhagic disease virus and HCV polymerases. In accordance with this hypothesis, we and others (35, 36) have recently shown that binding of metal ions to the NS5B protein is accompanied by a subtle conformational change.

The ability of ribavirin to be used by the NS5B protein and incorporated into viral RNA raised the question of whether RTP binds to the same active site as nucleotides. Based on competitive binding analysis, it clearly appears that both RTP and nucleotides bind to a common active site, suggesting that similar catalytic residues are used to direct the incorporation of RTP into nascent RNA molecules. However, recent crystallographic studies (29) identified the presence of an additional specific GTP-binding site, 30 Å away from the NS5B catalytic site. This site has been proposed to be an allosteric regulatory region that would be required to initiate transcription efficiently. Since the structure of RTP is related to GTP, we investigated the possibility that RTP might bind to the newly identified allosteric site or to any other region of the NS5B protein. The binding assays were performed over a wide range of RTP concentrations to examine the possibility of a second binding site. However, all of the binding studies performed in this work were adequately analyzed by using single exponential rate equations. In no case was there any evidence for another significant kinetic barrier during binding that would require the use of a second exponential equation to satisfactorily analyze the data. The binding of RTP to a region located outside of the active site could not be detected in our assays.

What is the biological relevance of the present findings? In vitro studies performed with the RNA polymerase of poliovirus showed that RTP incorporation in nascent RNA molecules is very low, i.e. similar to the rate of incorporation of an incorrect nucleotide (3000–6000-fold slower than for a correct nucleotide) (12). In accordance with these data, our binding assays indicate that the HCV polymerase has a much lower affinity for RTP in comparison to nucleotides. Furthermore, we demonstrated that RTP and nucleotides are binding to the same active site. Consequently, the active site of the enzyme is more likely to be occupied by nucleotides in the cytoplasm of an infected cell. However, it should be noted that the ability of ribavirin to inhibit inosine monophosphate dehydrogenase, which leads to a decrease in cellular GTP pools (5, 6), could increase the frequency of RTP incorporation. Finally, crystal structural analysis of ribavirin suggested that it adopts a conformation similar to that of guanosine, with the carbonyl oxygen and amino nitrogen of the 3-carboxamide group arranged spatially similarly to the O-6 and N-1 atoms of guanosine (37). A simple explanation for the weaker affinity of the HCV polymerase for RTP versus GTP is that the latter has an extra amino group that enables additional electrostatic or hydrogen-bonding contacts with the enzyme that is not available in RTP.

Our data demonstrate that RTP binds to the NS5B protein in the absence of RNA template. This is in agreement with a previous study demonstrating that binding of the RNA template is not required to promote the binding of nucleotides (29). In contrast to most other polymerases, in which large rearrangements are needed to contact the nucleotides, the HCV polymerase possesses a preformed active site (30, 31, 38). Numerous contacts have been detected from crystallographic studies between the enzyme and nucleotides (29). These contacts involved interactions with both the 2'-OH of the ribose (Asp-225) and the triphosphate moiety (Arg-158, Arg-394, Ser-367, Arg-386, and Thr-390). The importance of the triphosphate moiety for the binding of RTP has been demonstrated in our study. The presence of the triphosphate significantly contributes to the total free energy of binding, with a significant resultant free energy of stabilization (2805 J/mol). This high energy of stabilization clearly indicates that RTP binds with a higher affinity to the HCV polymerase than nonphosphorylated ribavirin. This may reflect the notion that unphosphorylated ribavirin has a reduced but detectable inhibitory activity compared with RTP in polymerization assays (39).

The incorporation of ribavirin triphosphate into nascent chains of RNA has been shown to be templated by both cytidine and uridine (12, 13). A rotation of the carboxamide moiety of ribavirin allows the pseudo base to form hydrogen bonds to both cytosine and uracil (12). Addition of a nucleotide to the NS5B protein saturated with a template-primer (TP) leads to the formation of a ternary complex NS5B·TP·NTP. Initial binding of ribavirin triphosphate to such a complex is differentiated from binding to the free enzyme by the presence of the template-primer. Our binding data clearly demonstrate that the association constant values (Ka) of ribavirin triphosphate are statistically similar regardless whether the enzyme is free or bound to a complementary or a noncomplementary template-primer. The failure of a complementary template-primer to increase the affinity of the NS5B protein for ribavirin triphosphate indicates that base pairing is clearly not involved in initial binding. Such a binding behavior has also been noted during the initial binding of deoxynucleotides to the Klenow fragment of the Escherichia coli DNA polymerase (40–42).

The use of fluorescence spectroscopy allowed us, for the first time, to separate the RTP binding activity from the concomitant hydrolysis and polymerization steps. Our study shows that although metal ions are required for catalytic activity, they are not required for the initial binding of ribavirin triphosphate to the polymerase. This is in agreement with our previous results that demonstrated that the presence of metal ions does not increase the affinity of the NS5B for free nucleotides (35). The role of metal ions in the NS5B polymerase activity probably resides in the catalytic portion of the reaction. In fact, a recent study demonstrated that the presence of manganese reduced the Km value for GTP and affect de novo initiation and/or the primer extension activities of NS5B (36).

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 technique. Many aspects of the binding activity, including characterization of the kinetics and thermodynamics parameters of the interactions, can easily be performed by fluorescence spectroscopy analysis. Analysis of the interaction between ribavirin analogs and the HCV polymerase by fluorescence spectroscopy is particularly appealing. Side effects resulting from the use of ribavirin have been noted and include defects in sperm development and hemolytic anemia (43–45). Improved versions of ribavirin are thus needed to improve the safety and efficacy of the therapies against HCV. Fluorescence spectroscopy appears as a tremendous tool for the development and screening of such compounds.


   FOOTNOTES
 
* 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 U.S.C. Section 1734 solely to indicate this fact. Back

{ddagger} New Investigator Scholar from the Canadian Institutes for Health Research. To whom correspondence should be addressed: Dépt. de Biochimie, Faculté de Médecine, Université de Sherbrooke, 3001 12e Ave., Sherbrooke, Québec J1H 5N4, Canada. Tel.: 819-564-5227; Fax: 819-564-5340; E-mail: Martin.Bisaillon{at}USherbrooke.ca.

1 The abbreviations used are: HCV, hepatitis C virus; RTP, ribavirin triphosphate; GdmHCl, guanidinium hydrochloride; TP, template-primer. Back


   ACKNOWLEDGMENTS
 
We thank Dr. Pierre Lavigne for his generosity and expert assistance with CD spectroscopy measurements.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Sidwell, R. W., Huffman, J. H., Khare, G. P., Allen, L. B., Witkowski, J. T., and Robins, R. K. (1972) Science 177, 705-706[Abstract/Free Full Text]
  2. De Clercq, E. (1993) Adv. Virus Res. 42, 1-55[Medline] [Order article via Infotrieve]
  3. Miller, J. P., Kigwana, L. J., Streeter, D. G., Robins, R. K., Simon, L. N., and Roboz, J. (1977) Ann. N. Y. Acad. Sci. 284, 211-229[Medline] [Order article via Infotrieve]
  4. Page, T., and Connor, J. D. (1990) Int. J. Biochem. 22, 379-383[CrossRef][Medline] [Order article via Infotrieve]
  5. Streeter, D. G., Witkowski, J. T., Khare, G. P., Sidwell, R. W., Bauer, R. J., Robins, R. K., and Simon, L. N. (1973) Proc. Natl. Acad. Sci. U. S. A. 70, 1174-1178[Abstract/Free Full Text]
  6. Muller, W. E., Maidhof, A., Taschner, H., and Zahn, R. K. (1977) Biochem. Pharmacol. 26, 1071-1075[CrossRef][Medline] [Order article via Infotrieve]
  7. Fang, S. H., Hwang, L. H., Chen, D. S., and Chiang, B. L. (2000) J. Hepatol. 33, 791-798[CrossRef][Medline] [Order article via Infotrieve]
  8. Zuckerman, E., Zuckerman, T., Sahar, D., Streichman, S., Attias, D., Sabo, E., Yeshurun, D., and Rowe, J. M. (2001) Blood 97, 1555-1559[Abstract/Free Full Text]
  9. Eriksson, B., Helgstrand, E., Johansson, N. G., Larsson, A., Misiorny, A., Noren, J. O., Philipson, L., Stenberg, K., Stening, G., Stridh, S., and Oberg, B. (1977) Antimicrob. Agents Chemother. 11, 946-951[Abstract/Free Full Text]
  10. Lau, J. Y., Tam, R. C., Liang, T. J., and Hong, Z. (2002) Hepatology 35, 1002-1009[CrossRef][Medline] [Order article via Infotrieve]
  11. Cameron, C. E., and Castro, C. (2001) Curr. Opin. Infect. Dis. 14, 757-764[Medline] [Order article via Infotrieve]
  12. Crotty, S., Maag, D., Arnold, J. J., Zhong, W., Lau, J. Y., Hong, Z., Andino, R., and Cameron, C. E. (2000) Nat. Med. 6, 1375-1379[CrossRef][Medline] [Order article via Infotrieve]
  13. Maag, D., Castro, C., Hong, Z., and Cameron, C. E. (2001) J. Biol. Chem. 276, 46094-46098[Abstract/Free Full Text]
  14. Crotty, S., Cameron, C. E., and Andino, R. (2001) Proc. Natl. Acad. Sci. U. S. A. 98, 6895-6900[Abstract/Free Full Text]
  15. McHutchison, J. G., Gordon, S. C., Schiff, E. R., Shiffman, M. L., Lee, W. M., Rustgi, V. K., Goodman, Z. D., Ling, M. H., Cort, S., and Albrecht, J. K. (1998) N. Engl. J. Med. 339, 1485-1492[Abstract/Free Full Text]
  16. Davis, G. L., Esteban-Mur, R., Rustgi, V., Hoefs, J., Gordon, S. C., Trepo, C., Shiffman, M. L., Zeuzem, S., Craxi, A., Ling, M. H., and Albrecht, J. (1998) N. Engl. J. Med. 339, 1493-1499[Abstract/Free Full Text]
  17. Sarbah, S., and Younossi, Z. (2000) J. Clin. Gastroenterol. 30, 125-143[CrossRef][Medline] [Order article via Infotrieve]
  18. Choo, Q. L., Kuo, G., Weiner, A. J., Overby, L. R., Bradley, D. W., and Houghton, M. (1989) Science 244, 359-362[Abstract/Free Full Text]
  19. Saito, I., Miyamura, T., Ohbayashi, A., Harada, H., Katayama, T., Kikuchi, S., Watanabe, Y., Koi, S., Onji, M., Ohta, Y., Choo, Q. L., Houghton, M., and Kuo, G. (1990) Proc. Natl. Acad. Sci. U. S. A. 87, 6547-6549[Abstract/Free Full Text]
  20. Alter, H. J. (1995) Blood 85, 1681-1695[Free Full Text]
  21. Houghton, M. (1996) in Virology (Fields, B. N., Knipe, D. M., and Howley, P. M., eds) pp. 1035-1058, Lippincott-Raven Publishers, Philadelphia
  22. Fried, M. W., Shiffman, M. L., Reddy, K. R., Smith, C., Marinos, G., Goncales, F. L., Jr., Haussinger, D., Diago, M., Carosi, G., Dhumeaux, D., Craxi, A., Lin, A., Hoffman, J., and Yu, J. (2002) N. Engl. J. Med. 347, 975-982[Abstract/Free Full Text]
  23. Lauer, G. M., and Walker, B. D. (2001) N. Engl. J. Med. 345, 41-52[Free Full Text]
  24. Lanford, R. E., Guerra, B., Lee, H., Averett, D. R., Pfeiffer, B., Chavez, D., Notvall, L., and Bigger, C. (2003) J. Virol. 77, 1092-1104[CrossRef][Medline] [Order article via Infotrieve]
  25. Eftink, M. R., and Ghiron, C. A. (1976) Biochemistry 15, 672-680[CrossRef][Medline] [Order article via Infotrieve]
  26. Flowers, S., Biswas, E. E., and Biswas, S. B. (2003) Biochemistry 42, 1910-1921[Medline] [Order article via Infotrieve]
  27. Pan, J. Y., Sanford, J. C., and Wessling-Resnick, M. (1995) J. Biol. Chem. 270, 24204-24208[Abstract/Free Full Text]
  28. Zhou, T., and Rosen, B. P. (1997) J. Biol. Chem. 272, 19731-19737[Abstract/Free Full Text]
  29. Bressanelli, S., Tomei, L., Rey, F. A., and De Francesco, R. (2002) J. Virol. 76, 3482-3492[Abstract/Free Full Text]
  30. Bressanelli, S., Tomei, L., Roussel, A., Incitti, I., Vitale, R. L., Mathieu, M., De Francesco, R., and Rey, F. A. (1999) Proc. Natl. Acad. Sci. U. S. A. 96, 13034-13039[Abstract/Free Full Text]
  31. Lesburg, C. A., Cable, M. B., Ferrari, E., Hong, Z., Mannarino, A. F., and Weber, P. C. (1999) Nat. Struct. Biol. 6, 937-943[CrossRef][Medline] [Order article via Infotrieve]
  32. Ng, K. K. S., Cherney, M. M., Vazquez, A. L., Machin, A., Alonso, J. M. M., Parra, F., and James, M. N. G. (2002) J. Biol. Chem. 277, 1381-1387[Abstract/Free Full Text]
  33. Doublie, S., Sawaya, M. R., and Ellenberger, T. (1999) Structure 7, R31-R35[Medline] [Order article via Infotrieve]
  34. Huang, H., Chopra, R., Verdine, G. L., and Harrison, S. C. (1998) Science 282, 1669-1675[Abstract/Free Full Text]
  35. Bougie, I., Charpentier, S., and Bisaillon, M. (2003) J. Biol. Chem. 278, 3868-3875[Abstract/Free Full Text]
  36. Ranjith-Kumar, C. T., Kim, Y. C., Gutshall, L., Silverman, C., Khandekar, S., Sarisky, R. T., and Kao, C. C. (2002) J. Virol. 76, 12513-12525[Abstract/Free Full Text]
  37. Prusiner, P., and Sundaralingam, M. (1973) Nat. New Biol. 244, 116-118[Medline] [Order article via Infotrieve]
  38. Ago, H., Adashi, T., Yoshida, A., Yamamoto, M., Habuka, N., Yatsunami, K., and Miyano, M. (1999) Structure 7, 1417-1426[Medline] [Order article via Infotrieve]
  39. Fernandez-Larsson, R., and Patterson, J. L. (1990) Mol. Pharmacol. 38, 766-770[Abstract]
  40. Kuchta, R. D., Mizrahi, V., Benkovic, P. A., Johnson, K. A., and Benkovic, S. J. (1987) Biochemistry 26, 8410-8417[CrossRef][Medline] [Order article via Infotrieve]
  41. Kuchta, R. D., Benkovic, P., and Benkovic, S. J. (1988) Biochemistry 27, 6716-6725[CrossRef][Medline] [Order article via Infotrieve]
  42. Ferrin, L. J., and Mildvan, A. S. (1986) Biochemistry 25, 5131-5145[CrossRef][Medline] [Order article via Infotrieve]
  43. Kochhar, D. M., Penner, J. D., and Knudsen, T. B. (1980) Toxicol. Appl. Pharmacol. 52, 99-112[CrossRef][Medline] [Order article via Infotrieve]
  44. Huggins, J. W., Hsiang, C. M., Cosgriff, T. M., Guang, M. Y., Smith, J. I., Wu, Z. O., LeDuc, J. W., Zheng, Z. M., Meegan, J. M., Wang, Q. N., Oland, D. D., Gui, X. E., Gibbs, P. H., Yuan, G. H., and Zhang, T. M. (1991) J. Infect. Dis. 164, 1119-1127[Medline] [Order article via Infotrieve]
  45. Saab, S., and Martin, P. (1999) J. Clin. Gastroenterol. 28, 289-290[CrossRef][Medline] [Order article via Infotrieve]

Add to CiteULike CiteULike   Add to Complore Complore   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us   Add to Digg Digg   Add to Reddit Reddit   Add to Technorati Technorati    What's this?


This article has been cited by other articles:


Home page
 J. Biol. Chem.Home page
J. Q. Hang, Y. Yang, S. F. Harris, V. Leveque, H. J. Whittington, S. Rajyaguru, G. Ao-Ieong, M. F. McCown, A. Wong, A. M. Giannetti, et al.
Slow Binding Inhibition and Mechanism of Resistance of Non-nucleoside Polymerase Inhibitors of Hepatitis C Virus
J. Biol. Chem., June 5, 2009; 284(23): 15517 - 15529.
[Abstract] [Full Text] [PDF]

Home page
 J. Virol.Home page
K. D. Ibarra and J. K. Pfeiffer
Reduced Ribavirin Antiviral Efficacy via Nucleoside Transporter-Mediated Drug Resistance
J. Virol., May 1, 2009; 83(9): 4538 - 4547.
[Abstract] [Full Text] [PDF]

Home page
 Clin. Microbiol. Rev.Home page
A. Wohnsland, W. P. Hofmann, and C. Sarrazin
Viral Determinants of Resistance to Treatment in Patients with Hepatitis C
Clin. Microbiol. Rev., January 1, 2007; 20(1): 23 - 38.
[Abstract] [Full Text] [PDF]

Home page
 J. Virol.Home page
M. A. Mir and A. T. Panganiban
The Hantavirus Nucleocapsid Protein Recognizes Specific Features of the Viral RNA Panhandle and Is Altered in Conformation upon RNA Binding
J. Virol., February 1, 2005; 79(3): 1824 - 1835.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
K. K.-S. Ng, N. Pendas-Franco, J. Rojo, J. A. Boga, A. Machin, J. M. M. Alonso, and F. Parra
Crystal Structure of Norwalk Virus Polymerase Reveals the Carboxyl Terminus in the Active Site Cleft
J. Biol. Chem., April 16, 2004; 279(16): 16638 - 16645.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
278/52/52471    most recent
M308917200v1
Right arrow Submit a Letter to Editor
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowRequest Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Bougie, I.
Right arrow Articles by Bisaillon, M.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Bougie, I.
Right arrow Articles by Bisaillon, M.
Social Bookmarking
Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati  
What's this?

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
 All ASBMB Journals   Molecular and Cellular Proteomics 
 Journal of Lipid Research   ASBMB Today 
Copyright © 2003 by the American Society for Biochemistry and Molecular Biology.
Advertisement
spacer
Advertisement
Advertisement