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J. Biol. Chem., Vol. 279, Issue 34, 35638-35643, August 20, 2004

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A Structural Basis for the Inhibition of the NS5 Dengue Virus mRNA 2'-O-Methyltransferase Domain by Ribavirin 5'-Triphosphate^*

Delphine Benarroch, Marie-Pierre Egloff, Laurence Mulard¶, Catherine Guerreiro¶, Jean-Louis Romette, and Bruno Canard||

From the Centre National de la Recherche Scientifique and Universités d'Aix-Marseille I et II, UMR 6098, Architecture et Fonction des Macromolécules Biologiques, École Supérieur d'Ingénieurs de Luminy-Case 925, 163 avenue de Luminy, 13288 Marseille cedex 9, France and the ^¶Institut Pasteur, Unité de Chimie Organique, 25 rue du Dr. Roux, 75724 Paris cedex 15, France

Received for publication, January 15, 2004 , and in revised form, May 13, 2004.

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Ribavirin is one of the few nucleoside analogues currently used in the clinic to treat RNA virus infections, but its mechanism of action remains poorly understood at the molecular level. Here, we show that ribavirin 5'-triphosphate inhibits the activity of the dengue virus 2'-O-methyltransferase NS5 domain (NS5MTase_DV). Along with several other guanosine 5'-triphosphate analogues such as acyclovir, 5-ethynyl-1--D-ribofuranosylimidazole-4-carboxamide (EICAR), and a series of ribose-modified ribavirin analogues, ribavirin 5'-triphosphate competes with GTP to bind to NS5MTase_DV. A structural view of the binding of ribavirin 5'-triphosphate to this enzyme was obtained by determining the crystal structure of a ternary complex consisting of NS5MTase_DV, ribavirin 5'-triphosphate, and S-adenosyl-L-homocysteine at a resolution of 2.6 Å. These detailed atomic interactions provide the first structural insights into the inhibition of a viral enzyme by ribavirin 5'-triphosphate, as well as the basis for rational drug design of antiviral agents with improved specificity against the emerging flaviviruses.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
The guanosine analogue ribavirin is a broad spectrum antiviral agent discovered almost 30 years ago (1). Since its discovery, many mechanisms of action have been proposed (reviewed in Refs. 2 and 3). Like most nucleoside analogues, ribavirin is phosphorylated by cellular kinases at its 5'-position upon entry into the cell. Ribavirin 5'-monophosphate is a potent inhibitor of the cellular enzyme inosine 5'-monophosphate dehydrogenase. This inhibition results in the depletion of the intracellular guanosine nucleotide pool, which feeds capping and polymerase enzymes from both viral and cellular origin. Consequently, the depressed guanosine nucleotide pool may exert an indirect antiviral effect, since viral enzymes would not compete advantageously for guanosine nucleotides with cellular enzymes. In addition, ribavirin nucleotides may have a viral target, such as RNA polymerization and RNA capping or induce lethal mutagenesis of viral genomes (4), accounting for the observed antiviral effect. Both direct and indirect mechanisms may thus contribute to the ribavirin mode of action. To date, ribavirin nucleotides have been crystallized with two cellular enzymes, namely inosine 5'-monophosphate dehydrogenase (5) and nucleoside diphosphate kinase (NDPK (6)) but not with any viral enzyme or protein.

The genus Flavivirus comprises important human pathogens such as West Nile, dengue, and yellow fever viruses, which are moderately sensitive to ribavirin (7–9). These mosquito-borne viruses are currently expanding their distribution throughout the world. The introduction of West Nile virus in North America may be an important milestone in the history of this virus, as exemplified by outbreaks in the New York area (10) followed by the gradual spread to 47 of the 49 continental states of the United States of America (www.cdc.gov/ncidod/dvbid/westnile/index.htm). The Camargue area in France has re-witnessed West Nile viral infection of horses after 40 years, and the first human cases were reported in the French Riviera in October 2003. Likewise, dengue virus, an agent responsible for hemorrhagic fever, infects more than 50 million persons annually with an increasing incidence in tropical areas around the world.

The single-stranded RNA genome of flaviviruses is of positive polarity, and is capped with a cap 1 structure ^Me7GpppA_2'OMe (11). The N-terminal domain of the dengue virus polymerase NS5 is a 2'-O-methyltransferase that is active on RNA cap structures (12). This enzyme, referred to as NS5MTase_DV, is able to bind a GTP molecule that may mimic the RNA cap structure prior to methylation. Thus, it was of interest to determine whether guanosine analogues could bind to the GTP binding site of NS5MTase_DV. If so, guanosine analogues may act as potential competitive inhibitors of RNA cap binding and aid in the rational design of inhibitors directed against flaviviruses.

In this report, we present biochemical evidence that ribavirin 5'-triphosphate (RTP)¹ inhibits the 2'-O-methyltransferase activity of dengue virus NS5MTase_DV. We also show that a series of RTP analogues, as well as EICAR 5'-triphosphate (EICAR-TP) and acyclovir 5'-triphosphate (acyclovir-TP), compete with GTP for binding to NS5MTase_Dv. In addition to show that RTP and GTP share a common binding site on NS5MTase_DV, the crystal structure of the RTP-NS5MTase_DV complex reveals a unique mode of binding for the ribavirin pseudobase that is consistent with a lack of discrimination of this antiviral molecule relative to GTP.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Enzymes and Reagents—The purification and crystallization of the NS5 capping domain of the dengue virus RNA-dependent RNA polymerase has been described (12). Synthesis and purification of RTP, ribavirin nucleotide analogues, and acyclovir 5'-TP to homogeneity, as determined by ¹H, ¹³C, and ³¹P NMR and HPLC, have been described (6). EICAR 5'-TP was a kind gift from P. Herdewijn (Leuven, Belgium). All other nonradioactive and ³²P-labeled nucleotides were of HPLC grade and purchased from Amersham Biosciences.

Inhibition of RNA 2'-O-Methyltransferase Activity by RTP—The methyltransferase assay was performed in 40 mM Tris-HCl, pH 7.1. 100 µM S-adenosylmethionine with 10 µCi of Ado[methyl-³H]Met (74 Ci/mmol; Amersham Biosciences) were incubated with 2 µg of purified NS5MTase_DV, 35 µl of RNA substrate, and various concentrations of ribavirin 5'-triphosphate (50, 100, 250, 300, 500 and 750 µM) in a 50-µl reaction mix (12). The RNA substrate mix was produced using purified T7 DNA primase in conjunction with the synthetic DNA oligonucleotide T₁₀CTG₅ (10 µM), 300 µM CTP, and 200 µM cap analogue to obtain capped AC₅ (GpppAC₅ and and ^m7GpppAC₅) as described (13). The methyl-transfer reaction was incubated at 30 °C and monitored from 30 min to 3 h. 8-µl aliquots were spotted onto DEAE-81 filter paper (Whatman) and washed with 20 mM sodium formate, pH 8.0 to remove any remaining S-adenosylmethionine. The experiment without ribavirin 5'-triphosphate served as a reference. Radioactively labeled RNA was quantified by liquid scintillation counting.

Determination of RTP Dissociation Constant for NS5MTase_DV—The dissociation constant, K_d, was determined using 58 µM [³²P]GTP and increasing concentrations of RTP (0, 100, 200, 500, 800, and 1000 µM) incubated with 2 µg of NS5MTase_DV. The bound radioactivity was quantitated after SDS-PAGE using photostimulatable plates and a FujiImager. Data were fit to a hyperbolic function from which the K_d was determined.

GTP-binding Inhibition by RTP and Its Analogues—Two µg of NS5MTase_DV was incubated with RTP or RTP analogues and with [³²P]GTP (50 µM, 10% volume of [³²P]GTP) in 50 mM Tris, pH 7.6, 5 mM dithiothreitol, and 5 mM MgCl₂. The reaction mixture was irradiated with UV light for 3 min using a UV lamp (40 watts, at 254 nm) at a distance of 12 mm, boiled for 5 min, and subjected to denaturing gel electrophoresis. The gel was stained using Coomassie Blue dye, and radioactive products were visualized using photostimulatable plates and a FujiImager.

Structure Determination and Refinement—Crystals of NS5MTase_DV were grown by vapor diffusion as described (12) and further soaked for 3 h in 2 mM RTP. A 2.6-Å data set was collected at 100 K using a charge-coupled device detector (ADSC Q4) at ID14-2 beamline, ESRF, Grenoble, France. Data were processed using DENZO (14), and intensities were merged with SCALA (15). Since the unit cell dimensions of the crystal were slightly different from those of the apo-form, the structure was solved by the molecular replacement program AmoRe, whereas polypeptide chain of the apo-form served as a model (PDB code 1L9K [PDB] ). A random set comprising 5% of the data was omitted from refinement for R_free calculation (16). A round of simulated annealing refinement, as implemented using crystallography CNS software, was performed (17). As in the case of the apo-form, no density was observed for the first 6 and the last 30 residues. Models for one molecule of S-adenosyl-L-homocysteine (SAHC) and one molecule of ribavirin 5'-monophosphate were built into the F_o – F_c and 2F_o – F_c SIGMAA-weighted electron density. The model was further built and refined through alternating cycles using the programs TURBO-FRODO (18) and CNS (crystallography NMR software), respectively. During this process seven sulfate ions, which were already present in the native structure, and 66 additional water molecules were refined. Refinement statistics are given in Table I. Atomic coordinates have been deposited in the Protein Data Bank under code 1R6A.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

View larger version (25K): [in this window] [in a new window]   FIG. 1. Inhibition of the RNA 2'-O-methyltransferase activity of NS5MTaseDV and mapping of the binding site of guanosine analogues. A, inhibition of RNA 2'-O-methyltransferase activity. The incorporation of 3H-labeled methyl groups into small capped RNAs was performed using a filter paper binding assay as described under "Experimental Procedures" and in Ref. 12. RTP was added at indicated concentrations immediately prior to the addition of purified NS5MTaseDV to start the reaction. The plot is an average of three independent experiments. B, determination of the dissociation constant (Kd) of RTP for NS5MTaseDV. The protein was incubated with a constant concentration of [32P]GTP (58 µM, the apparent Kd concentration) and increasing concentrations of RTP. The mixture was irradiated with UV light and analyzed using denaturing gel electrophoresis as described under "Experimental Procedures." The upper panel shows the Coomassie Blue-stained gel, and the lower panel depicts the corresponding autoradiographic analysis. Quantitation of the autoradiographic analysis is plotted, and the relative inhibition of GTP-binding as a function of RTP concentration is shown. Curve fitting was performed with a hyperbolic equation. C, same analysis as in B using acyclovir-5'-TP and EICAR 5'-TP. After the autoradiograms were analyzed and quantified, the data were plotted as inhibition of GTP binding as a function of acyclovir-5'-TP (upper plot) or EICAR 5'-TP (lower plot) concentration.

View larger version (53K): [in this window] [in a new window]   FIG. 2. Displacement of GTP from the nucleotide binding site of NS5MTaseDV by RTP and RTP analogues. NS5MTaseDV was incubated with 150 µM RTP (lane 1) before the addition of [32P]GTP (50 µM) to the reaction mixture. The mixture was irradiated with UV light and analyzed using denaturing gel electrophoresis as described under "Experimental Procedures." The same experiment was performed for the five RTP analogues: 2'-deoxy-RTP (lane 2), 3'-deoxy-RTP (lane 3), 2'3'-dideoxy-RTP (lane 4), 2'3'-dideoxy-2'3'-didehydro-RTP (lanes 5), and 2'3'-epoxy-RTP (lane 6). The upper panel shows lanes from a Coomassie Blue-stained gel of a typical experiment, and the lower panel shows the corresponding autoradiographic analysis. The diagram below represents the percentage of inhibition of [32P]GTP-NS5MTaseDV complex formation and is the average of three experiments with RTP and two experiments with RTP analogues, respectively.

View larger version (28K): [in this window] [in a new window]   FIG. 3. Structural features of the nucleotide binding site of NS5MTaseDV. A, the model of GDPMP in the nucleotide binding site of NS5MTaseDV has been described in Ref. 12. Residues interacting with the nucleotide are shown in ball-and-stick form. Main-chain carbon atoms are shown in dark blue except for the carbonyl oxygens, which are red. Side chains are colored according to atom type. For clarity, noninteracting side chains of residues 17, 19, and 20 are not shown. Dotted lines indicate hydrogen bonds. B,2Fo – Fc electron density map of the refined complex between RTP and NS5MTaseDV. The color code is the same as in A. The - and -phosphates of RTP are present but could not be modeled accurately because of poor electron density in this area, suggesting the coexistence of several conformations. Schematic diagrams show the interactions of the guanine moiety of GDPMP (C) and the ribavirin pseudobase with NS5MTaseDV residues (D). Main-chain carbonyl oxygens within hydrogen bonding distance (in Å) of the GDPMP and RTP 2-amino group are indicated by dotted lines (only two of the three displayed H-bonds can be fulfilled simultaneously). "O = C<" symbolizes main chain carbonyl groups. E, superimposition of ribavirin triphosphate and GDPMP as seen in the NS5MTaseDV domain. The structures of the two complexes (NS5MTaseDV-GDPMP-SAHC and NS5MTaseDV-RTP-SAHC) have been superimposed. For clarity, only a few amino acids from the binding site are shown in stick representation (K14, L17, N18, A19, L20). The main-chain carbon atoms are shown in dark blue, except for the carbonyl oxygens of Leu-17, Asn-18, and Leu-20 (which are within hydrogen bond distance of the NH2 group of the guanosine base and the ribavirin pseudobase; see A and B) and for the amino group of Lys-14 (which interacts via hydrogen bonding with the O-2 position of the ribose; see A and B). GDPMP is also represented in dark blue, except for oxygen and nitrogen atoms. Ribavirin triphosphate is represented in ball-and-stick form and colored according to atom type. F, schematic diagram showing the interactions of the ribavirin pseudobase with inosine 5'-monophosphate dehydrogenase (PDB code 1ME8 [PDB] ). Dotted lines indicate hydrogen bonds, with numbers corresponding to distance in Å.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
In this paper, we have show that RTP inhibits the RNA 2'-O-methyltransferase activity of dengue virus NS5MTase_DV domain. We further mapped the binding site of RTP binding to the GTP/RNA cap binding site using GTP competition experiments. The RTP binding site was then characterized at the atomic level using x-ray crystallography at 2.6 Å resolution.

The structure presented here is the first example of a viral enzyme complexed to a ribavirin nucleotide. Two cellular protein structures have been solved in complex with ribavirin nucleotides: the H122G mutant of nucleoside diphosphate kinase from Dyctyostelium discoideum (PDB code 1MN9 [PDB] (6)) and the inosine-5'-monophosphate dehydrogenase from Tritrichomonas foetus (PDB code 1ME8 [PDB] (5)). In the former complex, the binding of RTP is completely aspecific; the phosphate and ribose of RTP interact via hydrogen bonding with the protein, but the only ribavirin pseudobase-protein interaction is an aromatic stacking involving Phe-64, which could be achieved by any nucleobase (Fig. 3, B and E). The atomic contacts are different in the complex of RMP with inosine-5'-monophosphate dehydrogenase, as the ribavirin pseudobase establishes three hydrogen bonds with the protein (Fig. 3F). In the ternary complex with coenzyme A (PDB code 1ME7 [PDB] ), no structural rearrangements take place, as the CoA binds in a preformed binding site and the interactions of ribavirin with the protein are not modified. However, a comparison of the relative position of RMP and IMP in complex with inosine 5'-monophosphate dehydrogenase reveals that ribavirin superimposes with IMP perfectly, contrary to what is observed in the NS5MTase_DV-RTP complex. This is because of the intrinsic nature of both nucleotides; with IMP, the NH₂ group of ribavirin could have been mimicked by the NH group in the 1-position, whereas with GMP, either the 1- or the 2-position could have been mimicked by the NH₂ group of ribavirin.

It is not known whether ribavirin targets NS5MTase_DV in flavivirus-infected cells. It has been demonstrated that ribavirin significantly reduces the growth of several flaviviruses, with no information about the actual viral target (7–9, 19, 20). In the case of dengue serotypes 1, 2, and 3, West Nile, and yellow fever viruses, the reported IC₅₀ values are between 81 and 171 µM (7, 9, 20). The apparent inhibition parameters observed in this report for RTP (IC₅₀ 100 µM) are in good agreement with these IC₅₀ values. Because ribavirin decreases the intracellular GTP concentration drastically, it potentiates the successful competition of RTP against GTP for NS5MTase_DV binding. Indeed, the average intracellular GTP concentrations in various mammalian cell types have been measured and found to be 0.48 mM (compiled in Ref. 21). Treatment of murine leukemia L1210 cells with 20 µM ribavirin decreases the GTP concentration to a value equal to 12% of that of the untreated control, i.e. around 60 µM (22). This latter value is precisely the same as that of the K_D (GTP) for NS5MTase_DV (58 ± 14 µM (12)). This observation suggests that with ribavirin treatment, which involves ribavirin concentrations of 600–800 µM in biological fluids (23), the down-regulated GTP concentration is not likely to saturate NS5MTase_DV, leaving open the possibility that the binding of RTP to NS5MTase_DV may be relevant to the known ribavirin anti-lavivirus activity. This discussion may also be relevant to other viruses. Indeed, RTP has been reported to be an inhibitor of the (guanine-N7)-methyltransferase of vaccinia virus, as well as to lead to abnormal RNA cap formation in alphaviruses (reviewed in Ref. 24). To date, there is little evidence that ribavirin actually targets viral enzymes other than RNA polymerases, as in the case of hepaciviruses (3, 25). The structural data presented here suggest a model for the inhibition of the RNA 2'-O-methyltransferase activity by RTP. RTP may compete with viral RNA cap structures to bind the NS5MTase_Dv and prevent efficient RNA cap methylation.

Whether or not RTP causes flavivirus growth inhibition through the targeting of NS5MTase_DV in vivo, our results still have implications in terms of drug design. First, screening of the Protein Data Bank revealed that no specific recognition of GTP with only the purine 2-position (as shown in Fig. 3, B and D) has been reported before that of NS5MTase_DV (12). The great majority of cellular NTP-binding enzymes appear to contact at least two of the purine at position 1, 2, or 6 (12). Among over 60 protein-GTP or protein-RNA cap complexes examined, only two (PDB codes 1KHB [PDB] and 1DOA [PDB] ) bind GTP using a single position (O-6 in this case) in addition to base-stacking contacts. In theory, since RTP and GTP have equivalent H-bond donors and acceptors at positions 1 and 6 but not position 2, RTP could bind to GTP-binding proteins using positions 1 and 6. To the best of our knowledge, there is no example of such binding in the Protein Data Bank involving a protein of human origin. The observation of an antiviral selectivity for ribavirin may indicate that ribavirin either targets viral proteins through this position 2 alone (amine of RTP) or binds both viral and cellular GTP-binding proteins through positions 1 and 6 (carbonyl and the NH of RTP) but has a more profound effect on viral targets, hence the observed selectivity. Second, the same series of RTP analogues has been used to inhibit HCV NS5B, a hepacivirus RNA-dependent RNA polymerase (6). Hepaciviruses do not have an RNA capping machinery, but their RNA-dependent RNA polymerase domain is 10% identical (22% amino acid similarity) to that of flaviviruses. We note that the most important ribose groups for binding/inhibition are not similar between the hepatitis C virus NS5B polymerase and NS5MTase_DV. For hepatitis C virus NS5B the 3'-hydroxyl is critical (6), whereas our present results indicate that the 2'-hydroxyl is most important in the case of NS5MTase_DV. Therefore, if the most important group is also the 3'-hydroxyl for the flavivirus RNA polymerase, it seems difficult to design a simple ribose modification that would target both the RNA polymerase and the cap-binding enzyme of flaviviruses simultaneously. Third, only main-chain contacts are involved in the binding of RTP to NS5MTase_DV. A mere substitution of an amino acid side chain cannot directly discriminate RTP relative to GTP in order to provide potential drug resistance.

Since RNA capping is essential for various viruses (18), the mechanism for ribavirin-mediated inhibition of RNA capping presented here may account in part for the antiviral activity of ribavirin against flaviviruses, but it does not exclude the inhibition of additional viral enzymatic activities. Our results would then provide a basis for rational drug design against human pathogens of viral origin, of which the emerging flaviviruses are a timely example.

	FOOTNOTES

 
^* This investigation was supported in part by the French Ministry Program "Maladies Infectieuses" and a grant from the Direction Générales des Armées and in part by the European Economic Community program "Flavitherapeutics" (QLK3-CT-2001-00506). 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.

Supported by a predoctoral fellowship from the Direction Générales des Armées and the Fondation pour la Recherche Medicale.

^|| To whom correspondence should be addressed. Tel.: 33-491-82-86-44; Fax: 33-491-82-86-46; E-mail: bruno{at}afmb.cnrs-mrs.fr.

¹ The abbreviations used are: RTP, ribavirin 5'-triphosphate; RMP, ribavirin 5'-monophosphate; SAHC, S-adenosyl-L-homocysteine; PDB, Protein Data Bank; EICAR, 5-ethynyl-1--D-ribofuranosylimidazole-4-carboxamide; HPLC, high pressure liquid chromatography; GDPMP, --methylene GTP.

	ACKNOWLEDGMENTS

 
We thank Sonia Longhi, Zhi Hong, Cindy Yee, and Holli Conway for critical reading of the manuscript.

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TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 

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