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Originally published In Press as doi:10.1074/jbc.M405465200 on August 3, 2004

J. Biol. Chem., Vol. 279, Issue 45, 47212-47221, November 5, 2004
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Structure of Foot-and-Mouth Disease Virus RNA-dependent RNA Polymerase and Its Complex with a Template-Primer RNA*

Cristina Ferrer-Orta{ddagger}§, Armando Arias¶||, Rosa Perez-Luque{ddagger}, Cristina Escarmís¶, Esteban Domingo¶, and Nuria Verdaguer{ddagger}**

From the {ddagger}Institut de Biologia Molecular de Barcelona (CSIC), Parc Científic de Barcelona, Josep Samitier 1–5, E-08028 Barcelona, Spain and the Centro de Biologia Molecular "Severo Ochoa" (CSIC-UAM), Cantoblanco, E-28049 Madrid, Spain

Received for publication, May 17, 2004 , and in revised form, August 2, 2004.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Genome replication in picornaviruses is catalyzed by a virally encoded RNA-dependent RNA polymerase, termed 3D. The enzyme performs this operation, together with other viral and probably host proteins, in the cytoplasm of their host cells. The crystal structure of the 3D polymerase of foot-and-mouth disease virus, one of the most important animal pathogens, has been determined unliganded and bound to a template-primer RNA decanucleotide. The enzyme folds in the characteristic fingers, palm and thumb subdomains, with the presence of an NH2-terminal segment that encircles the active site. In the complex, several conserved amino acid side chains bind to the template-primer, likely mediating the initiation of RNA synthesis. The structure provides essential information for studies on RNA replication and the design of antiviral compounds.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Foot-and-mouth disease virus (FMDV)1 is the prototype member of the Aphthovirus genus of the Picornaviridae family, and the etiological agent of the economically most important disease of farm animals (1, 2). The cost of the FMD outbreak that took place in 2001 in the United Kingdom has been evaluated in 6 billion pounds sterling (3), and it has been estimated that a large FMD outbreak in northern Europe or the United States would entail losses exceeding 100 billion dollars (4). Probably its most devastating effects are felt in underdeveloped countries because of the severe trade restrictions imposed by the disease (5). Difficulties for the control of FMD stem from the wide host range of FMDV, variability in pathogenic manifestations, antigenic diversity, high infectivity and transmissibility, capacity to establish persistent, asymptomatic infection affecting farm ruminants and also wildlife species, and the limited efficacy of current vaccines (1, 2). Some of these difficulties have their origin in a basic feature of FMDV biology, namely the error-prone nature of FMDV RNA replication, determined by the limited template copying fidelity of the RNA-dependent RNA polymerase (RDRP) 3D (6). To understand FMDV RNA synthesis at the molecular level, to interpret the copying-fidelity properties of the replication machinery, and to design antiviral compounds against FMD as a potential new approach to control the disease, a knowledge of the structure of the polymerase 3D is essential.

Here, we report the structure of the RDRP of FMDV in a free form at 1.9-Å resolution and in complex with a template-primer RNA decanucleotide at 3.0-Å resolution. These structures represent the first complete three-dimensional structure of a picornavirus polymerase and provide new insights into the structural basis for the FMDV 3D function and the structure-based design of antiviral compounds against an important group of animal pathogens.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Cloning of FMDV C-S8c1 3D Polymerase in Plasmid pET-28a—The genomic region coding for the 3D polymerase of FMDV was amplified by PCR from the infectious plasmid pMT28,2 containing a complete copy of the genome of our standard FMDV C-S8c1, and cloned in plasmid pET-28a (Novagen). An NcoI restriction site present in the 3D coding region was eliminated to allow this restriction site to be used for cloning. For this purpose, two amplifications were prepared: the region between positions 6610 and 6953 (numbering of genomic residues as in Ref. 7) was amplified using a sense primer spanning residues 6610–6631 and an antisense primer complementary to nucleotides 6953–6929. The sense primer contained an initiating AUG preceding the 3D coding sequence and a NcoI restriction sequence upstream of the AUG. The antisense primer had the mutation G6939 -> A to eliminate the NcoI internal restriction site without affecting the amino acid sequence of the protein. The second amplicon was obtained by primers spanning residues 6925–6947 (sense) containing the mutation C6939 -> T, and 8019–7998 (antisense), which included the restriction sequence for NotI after the last codon. These two amplicons were shuffled in vitro in the presence of the sense primer of the first amplicon and the antisense primer of the second amplicon, and PfuI DNA polymerase. After phenol extraction the recombined DNA was digested with NcoI and NotI, the fragment obtained was purified by electrophoresis through agarose gel and ligated to the vector (pET28a) digested with the same enzymes. The ligation product was used to transform Escherichia coli BL21, and the plasmid of a positive colony was sequenced to confirm that it contained the expected 3D sequence.

Expression and Purification of Recombinant 3D Polymerase—Plasmid pET-28a provided a tract of AAALE and 6 histidines at the carboxyl terminus of the protein. Kanamycin-resistant colonies were grown in L broth medium until an OD of 0.8–1 at 595 nm was reached. Then isopropyl 1-thio-{beta}-D-galactopyranoside was added to a final concentration of 0.5 mM and the culture grown for two additional hours. Cells were pelleted by centrifugation at 4000 x g for 15 min and then kept at -20 °C. For lysis, the cells were suspended in 50 mM phosphate buffer, pH 7.8, 0.3 M NaCl and incubated with 20 mg/ml lysozyme for 15 min in the presence of 1 mM phenylmethylsulfonyl fluoride. The cells were then sonicated for 5 cycles of 30 s alternating with 5 cycles of 30 s in ice. Cellular debris was pelleted and the supernatant was loaded on a nickel-nitrilotriacetic acid column. Elution was carried out with an imidazol gradient (50 to 500 mM)in50 mM phosphate buffer, pH 6.0, 0.5 M NaCl. Fractions of 2 ml were collected and analyzed for the presence of the protein. Fractions containing the protein were pooled and dialyzed against 50 mM Tris-HCl, pH 7.5, 100 mM NaCl, 10% glycerol, 1 mM EDTA, 1 mM dithiothreitol and stored in aliquots at -20 °C. The enzyme was >95% pure, its specific activity was 199 pmol of UTP incorporated/min per µg of protein (Table I) and the activity was inhibited specifically by monoclonal antibodies directed to FMDV 3D. An additional chromatographic step was performed before the crystallization trials on a size exclusion Superdex 200 HR column (Amersham Biosciences). Preparation of seleniomethionine-labeled 3D polymerase was carried out as described above except that the E. coli harboring the plasmid containing 3D was grown in a minimal medium lacking methionine and supplemented with seleniomethionine (8).


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  		TABLE I
Purification of FMDV 3D Determination of the -fold and yield of the purification of 3D polymerase.

 
Crystallization, Data Collection, and Processing—FMDV 3D proteins, both the native form and the seleniomethionine derivative used for the structure determination, were crystallized using the hanging drop vapor diffusion method at room temperature. Typically 4 µl of protein solution (4.6 mg/ml) in 40 mM Tris-HCl, pH 7.5, 0.5 M NaCl, 8% glycerol were mixed with an equal volume of a reservoir solution. Tetragonal crystals, space group P41212(a = b = 93.04 Å, c = 119.05 Å), were obtained in 35% PEG 4K, 0.2 M sodium acetate, 0.1 M sodium citrate, pH 5.6, and 4% {gamma}-butirolactone.

The 3D-RNA complex was obtained and crystallized as follows. The oligonucleotide r5'-GCAUGGGCCC-3' (NWG-Biotech) was annealed following the procedure described in Ref. 9. Briefly, 8 nmol of the oligonucleotide was heated to 90 °C for 1 min and slowly cooled to 10 °C at a rate of ~5 °C/min. Then the 3D polymerase was slowly added in an equimolar proportion in the presence of 2 mM MgCl2. The crystallization trials were performed by the hanging drop vapor diffusion method. Trigonal P3221 crystals (a = b = 94.07 Å, c = 102.89 Å) were obtained with 30% PEG 4K, 0.2 M magnesium acetate and sodium cacodylate, pH 6.0.

X-ray data of native and seleniomethionine-3D crystals were collected at the synchrotron beamlines ID13 and ID29, respectively (ESRF, Grenoble, France). Three data sets were recorded at 100 K from a single seleniomethionine crystal at wavelengths corresponding to the selenium absorption maximum, inflection point, and a hard remote (Table II). For the 3D-RNA crystals, data were collected at 100 K at beamline ID13 (ESRF) to 3.0-Å resolution. All data were processed and reduced using the DENZO/SCALEPACK package (10).


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  		TABLE II
Summary of the data collection and refinement statistics

 
Structure Solution and Refinement—The structure of the isolated 3D protein was determined using the multiwavelength anomalous dispersion technique. Local scaling, Patterson searches, and phasing calculations were performed using SOLVE (11). All 14 selenium atoms predicted from the amino acid sequence were located from Patterson searches. Phase improvement in RESOLVE (12, 13) allowed an automatic tracing of 320 residues of the 476 total residues. The initial model was manually rebuilt using TURBO (14) and refined with CNS (15) against a wavelength-merged data set. The structure built from the multiwavelength anomalous dispersion data was further refined against the 1.9-Å data from native crystals using CNS. Additional rounds of manual rebuilding and refinement produced the present model containing the whole protein molecule, plus the five amino acid linker residues and two of the His6 tag at the COOH terminus that participates in crystal contacts, and 237 water molecules. The refinement converged to an R-factor of 20.2% (Rfree = 24.1%) with 90% of the residues lying in the most favored regions in the Ramachandran plot (Table II).

The structure of the FMDV 3D in complex with the template-primer RNA was solved by molecular replacement with AMoRe (16) using the native 3D protein as starting model. The initial maps clearly showed the presence of extra density that would correspond to the RNA molecule. This electron density was well defined for a 5' overhang region in particular C2A3U4 of the template strand plus the four following base pairs (G5 to C9 and their complementary G16 to C20 residues), but weakened toward the 3' end. Refinement and manual model correction proceeded as for the native crystal. The final model, refined to 3.0-Å resolution, included 476 protein residues, 23 solvent molecules, one Mg2+ ion, and the region: 5'-CAUGGGCC-3' of the template plus the complementary 3'-CCCGG-5' stretch of the primer RNA oligonucleotide (Table II).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Overall Structure of the FMDV 3D Polymerase in Its Free and RNA-bound Forms—Crystals of recombinant 3D polymerase of FMDV were obtained and analyzed by x-ray crystallography both isolated and in complex with a self-annealing RNA decanucleotide in the presence of Mg2+. The structure of the unliganded FMDV 3D polymerase was determined by multiwavelength anomalous dispersion of seleniomethionated protein and the structure of FMDV 3D-RNA complex was solved by molecular replacement using the isolated 3D structure as starting model.

The 476 residues of the polypeptide chain (including part of the COOH-terminal His tag) fold in 17 {alpha}-helices and 16 {beta}-strands that form the canonical structure of a right hand with the "fingers," "palm," and "thumb" subdomains as seen in the three-dimensional structure of most other polynucleotide polymerases (17). In addition, there is an NH2-terminal segment (residues 1–96) bridging the fingers and the thumb subdomain that encircles the active site of the enzyme (Fig. 1, A and B). This region, also termed the fingertips, seems to be unique to the RDRP compared with other classes of nucleic acid polymerases (18, 19).



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  		FIG. 1.
The structure of the FMDV 3D polymerase isolated and bound to a template-primer. A, stereoscopic view of a ribbon diagram showing the secondary structural elements explicitly labeled. The NH2-terminal segment (residues 1–96) is shown in pink. The fingers subdomain (residues 97–207 and 249–302) consist of eight {alpha}-helices ({alpha}3-{alpha}9) and 7 {beta}-strands ({beta}3-{beta}7 and {beta}9-{beta}10). The palm (residues 230–248 and 303–403) consists of a {beta}-sheet ({beta}8, {beta}11, {beta}12) sandwiched between helices {alpha}11 and {alpha}12 and followed by a pair of {beta}-strands ({beta}14, {beta}15). The six different structure-sequence motifs of the palm and fingers are colored as follows: A, red; B, green; C, yellow; D, magenta; E, dark-blue; and F, orange. The thumb subdomain (residues 403–470) comprises four {alpha}-helices ({alpha}13 to {alpha}16) and two long loops. B, stereoscopic view of the {alpha}-carbon trace of the unbound FMDV 3D (blue) with every 20th residues numbered. The partial structure of the 3D polymerase of PV is superimposed (red). The comparison between both structures, performed with the program SHP (20), gave a root mean square deviation of 1.3 Å for the superimposition of 241 C{alpha} atoms. C, surface representation of the FMDV 3D polymerase in complex with a template-primer. The bound RNA oligonucleotide is shown as stick representation in yellow (template chain) and green (primer chain). For clarity, the thumb subdomain is omitted, motifs A and C of palm are shown in a ribbon representation (magenta) and the molecule is placed in a different orientation compared with A and B. The acidic residues Asp238 and Asp240 that coordinate the Mg2+ ion and the catalytic aspartates, 338 and 339, are shown as sticks in magenta. The Mg2+ ion is represented as an orange ball.

 
The free and RNA-bound structures are in excellent agreement. The root mean square deviation of the superimposition of all 476 amino acid residues, carried out with the program SHP (20), is only 0.5 Å, showing that no important rearrangements of the 3D polymerase conformation were induced by the RNA binding.

The Conserved Structural Motifs of the Palm Subdomain— The palm subdomain residues consist of a three-stranded {beta}-sheet ({beta}8, {beta}11, and {beta}12) sandwiched between helices {alpha}11 and {alpha}12 and followed by a pair of {beta}-strands ({beta}14, {beta}15) that form an interface with the mostly helical thumb (Fig. 1A). The architecture of this region is the most highly conserved feature of all known polymerases (21). It is composed of five conserved motifs A to E, involved in nucleotide binding, phosphoryl transfer, structural integrity of the palm subdomain, and priming nucleotide binding, respectively (18) (Figs. 1A and 2). Strand {beta}8 contains the conserved acidic residues Asp240 and Asp245 and forms motif A. The aphthovirus RDRP has an additional acidic residue within this region (Asp238). In the structure of the complex, the side chain of Asp238 is reoriented toward the active site close to the catalytic aspartic acid residues 338 and 339 of motif C (Fig. 3, A and B). Motif C (the {beta}11-turn-{beta}12 structure) contains the sequence GDD (Gly337-Asp338-Asp339), almost universally conserved in RDRPs. Metal ions that are likely to be involved in the nucleotidyl transfer reaction interact with these aspartate residues. In the structure of the FMDV 3D-RNA complex that was obtained in absence of NTP, only one Mg2+ ion was located at a site ~6 Å from the expected catalytic positions. This finding is similar to that reported in the structure of the RDRP of bacteriophage {varphi}6 crystallized in the absence of an incoming nucleotide (22). Both ions occupied similar sites. In FMDV 3D, the Mg2+ coordinates the O{delta} atoms of Asp238 and Asp240 (motif A), Asp339 (motif C), and the carboxylate oxygen atoms of Val239, Ile340, and Thr384 (Figs. 1C and 3B).



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  		FIG. 2.
Structure based sequence alignment of the four ssRDRPs of known structure. The strictly conserved residues are in red blocks and similar residues in blue boxes. Secondary structure definitions for FMDV 3D are above the sequence alignment and the conserved sequence motifs are marked by bars below the sequences. Motif A is colored red; B, green; C, yellow; D, magenta; E, gray; and F, orange. Catalytic aspartates are marked by blue spheres, residues interacting with the RNA in the FMDV 3D complex are marked by green triangles and residues predicted to interact with the incoming NTP substrate by yellow squares. Despite FMDV RDRP sharing only 29% of sequence identity with PV polymerase and about 12% identity with caliciviral RDRPs, the RNA interacting residues are highly conserved. The amino acids that are in contact, either with the template-primer or the incoming NTP, showed 69% of identity with PV polymerase and about 50% with the calicivirus enzymes. The charged amino acids interacting with the template-primer or with the NTP substrate are conserved in 88% for PV RDRP and about 60% for calicivirus polymerases.

 



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  		FIG. 3.
Electron density maps around the FMDV 3D active site. A, stereoview of the final {sigma}A-weighted 2Fo - Fc Fourier map, contoured at 1.5 {sigma}, in the isolated 3D structure with the model placed inside (ball and sticks colored in atom type code). B, {sigma}A-weighted 2Fo - Fc map, shown at 1.0 {sigma}, in the FMDV 3D-RNA complex structure. A portion of the RNA oligonucleotide is shown in the picture in a stick representation in light green, the template strand, and dark green, the primer strand. Only two nucleotides of the template and three of the primer are shown for clarity. The Mg2+ ion, located close to acidic residues Asp238, Asp240, and Asp339, is shown as an orange ball. Water molecules are shown as red balls and labeled as w.

 
The helix {alpha}11 packs adjacent to the central {beta}-sheet near the catalytic site and forms the motif B, and the helix {alpha}12 that supports this central ({beta}8, {beta}11, {beta}12) sheet conforms the motif D. Asn307 in motif B is conserved in all picornavirus and also in hepatitis C virus RDRPs. It has been described that this residue, together with Asp245 in motif A would be important for rNTP selection (23, 24) (see "NTP Site" section).

Strands {beta}14 and {beta}15 are located at the junction between the palm and the thumb subdomains and form the structural motif E (Fig. 1A). In the structure of HIV-RT, the residues immediately following motif E act as a pivot point for the thumb subdomain movement upon template-primer binding (25). The comparison of the FMDV 3D in its free and RNA-bound forms shows that no domain movements are required in FMDV 3D to accommodate the template-primer RNA.

Interactions of the Complex 3D-RNA Template-Primer—The RNA decanucleotide used in the co-crystallization work was originally designed for the analysis of the kinetics and mechanism of nucleotide incorporation by the poliovirus 3D polymerase (9). This RNA sym/sub decanucleotide (sequence 5'-GCAUGGGCCC-3') is able to form a 6-base pair duplex flanked by two, 4-nucleotide 5' overhangs and can act as both RNA template and primer.

The final refined electron density (Fig. 3B) was clear for residues, from C2 to C9 of the template strand plus the complementary G16 to C20 stretch of the primer strand. Density for the last base pairs was too weak and these residues were not included in the final model. The 5' template overhang binds in a deep groove, with a moderately positive charge, which extends across the face of the fingers domain toward the active site cleft, making numerous interactions with residues on the NH2 terminus and fingers (Figs. 1C and 4). The nucleotide bases A3 and U4 pack against the solvent exposed hydrophobic residues Val181 and Phe162. The presence of a hydrophobic amino acid at these positions is a conserved feature of RDRPs in picornaviruses. In addition, Asp165 is hydrogen bonded to atom N6 of the adenine and the amino group of the Lys164 side chain contacts the uridine atom O4. The basic side chains Arg17, Lys20, and Arg128, mostly conserved in picornaviral RDRPs, and Thr115 are hydrogen bonded to the phosphodiester backbone of A3 and U4 driving the single-stranded RNA toward the active site cavity. In the unbound 3D structure this groove is filled with water molecules that mimic the oxygen atoms of the phosphodiester backbone of the template.



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  		FIG. 4.
Interactions between the FMDV 3D and its template-primer and NTP substrates. A schematic diagram of the template-primer-nucleotide chains, indicating the amino acid residues seen in contact with the RNA in the structure, yellow (template chain) and green (primer chain), and with the modeled ATP molecule (red). Residues making polar interactions with substrates are highlighted in red and those involved in hydrophobic contacts in black. The model of the ATP bound to the active site was obtained by comparison of the HIV-RT/dsDNA and {varphi}6/ssDNA models with the FMDV 3D-template-primer complex. The two putative Mg2+ ions are highlighted in blue. Polar interactions were considered when distances were shorter that 3.5 Å.

 
The structural superimpositions of the five conserved motifs in the palm subdomain of FMDV 3D with the equivalent residues of {varphi}6/ssDNA and HIV1-RT/dsDNA (22, 25) showed that the course of the bound template overhang in its entry to the active site is in good agreement between the three structures. However, there are some differences in the degree of bending of the phosphate backbones at the 5' end (Fig. 5, A and B).



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  		FIG. 5.
Ribbon diagrams showing the trajectory of the RNA template-primer in the FMDV 3D structure compared with other polymerases. The FMDV 3D polymerase is shown in a blue ribbon and the template-primer RNA in green. Comparisons were done by the structural superimpositions of the palm subdomain in the FMDV 3D with the corresponding palm residues (yellow) in: A, {varphi}6 RDRP plus the initiation replication complex (Protein Data Bank entry 1HI0 [PDB] ), where the ssDNA is shown in ribbon representation in brown and the NTP substrates in brown sticks, and B, HIV1-RT plus the covalently trapped catalytic complex (Protein Data Bank entry 1RTD [PDB] ), where the template-primer DNA is shown in red. The root mean square deviations of the superimpositions were 1.4 Å in {varphi}6/ssDNA and 1.5 Å in HIV1-RT/dsDNA for 116 and 55 C{alpha} atoms superimposed, respectively.

 
The short double-stranded stem seen in the structure forms a right-handed A-like helix with Watson-Crick base pairing from G5 to C9 on the template strand and from G16 to C20 on the primer strand. This dsRNA region stretches from the polymerase active site to the COOH-terminal end of the protein following a path that is slightly different to that taken by the template-primer duplex in the HIV1-RT/dsDNA complex (Fig. 5B). The different trajectories observed in both complexes might be explained by the different interactions with the protein residues in the fingers and thumb subdomains that are not structurally conserved in the different polymerases.

The template strand of the duplex in the FMDV 3D complex contacts mainly residues in the fingers, whereas the primer strand interacts with the thumb and with motifs C and E in the palm subdomain (Fig. 4). The sugar phosphate backbone of the template chain contacts the protein by four hydrogen bonds (with residues Arg193, His204, Asn218, and Ser301) and three hydrophobic contacts (with residues Leu108, Asp109, and Ile189). The primer backbone interacts with the protein through: (i) five salt bridges between the phosphate oxygen atoms of G16 to G19 and the basic residues, Lys387, Arg388, Arg416, and Lys423; (ii) two hydrogen bonds (with residues Asp338 and Glu442); and (iii) one hydrophobic contact between the ribose ring of G17 and Val427 (Fig. 4).

The nucleotide bases, in both template and primer strands, are also participating in interactions with the polymerase. Two Van der Waals contacts between Pro219 and Tyr336 with the base G7 and six hydrogen bonds involving residues Ser304, Tyr336, and Ser426 are observed (Fig. 4).

The NTP Site—The FMDV 3D-RNA structure was determined in the absence of an incoming nucleotide complementary to the template base U4. Despite this absence, U4 lies close to its theoretical binding site, predicted from the superimpositions of HIV-RT/dsDNA and {varphi}6/ssDNA models onto the FMDV 3D complex structure. These superimpositions have permitted the modeling of an incoming ATP, and the identification of residues in FMDV 3D that come into proximity of this substrate (Fig. 4). In this model, the triphosphate moiety is coordinated by Arg168, Lys172, Arg179, and two putative metal ions. These positively charged amino acids belong to structural motif F in the fingers subdomain, which is conserved in all RDRPs (19). The guanidinium group of Arg168 interacts with {beta}- and {gamma}-phosphates, Lys172 also contacts the {gamma}-phosphate and the guanidinium group of Arg179 is hydrogen bonded to the {alpha}-phosphate and makes stacking interactions with the base (Fig. 4B). The model of the ATP in the FMDV 3D-RNA complex suggests that amino acids Asp245 (motif A) and Asn307 (motif B) play a critical role in rNTP selection by direct hydrogen bonding its side chain to the ribose 2' hydroxyl group of the incoming nucleotide. In addition, Ser298 and Thr303 might contact the adenine base. Finally, the side chains of catalytic amino acids Asp338 and Asp339 are in the expected position for the in-line attack of the {alpha}-phosphate (25, 26).

Structural Comparison with Other RDRPs—The overall architecture of FMDV 3D is similar to that of the partial structure reported for the RDRP of poliovirus (PV) (Fig. 1B (23)), with which it shares 29% of sequence identity (Fig. 2), to the caliciviral polymerases (27, 28), sharing only 12% of sequence identity (Fig. 2), and to a lesser extend to the bacteriophage {varphi}6 (22) and to the Flaviviruses (hepatitis C virus and bovine viral diarrhea virus (29–33)) RDRPs, with less than 10% of sequence identity. However, significant differences are observed when comparing them in detail.

The main structural differences between RDRPs of FMDV and PV are in the NH2-terminal subdomain (Fig. 1B). In FMDV 3D, this region consists of two short {beta}-strands ({beta}1, {beta}2), and two {alpha}-helices ({alpha}1, {alpha}2), connected by two long loop segments, which form a bridge between fingers and thumb. The 3D polymerase NH2-terminal domain in PV appeared mostly disordered in the crystal structure (23) and residues 1 to 11 and 38 to 66 were not determined. Residues 12 to 37 formed a strand that contacted part of the thumb subdomain, and residues 67 to 97 formed a helix located beneath the fingers subdomain. Based in the PV structure reported, it was not possible to determine how the strand and helix were connected. The FMDV 3D structure and sequence comparisons (Fig. 2) of the different RDRPs suggest that the PV enzyme may also have an encircled active site. However, this can be achieved only by the repositioning the 12 to 37 region (Fig. 1B). The possible existence of an interaction between the fingertips and the thumb subdomain in PV was also suggested in a previous work (18, 24).

Biochemical studies in PV suggested that polymerase-polymerase interactions might be important for polymerase function (34) and the crystal structure of PV polymerase revealed two regions of extensive intermolecular interactions, named interfaces I and II (23). In addition, electron microscopy analyses showed that purified PV RDRP formed planar and tubular oligomeric arrays and that the structural integrity of these arrays correlated well with cooperative RNA binding and elongation (35). Isolated FMDV 3D and the 3D-RNA complex have crystallized in two different space groups: P41212, the isolated polymerase, and P3221, the 3D-RNA complex. The availability of two crystal forms revealed two different packing environments for the enzyme. Both forms exhibited an extensive network of interactions, where the reference molecule contacted with eight and nine symmetrically related molecules, within the tetragonal and trigonal crystal lattices, respectively. The largest interface of protein-protein interactions, which is conserved in the two crystal forms, is formed between residues from 451 to the COOH-terminal end of one molecule, which contacts with residues: (i) from 45 to 90 at the NH2 terminus; (ii) from 255 to 265 on the fingers; and (iii) from 320 to 327 on the palm of the adjacent molecule (Fig. 6, A and B). The buried surface areas of these interfaces are 1430 and 1383 A2 for the P41212 and P3221 crystals, respectively, and combines both, hydrophobic and polar interactions. The interacting surfaces on the front of the thumb (residues 451–470) in FMDV 3D are mostly coincident with the thumb residues that form part of the interface I in PV polymerase (residues 446–461 (23)). However, the counterpart is not coincident and no high order structures, like the head-to-tail fibers, described for PV are apparent from the FMDV crystal packing.



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  		FIG. 6.
Protein-protein interactions in FMDV 3D polymerase. A, ribbon representation of the largest interface of interactions in the P41212 crystal lattice, also conserved in P3221 crystals; B, close up of the interacting surfaces.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
The comparison of the three-dimensional structures of free FMDV 3D, and 3D complexed with a template-primer RNA has identified 27 amino acids, in direct contact with RNA residues (Fig. 4). Eleven of them are from the conserved structural motifs B, C, and E in the palm and F in the fingers, and the remaining 16 residues belong to different regions on the NH2 terminus, fingers and thumb subdomains. The amino acid sequence and the three-dimensional structure of the FMDV 3D reported here do not provide any evidence of a domain that could correspond to proofreading-repair activity, such as an Exo N domain encoded by some coronaviruses (36). Therefore, the limited fidelity of copying by 3D is determined by the substrate-template-protein interactions in the template-primer binding groove (Fig. 4).

Superimposition of the conserved structural motifs with the equivalent motifs in HIV-RT/dsDNA and {varphi}6/ssDNA RDRPs allowed the modeling of an incoming nucleotide substrate in the structure. This nucleotide likely interacts with residues of motifs B, C, and F (Fig. 4). Site-directed mutagenesis of residues around the critical amino acids involved in nucleotide recognition and positioning of the 3'-OH primer residue should help in defining the molecular basis of the error-prone replication properties of FMDV, as currently investigated for HIV-RT (37).

The structures of the bacteriophage {varphi}6 and hepatitis C virus RDRPs determined previously revealed the presence of an additional COOH-terminal domain that blocks part of the active site cleft and seems to stabilize short primers for the initiation of RNA synthesis (22, 31, 32, 38, 39). It appears that this COOH-terminal segment may play a role in initiating RNA replication, and large conformational rearrangements are predicted to occur during the process of elongation of the RNA product. This COOH-terminal extra domain is absent in the structure of the polymerase of FMDV allowing the accommodation of long RNA products without significant conformational rearrangements in the RNA binding groove. This comparison suggests a different mechanism of control of the initiation of RNA replication between these two groups of viruses. In Flavivirus and F6 polymerases the initiation of the replication follows a "de novo" synthesis mechanism (40, 41) copying the template from its 3' terminus. In picornaviral polymerases this initiation of replication is primed by the VPg peptide (42), so the groove should be accessible for larger primed-template molecules. In the absence of this VPg no RNA synthesis is detected during a viral infection (43).

The structure of FMDV 3D will be of value to interpret the anti-FMDV activity shown by some base and nucleotide analogues, in particular 5-fluorouracil (44–46) and ribavirin (1-{beta}-D-ribofuranosyl-1,2,3-triazole-3-carboxamide) (47, 48). 5-Fluorouracil, a pyrimidine analogue used in cancer chemotherapy (49, 50), is mutagenic for a number of RNA viruses, including FMDV (44). 5-Fluorouracil alone and in combination with antiviral inhibitors can drive FMDV to extinction through enhanced mutagenesis (virus entry into error catastrophe (45)). Ribavirin is mutagenic for a number of viral RNA polymerases (51, 52) and is active in eliminating FMDV from persistently infected cells via enhanced mutagenesis (47, 48). A case for pharmacological intervention can be made for the treatment of ruminants persistently infected with FMDV, which represent a reservoir of FMDV variants, and a potential source of acute outbreaks of disease (53). Ribavirin or related analogues that can penetrate epithelial cells of the oropharynx are candidates to cure animals from persistent FMDV, opening the possibility of solving one of the most severe problems for the control of FMD (5, 53). Work is now in progress to elucidate the structure of 3D complexed with nucleotide substrates and substrate analogues. The structural information on 3D and its complexes with template-primers and substrates or inhibitors will be instrumental to understand the molecular basis of low fidelity copying, entry into error catastrophe, and for the possibility to design new specific mutagenic and antiviral agents targeted to this important animal pathogen.


   FOOTNOTES
 
The atomic coordinates and structure factors (codes 1U09 [PDB] and 1WNE [PDB] ) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).

* This work was supported in part by Grants BMC2001-1823-C02-01 of Comisión Interministerial de Ciencia y Tecnología, Comunidad Autónoma de Madrid 08.2/0015/2001 and Fundación R. Areces (to E. D.) and Grant BIO2002-00517 of the Comisión Interministerial de Ciencia y Tecnología (to N. V.). Data were collected at the EMBL protein crystallography beam lines ID13 and ID29 at ESRF (Grenoble) within a Block Allocation Group (BAG Barcelona) with financial support provided by the ESRF. 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

§ Supported by a fellowship from the Ministerio de Ciencia y Tecnología. Back

|| Supported by a fellowship from the Comunidad Autónoma de Madrid. Back

** To whom correspondence should be addressed. Tel.: 34-93-403-49-52; Fax: 34-93-403-49-79; E-mail: nvmcri{at}ibmb.csic.es.

1 The abbreviations used are: FMDV, foot-and-mouth disease virus; RDRP, RNA-dependent RNA polymerase; PV, poliovirus; ds, double-stranded; ss, single-stranded; MOPS, 4-morpholinepropanesulfonic acid. Back

2 García-Arriaza, J., Manrubia, S. C., Toja, M., Domingo, E., and Escarmís, C. (2004) J. Virol. 78, in press. Back


   ACKNOWLEDGMENTS
 
We are indebted to E. Brocchi for generous supply of FMDV 3D-specific monoclonal antibodies and I. Fita for critically reading the manuscript.



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 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
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