Characterization of the Nucleoside Triphosphatase Activity of Poliovirus Protein 2C Reveals a Mechanism by Which Guanidine Inhibits Poliovirus Replication*

Thomas PfisterDagger and Eckard Wimmer§

From the Department of Molecular Genetics and Microbiology, State University of New York at Stony Brook, Stony Brook, New York 11794-5222

    ABSTRACT
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Abstract
Introduction
References

The highly conserved non-structural protein 2C of picornaviruses is involved in viral genome replication and encapsidation and in the rearrangement of intracellular structures. 2C binds RNA, has nucleoside triphosphatase activity, and shares three motifs with superfamily III helicases. Motifs "A" and "B" are involved in nucleotide triphosphate (NTP) binding and hydrolysis, whereas a function for motif "C" has not yet been demonstrated. Poliovirus RNA replication is inhibited by millimolar concentrations of guanidine hydrochloride (GdnHCl). Resistance and dependence to GdnHCl map to 2C. To characterize the nucleoside triphosphatase activity of 2C, we purified poliovirus recombinant 2C fused to glutathione S-transferase (GST-2C) from Escherichia coli. GST-2C hydrolyzed ATP with a Km of 0.7 mM. Other NTPs, including GTP, competed with ATP for binding to 2C but were poor substrates for hydrolysis. Mutation of conserved residues in motif A and B abolished ATPase activity, as did mutation of the conserved asparagine residue in motif C, an observation indicating the involvement of this motif in ATP hydrolysis. GdnHCl at millimolar concentrations inhibited ATP hydrolysis. Mutations in 2C that confer poliovirus resistant to or dependent on GdnHCl increased the tolerance to GdnHCl up to 100-fold.

    INTRODUCTION
Top
Abstract
Introduction
References

Poliovirus is the prototypical member of the genus Enterovirus which is one of six genera of Picornaviridae, a family of small, icosahedral, positive strand RNA viruses. The genome is typically 7.5 kilobases in length and codes for one polyprotein that is co- and post-translationally processed to give rise to functional proteins. During processing, precursor polyproteins arise that have functions which are distinct from their final cleavage products. In addition, many proteins have been shown to carry out several functions. These features allow picornaviruses to make maximal use of the genetic information stored in their small RNA genome but they make it difficult to address the roles and functions of a particular protein.

A key event in the picornavirus life cycle is the replication of the RNA genome. This process requires all non-structural proteins (1) and is confined within cytoplasmic replication complexes (RCs)1 (2, 3). RCs isolated from poliovirus-infected cells are active in RNA replication in vitro (4). Membranes appear to be crucial constituents of the RCs since detergents inhibit several steps of RNA replication (5-7). It appears that many viral, non-structural proteins are membrane associated and are abundant constituents of the RC (4, 8-10). Among them, 2BC has been shown to induce membrane proliferation from cytoplasmic membranes (11-13) leading to an enormous mass of virus-induced vesicles to which the RCs are attached (14-16). 2BC and its cleavage product 2B, but not 2C, block protein transport from the endoplasmic reticulum to the Golgi apparatus and increase the permeability of the cell membrane (17-19). 2C, a protein of 38 kDa and 329 amino acid residues in length, is also an abundant constituent of the RC, and it contains determinants for membrane association and vesicle induction (13, 20) (Fig. 1). Expression of a fragment encompassing residues 1-274 is sufficient to induce the formation of vesicles morphologically similar to those induced by 2BC and those observed in poliovirus-infected cells (21).


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Fig. 1.   Functional domains of poliovirus protein 2C. RNA-binding (22), membrane associating (20), amphipathic (40), and cysteine-rich (79) regions are indicated. The black triangles indicate the location of motifs A and B of the NTP binding motif (26). Motif C, the hallmark of superfamily III helicases (28), is represented by the white triangle. Conserved amino acid residues are depicted in capital letters preceded by the position number of the first residue of the sequence. The sequence of motif B (underlined) has been extended to include the residues that are changed in response to selection at <= 2 mM GdnHCl to class N (*) and class M (**) mutants resistant or dependent on the drug (35-37).

2C has RNA binding properties. UV irradiation of membrane fractions containing RCs revealed that 2C and 2BC can be cross-linked to RNA (16). Northwestern blotting with 2C fused to maltose-binding protein (MBP-2C) that was purified from recombinant Escherichia coli, identified amino- and carboxyl-terminal sequences required for RNA binding (22). Recently, it has been reported that 2C binds to the 3' end of negative strand RNA (23), an intermediate during RNA replication (24).

Although protein 2C is highly conserved among picornaviruses, little sequence similarity between 2C and non-picornaviral proteins exists. Similarities are restricted to three small motifs, called "A," "B," and "C" (Fig. 1). Motifs A and B belong to the well known "Walker" NTP binding motifs that appear in a variety of ATP- and GTP-binding proteins with a broad range of functions (25-27). Motif C has been found in members of the helicase superfamily III, the most prominent members being the DNA helicases SV40 large T antigen and papillomavirus protein E1 (28). Motif C consists of an invariant Asn residue preceded by a stretch of moderately hydrophobic residues and is located downstream at a distinct distance from motif B (28). The function of motif C is not known. ATPase (29, 30) and GTPase (30) activities of protein 2C have been reported, whereas helicase activity was not detectable (30).

Besides the co-localization of protein 2C with the RC and its RNA binding activities, genetic evidence has implicated 2C in RNA replication. Millimolar concentrations of guanidine hydrochloride (GdnHCl) efficiently inhibit poliovirus replication at the level of RNA synthesis (31, 32). In an in vitro system that allows de novo synthesis of poliovirus (33), GdnHCl was reported to prevent the initiation of minus strand RNA synthesis (34). Guanidine-resistant (gr) and -dependent (gd) mutants of poliovirus carry mutations in protein 2C indicating that GdnHCl inhibits RNA replication through an adverse effect on 2C (35-37). Because the mutations found in gr and gd mutants were located in the vicinity of motifs A, B, and C of the NTP-binding motif, GdnHCl was suspected to interfere with the NTP binding and/or hydrolysis activity (37). However, 2 mM GdnHCl did not appear to inhibit the NTPase activity of MBP-2C (30) or baculovirus-expressed 2C (29).

Mutations directed to the coding region of 2C affected RNA replication of the mutant viruses. Mutation of conserved residues in motifs A and B resulted in quasi-infectious and lethal phenotypes (38, 39). Similar phenotypes were obtained when mutations were introduced within a predicted amphipathic helical region near the amino terminus (40). Insertion of additional amino acid residues at positions 255 or 263, downstream of motif C yielded temperature-sensitive mutants that appeared to be defective in RNA replication at the non-permissive temperature (41). This study lead to the discovery of a revertant virus, carrying two additional mutations in 2C and exhibiting an uncoating defect (42). Recently, mutations in 2C were discovered that resulted in a virus resistant to 5-(3,4-dichlorophenyl)methylhydantoin, an antiviral drug that interferes with RNA encapsidation (43). Taken together, these findings indicate a role for 2C in RNA replication and in subsequent RNA encapsidation.

The multiple roles of 2C in virus replication are complex as they may depend on specific interactions not only with cellular membranes, but also with viral RNA, and with viral and cellular proteins. Oligomerization of 2C has been suggested based on complementation experiments with guanidine mutants (37). A physical interaction of 2C with 2C, 2B, and 2BC has been demonstrated in the yeast two-hybrid system and in a GST pull-down assay (44). Protein 2C has also been shown to be part of a detergent-resistant complex containing 2B and 2BC, as well as 3AB (the precursor of the genome-linked protein VPg) and capsid proteins (9). Considering the simultaneous membrane and RNA binding activities, 2C and/or 2BC may contribute to the structural integrity of the RC (6, 16). This model has been substantiated by the observation that the association of the RC with the virus-induced membranes was weakened in the presence of GdnHCl in vivo (16). However, the ability of 2C to associate with membranes was not affected by GdnHCl in the absence of other viral proteins (20).

Despite the wealth of the observations described above, little is known about the biochemical mechanisms by which protein 2C engages in virus replication. In particular, the NTPase activity has been only poorly characterized biochemically and its role in 2C functioning is not understood. In this study, the biochemical characterization of the NTPase activity of protein 2C has been addressed to better understand the functioning of this protein. We have purified soluble recombinant 2C fused to GST from E. coli lysate and characterized the NTPase activity of the protein. GST-2C hydrolyzed ATP to ADP and inorganic phosphate (Pi) with Michaelis-Menten kinetics. Other NTPs including GTP, were very poor substrates but competed with ATP at the NTP-binding site. We found that the ATPase activity required all three motifs A, B, and C. Moreover, the ATPase activity was found to be a target of the anti-poliovirus drug GdnHCl. The latter observation provides for the first time an explanation for the inhibitory activity of low concentrations (<= 2 mM) of GdnHCl on poliovirus replication and that of many other picornaviruses by inference.

    EXPERIMENTAL PROCEDURES

Construction of Plasmids for the Expression of GST Fusion Proteins-- The coding sequence of poliovirus type 1 (Mahoney) 2C was amplified from plasmid pT7PVM (45) by polymerase chain reaction using Pfu polymerase (Stratagene, La Jolla, CA). The sequence of the 5' oligonucleotide was 5'-GGAATTCTAGAAGCGCTGTTCCAAGGTGACAGTTGGTTG-3', and that of the 3' oligo was 5'-GCGCAAGCTTACTATTGAAACAAAGCCTCCATAC-3'. The latter contained two stop codons that resulted in an authentic COOH terminus of the encoded protein. The polymerase chain reaction product was digested with the restriction enzymes EcoRI and HinDIII and ligated into the corresponding sites of plasmid pGEX-KG (46). The plasmid was named pGEX-2C and propagated in E. coli DH5alpha . The 2C coding sequence was confirmed by sequencing (Sequenase, U. S. Biochemical Corp., Cleveland, OH).

Mutants pGEX-2C mA3 and mB4 were constructed by replacing the XhoI-MluI fragment in pGEX-2C with the XhoI-MluI fragment of pT7XL2C-M3 and pT7XL2C-M4, respectively (39). Mutants pGEX-2C mC1, mC2, and R111 were constructed by the mega-primer method (47). The sequences of the mutant oligonucleotides were 5'-GCATCCACAGACTCAAGCAG-3' for mC1, 5'-GCATCCACAGGCTCAAGCAG-3' for mC2, and 5'-GGTGCGGACCTCAAGCTGTTCTG-3' for R111. Mutant pGEX-2C GR1 was constructed by replacing the XhoI-MluI fragment in pGEX-2C with the XhoI-MluI fragment of pT7PVMgr (48). Mutant pGEX-2C GD1 was generated by the mega-primer method using the mutant primer 5'-CTCAAGCAGAATGTCCCCCC-3' with pGEX-2C GR1 as a template. All sequences derived from polymerase chain reaction were verified by sequencing. Table I lists the plasmids for the expression of wild-type (wt) and mutant GST-2C used in this study.

Expression and Purification of GST-2C-- Plasmid pGEX-2C and its derivatives were transformed into E. coli strain BL21 (DE3). A single colony was picked and grown in 250 ml of 2 × YT media (49) containing 100 mg/liter ampicillin at 37 °C until OD600 was between 0.6 and 0.7. The culture was cooled to 20 °C and isopropyl-1-thio-beta -D-galactopyranoside and ampicillin were added to final concentrations of 0.1 mM and 100 mg/liter, respectively. The culture was kept shaking at 20 °C for 7-8 h. The pelleted cells were stored at -80 °C. After thawing at room temperature, the cells were resuspended in lysis buffer (20 mM HEPES/KOH, pH 7.5, 140 mM NaCl, 10 mM 2-mercaptoethanol, 5 µg/ml leupeptin, and 2 µg/ml pepstatin A (Boehringer-Mannheim)). Cells were lysed by two cycles in a French Press at a pressure of 15,000 psi for 40 s each. The lysate was solubilized in the presence of 1% Triton X-100 (Sigma) at 4 °C for 30 min and subsequently clarified by centrifugation at 12,000 × g at 4 °C for 10 min. GST-2C was batch-immobilized on 125 µl (bed volume) of glutathione-Sepharose (Pharmacia Biotech, Piscataway, NJ) equilibrated in wash buffer I (20 mM HEPES/KOH, pH 7.5, 140 mM NaCl, 5 mM 2-mercaptoethanol, 0.05% Triton X-100) at 4 °C for 45 min. The Sepharose beads were washed three times with 12.5 ml of wash buffer I each and once with 12.5 ml of wash buffer II (wash buffer I containing 10 mM NaCl). GST-2C was eluted by incubating the Sepharose in 125 µl of elution buffer (50 mM HEPES/KOH, pH 8.2, 10 mM reduced glutathione (Sigma), 20 mM 2-mercaptoethanol, 0.05% Triton X-100) at 4 °C on a rotating device for 10 min. Elution was repeated three times: once for 10 min and twice for 1 h. Glycerol was added to the pooled eluates at a final concentration of 50%. Such GST-2C preparations were used in the experiments described below. Purification was monitored by SDS-PAGE. Total protein concentration was measured using the Bio-Rad Protein Assay (Bio-Rad). Relative amounts of proteins were measured by laser scanning densitometry (Ultrascan XL, LKB, Bromma, Sweden) of a Coomassie-stained SDS-PAGE gel. GST-2C was identified by Western blot analyses using monoclonal antibodies against GST or 2C. Anti-2C antibodies were produced in our laboratory.2 Anti-GST is commercially available from Pharmacia Biotech.

Detection of ATP Hydrolysis by Thin Layer Chromatography (TLC)-- ATPase reaction mixtures of 20 µl contained 20 mM HEPES/KOH, pH 6.8, 2 mM magnesium acetate, 5 mM dithiothreitol, 0.1 mM cold ATP (Pharmacia Biotech), 10 µM [alpha -33P]ATP (NEN Life Science Products Inc., Boston, MA), and 0.67 µg of protein. Reactions were performed at 37 °C for 10 min and stopped on ice by the addition of 1 µl of 0.1 M EDTA. 2 µl of the reaction mixture were applied on polyethyleneimine cellulose-coated TLC plastic sheets (EM Separations, Gibbstown, NJ). The chromatogram was developed in 0.75 M NaH2PO4, dried under an infrared lamp, and exposed to Biomax MR radiography film (Kodak, Rochester, NY).

NTPase Reactions and Colorimetric Phosphate Detection Assay-- NTPase reaction mixtures of 60 µl contained 20 mM HEPES/KOH, pH 6.8, and 5 mM dithiothreitol. The concentration of NTP, magnesium acetate, and protein as well as the reaction time and temperature varied among different experiments (cf. "Results" and figure legends). Reactions were stopped on ice and by the addition of 60 µl of ice-cold 16% trichloroacetic acid. The samples were left on ice for 30 min and subsequently centrifuged at top speed in a microcentrifuge at 4 °C. This trichloroacetic acid precipitation step removed protein that otherwise may have precipitated in the subsequent color reaction rendering optical density measurements inaccurate. Inorganic phosphate (Pi) released during the NTPase reaction was measured by a colorimetric assay as described previously (50). 100 µl of the supernatant from the trichloroacetic acid-precipitation step was added to 100 µl of colorimetric reagent (3 volumes of 0.8% ammonium molybdate, 1 volume of 6 N sulfuric acid, 1 volume of 10% (w/v) ascorbic acid) in a microtiter plate on ice. The color reaction was incubated at 37 °C for 30-40 min and subsequently measured in a microplate reader (Dynatech Laboratories, Chantilly, VA) at a wavelength of 630 nm. The measurements were blanked with NTPase reactions that were run in parallel and contained identical amounts of ingredients except that protein was replaced by an equal volume of 50% elution buffer in glycerol. Each microtiter plate included a dilution series of K2HPO4 that allowed quantitation of the amount of phosphate released in the NTPase reactions.

    RESULTS

Purification of GST-2C-- Poliovirus 2C was expressed in E. coli as a fusion polypeptide containing GST at the amino-terminal (46). Expression and purification of GST-2C was monitored on a Coomassie-stained SDS-PAGE gel (Fig. 2A). GST-2C was expressed to high yields and was reasonably soluble in the presence of 1% Triton X-100. GST-2C was batch purified on glutathione-Sepharose beads. The eluate contained two discrete bands that migrated with apparent molecular masses of 64 and 28 kDa presumably representing GST-2C and GST, respectively. Besides these strong bands, a few weak bands were visible as well. Western blot analysis revealed that the majority of protein bands that could be stained with India ink were recognized by anti-2C or anti-GST monoclonal antibodies (Fig. 2B). The additional proteins present in the GST-2C preparation are therefore related to GST-2C, most likely resulting from proteolytic degradation of GST-2C or premature termination of translation. These phenomena have been observed earlier when 2C was expressed in E. coli (51) or insect cells (29). A fast migrating band could not be stained with either antibody. The identity of this protein is unknown but could be a truncation of GST-2C that is devoid of the epitopes recognized by the antibodies. Laser scanning densitometry of Coomassie-stained SDS-PAGE gels showed that typically 55% of the total protein present in the eluate was full-length GST-2C (not shown). The GST band represented approximately 25% of total protein content. Our data do not exclude the presence of bacterial proteins in the GST-2C preparation, although all evidence suggests that the amount of such contaminants would be small.


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Fig. 2.   Purification of GST-2C. A, Coomassie-stained SDS-PAGE gel. Lane 1, marker. Molecular masses in kDa are given to the left. Lane 2, total lysate, before induction with isopropyl-1-thio-beta -D-galactopyranoside. Lane 3, total lysate, after induction. Lane 4, pellet, insoluble fraction. Lane 5, supernatant, soluble fraction. Lane 6, soluble fraction after adsorbtion to glutathione-Sepharose, unbound proteins. Lane 7, eluate. Lanes 2-6 correspond each to 100 µl of bacterial culture volume. Lane 7 correspond to 1-ml culture volume. B, Western blot analysis of eluate. Eluate was separated in a wide slot on an SDS-PAGE gel. After electrotransfer to nitrocellulose, the nitrocellulose was cut in stripes and stained individually. Lanes 1-3, immunostained with anti-GST (lane 1), anti-2C (clone 3.20; lane 2), anti-2C (clone 91.10; lane 3). Lane 4, stained with India ink.

GST-2C Has ATPase Activity-- Incubation of GST-2C with ATP at 37 °C in the presence of buffer and magnesium acetate led to an increase in the concentration of inorganic phosphate (Pi). This result indicated that GST-2C had ATPase activity. The release of phosphate was dependent on magnesium ions (Fig. 3A). A concentration of 2 mM magnesium acetate appeared to be optimal for ATP hydrolysis at an ATP concentration of 1 mM. ATP hydrolysis was also observed in the presence of manganese acetate, although less phosphate was released than in the presence of magnesium acetate (Fig. 3A). Zn2+ and Ca2+ did not serve as cofactors for the hydrolysis of ATP (not shown). The optimal pH for ATP hydrolysis was found to be 6.8 (Fig. 3B). NaCl, even at low concentrations, inhibited the hydrolysis of ATP (Fig. 3C). These initial experiments showed that the GST-2C preparation contained an ATPase activity that was strongest in the presence of magnesium as a cofactor, in the absence of salt, and at pH 6.8. 


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Fig. 3.   ATPase activity of GST-2C. All reactions contained 1 mM ATP, 2 µg of protein, 5 mM dithiothreitol, 20 mM Hepes/KOH and were performed in a volume of 60 µl, at 37 °C for 1 h. The release of inorganic phosphate was measured by a colorimetric assay (see "Experimental Procedures"). A, variable concentrations of either magnesium acetate (open circle ) or mangesese acetate (triangle ) assayed at pH 6.8. B, variable pH at a constant concentration of magnesium acetate of 2 mM. C, increasing concentrations of NaCl in the presence of 2 mM magnesium acetate, pH 6.8.

It has been shown that poliovirus 2C catalyzes the reaction of ATP right-arrow ADP + Pi (29, 30). The colorimetric assay used does not distinguish between Pi and pyrophosphate (PPi). Therefore, we used alpha -labeled [33P]ATP as substrate and separated the products by TLC. As can be seen in Fig. 4, lane 4, the only product of GST-2C mediated ATP hydrolysis was [alpha -33P]ADP indicating that the unlabeled gamma -phosphate has been cleaved off. [alpha -33P]ADP was not observed in a reaction containing no protein (lane 2) or GST that has been expressed and purified the same way as GST-2C (lane 3). Therefore, the co-purification of an E. coli ATPase is highly unlikely. This is strongly supported by the observation that mutations in the three conserved motifs A (lane 5), B (lane 6), and C (lanes 7 and 8) of 2C (for the precise definition of mutations see Table I) abolished ATP hydrolysis nearly completely. The same mutations also prevented the release of Pi as assessed by the colorimetric phosphate detection assay (Table II). Therefore, all three conserved motifs in 2C are involved in either binding of the NTP or its hydrolysis. These data provide the first evidence that motif C, the hallmark of superfamily III helicases (28), is involved in ATP hydrolysis and thus part of the NTP binding/hydrolysis motif.


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Fig. 4.   ATPase activity of wt and mutant GST-2C (Table I). Thin layer chromatogram of ATPase reactions containing [alpha -33P]ATP. A reaction containing alkaline phosphatase was included (lane 1) and served as a migration marker for ADP, AMP, and inorganic phosphate (Pi). It appeared that AMP and Pi co-migrated. Lane 2, without protein; lane 3, GST; lane 4, GST-2C; lane 5, GST-2C mA3; lane 6, GST-2C mB4; lane 7, GST-2C mC1; lane 8, GST-2C mC2.

                              
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Table I
Genetic variants of poliovirus protein 2C used in this study

                              
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Table II
ATPase activity of GST-2C mutants

Hydrolysis of ATP by GST-2C follows Michaelis-Menten Kinetics-- In order to set up the conditions for linear ATPase reactions, we first determined the saturating substrate concentration. ATP concentrations of 3 mM and higher appeared to be saturating since no further increase of product, i.e. Pi, was observed above 3 mM ATP (Fig. 5A). It was important to keep the concentration of magnesium acetate 1 mM above the concentration of ATP. Other concentrations of magnesium acetate resulted in a decrease of ATPase activity (not shown). GST-2C appeared to be most active at a temperature of 37 °C (Fig. 5B). At 37 °C and saturating substrate concentration, the rate of ATP hydrolysis by 1 µg of GST-2C was constant for at least 30 min (Fig. 5C). At these reaction conditions, the amount of enzyme could be varied from 0.5 to 1.5 µg/reaction resulting in a nearly linear response of the amount of product (Fig. 5D). Linear reaction conditions were thus achieved in a volume of 60 µl containing 1 µg of GST-2C, 3 mM ATP, 4 mM magnesium acetate, at pH 6.8, at a reaction temperature of 37 °C and for 30-min reaction times. Finally, the relationship between substrate concentration and the velocity of the reaction was displayed on a Lineweaver-Burk plot (Fig. 5E). Velocity was expressed as nanomoles of Pi produced per nanomole of GST-2C per second. Using a best-fit program (Cricket Graph, Cricket Software, Malvern, PA), the equation of the curve that describes the relationship between substrate concentration and velocity was determined (Fig. 5E). The equation allowed the calculation of the Michaelis-Menten concentration (Km) and the maximal velocity (Vmax) of the reaction. Km and Vmax appeared to be 0.7 mM and 0.9 s-1, respectively.


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Fig. 5.   Michaelis-Menten kinetics of ATP hydrolysis of wt GST-2C. Reactions of 60 µl contained 20 mM Hepes/KOH, pH 6.8, and 5 mM dithiothreitol. If not stated otherwise, reactions were carried out with 1 µg of protein at 37 °C for 30 min. A, variable concentrations of ATP and magnesium acetate, the latter was always 1 mM higher than the former. The average (solid symbols) of two experiments (open symbols) is displayed. B, reactions contained 3 mM ATP and 4 mM magnesium acetate and were performed at various temperatures. The average of three reactions are shown. Error bars indicate standard deviations. C, reactions as in B at 37 °C. The reactions were stopped at various time points. D, reactions as in B at 37 °C. The reaction contained various amounts of protein. The average (solid symbols) of two experiments (open symbols) is displayed. E, Lineweaver-Burk blot of an experiment similar to that in A. The correlation coefficient of the best-fit curve is 0.986. Km, Michaelis-Menten concentration; Vmax, maximal velocity.

ATP Is the Preferred Substrate for GST-2C-- Many NTPases can use more than one kind of nucleotide as substrate. NTPase reactions with GST-2C were carried out in the presence of either one of the common ribo- and deoxyribo-NTPs (Table III). The velocity of NTP hydrolysis was highest for ATP. Only a negligible amount of phosphate was released from GTP, dATP, and dGTP. Surprisingly, we reproducibly observed that dTTP was slowly hydrolyzed whereas CTP, UTP, and dCTP did not serve as substrate whatsoever.

                              
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Table III
Substrates of GST-2C

In order to find out whether non-ATP rNTPs have an effect on ATPase activity, ATPase assays were performed in the presence of rNTPs and the non-hydrolyzable analogs ATPgamma S and GTPgamma S (Table IV). Since the ATPase assay was done at saturating concentration of ATP, addition of ATP changed the amount of Pi released insignificantly. GTP, CTP, and UTP inhibited ATP hydrolysis. GTPgamma S and GTP inhibited the ATPase activity to a similar degree exemplifying again the poor ability of GST-2C to hydrolyze GTP. Inhibition was strongest in the presence of ATPgamma S, which presumably indicates that ATPgamma S competes with ATP for binding to the NTP-binding site of 2C.

                              
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Table IV
Inhibitory effect of rNTPs on ATP hydrolysis by GST-2C

Inhibition of ATP hydrolysis by ATPgamma S and GTPgamma S was compared using Lineweaver-Burk and Dixon plots (Fig. 6). On a Lineweaver-Burk plot, Vmax appeared to be unchanged in the presence or absence of ATPgamma S, whereas the slope of the curve increased in the presence of inhibitor (Fig. 6A). These effects are indicative for competitive inhibition as is expected for ATPgamma S. Inhibition of ATP hydrolysis by GTPgamma S was also competitive (Fig. 6B), indicating that GTPgamma S binds to the ATP-binding site of 2C. A set of reactions in which the substrate concentration was kept constant and the inhibitor concentration was varied, was displayed on Dixon plots, allowing easy determination of the inhibitor-specific constant Ki. The value for Ki was found to be 0.13 mM for ATPgamma S (Fig. 6C) and 0.25 mM for GTPgamma S (Fig. 6D). Since Ki represents the dissociation constant of the inhibitor-enzyme complex, the results suggest that ATPgamma S binds 2-fold stronger than GTPgamma S to protein 2C. Whereas the Ki of NTPs may differ from the Ki of the non-hydrolyzable analogs, the relative binding affinity may be similar. Therefore, it is feasible to assume that the affinity of ATP to 2C is about 2-fold higher than the affinity of GTP. The factor of two in binding seems to be in contrast with the factor of approximately 70 in velocity of hydrolysis (Table III). Therefore, substrate specificity may be achieved only marginally by NTP binding but rather by NTP hydrolysis.


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Fig. 6.   Inhibition of ATP hydrolysis by the non-hydrolyzable NTPs ATPgamma S (A and C) and GTPgamma S (B and D). A and B, Lineweaver-Burk plots of reactions containing 0.1 mM (triangle ) or 0.2 mM () inhibitor and variable concentrations of substrate (ATP). Reaction conditions were as for Fig. 5E. The curve of Fig. 5E (open circle ) is superimposed for comparison with an uninhibited reaction. C and D, Dixon plots of reactions containing 1 mM () or 2 mM (black-triangle) of substrate and variable concentrations of inhibitor. The dashed line marks the position of (-Ki) on the x axis. Each data point represents the average of duplicate reactions.

Guanidine HCl Inhibits ATPase Activity of GST-2C-- The gr and gd mutants of poliovirus have been shown to map to protein 2C (35-37). Therefore, we tested whether GdnHCl has an effect on ATP hydrolysis. Increasing concentrations of GdnHCl in the ATPase reactions resulted in a decrease in the amount of Pi released (Fig. 7A). At a concentration of 2 mM at which poliovirus RNA replication is completely inhibited, ATPase activity of GST-2C is abolished. In order to find out whether the ATPase activities of 2C of guanidine-resistant and -dependent mutants are tolerant to the drug, we expressed and purified the 2C mutants GST-2C GR1, GD1, and R111 (Table I) and tested their ATPase activity at different concentrations of GdnHCl. GR1 had one amino acid replaced at position 179 from Asn to Gly (N179G), a variation that renders poliovirus resistant to 2 mM GdnHCl (35, 36). This mutation increased the tolerance of the ATPase activity to GdnHCl approximately 100-fold (Fig. 7A). GD1 has not only the N179G mutation but in addition a I227M mutation that, combined, confers a gd phenotype in vivo. GST-2C GD1 exhibited a tolerance to GdnHCl similar to that of GR1. R111 was also found in a gd poliovirus mutant; it contained a single mutation at position 187 (Met to Leu) (52). GST-2C R111 was 10-fold more tolerant to GdnHCl than wt GST-2C. These results show for the first time that the ATPase activity of poliovirus 2C is a target for the inhibitory effect of low concentrations of GdnHCl. In addition, the gr and gd phenotypes of poliovirus mutants correlate with an increased tolerance to GdnHCl of the ATPase function of protein 2C. The ATPase activity of GST-2C GR1 and GD1 appeared to be stimulated by up to 20% in the presence of GdnHCl (Fig. 7A). Stimulation of virus production of a gr mutant by GdnHCl in vitro has been reported in earlier studies (33, 48). Whether a correlation between the two observations exists is beyond current knowledge. Surprisingly, the ATPase activities of the gd mutants GD1 and R111 were not dependent on GdnHCl. In fact, the rates of ATP hydrolysis of the gr and gd mutants in the absence of GdnHCl were indistinguishable from the rate of ATP hydrolysis of wt GST-2C (not shown).


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Fig. 7.   Inhibition of the ATPase activity by GdnHCl. A, amount of released phosphate by wt GST-2C (), GST-2C R111 (black-triangle), GST-2C GR1 (black-square), and GST-2C GD1 () at various GdnHCl concentrations. Reactions were performed as for Fig. 3C (in the absence of NaCl). The amount of released phosphate in the presence of GdnHCl is expressed as percentage of the amount of released phosphate in the absence of GdnHCl. The characteristics of the mutants are summarized in Table I. B, Lineweaver-Burk plot of ATPase reactions containing wt GST-2C and 0.05 mM (triangle ) or 0.085 mM () GdnHCl. Reaction conditions were as for Fig. 5E. The curve of Fig. 5E (open circle ) is superimposed for comparison with an uninhibited reaction. Each data point represents the average of duplicate reactions.

The inhibitory effect of GdnHCl on the ATPase activity of GST-2C was further investigated by using variable substrate concentrations at a fixed inhibitor concentration. The data were plotted on a Lineweaver-Burk diagram (Fig. 7B). The presence of GdnHCl in the reactions resulted in a lower Vmax and in slightly higher values for the slopes. These parameters are typical for non-competitive inhibitors. Thus, a feasible model for the inhibition of ATPase activity by GdnHCl would be that GdnHCl induces conformational changes in 2C that affect substrate binding and/or hydrolysis. These conformational changes may, theoretically, also affect other functions of protein 2C.

    DISCUSSION

This study was aimed at the biochemical characterization of the NTPase activity of protein 2C, a highly conserved protein among picornaviruses (1). Recombinant 2C has been purified from different sources in several laboratories. However, low expression levels, cytotoxicity, and poor solubility of 2C hampered detailed biochemical studies. Recently, efficient expression in E. coli and subsequent purification under denaturing conditions yielded a pure preparation of 2C that, following renaturation, specifically bound to the 3' end of minus strand poliovirus RNA (23). No biological function other than RNA binding, however, was reported. Particularly, it was not tested whether the renatured protein 2C possessed NTPase activity (23). Indeed, in earlier experiments we failed to regenerate ATPase activity from a "renatured" preparation of hexahistidine-tagged protein 2C expressed in E. coli.3 We took advantage of the GST expression system as the fusion of GST to a polypeptide promotes protein solubility and allows one-step purification (46). In different studies, purified GST-2C was used for the immunization of rabbits (20) and in protein-protein binding studies (44, 53). GST-2C, but not GST, hydrolyzed ATP to ADP and Pi in the presence of magnesium ions and at neutral pH. The ATPase activity followed Michaelis-Menten kinetics with a Km of approximately 0.7 mM. The Km of 2C-mediated ATP hydrolysis is thus slightly higher than the Km values of SV40 large T and papillomavirus E1 protein which were reported to be 0.07 mM (54) and 0.23 mM (55), respectively. We also expressed and purified GST-2BC, a fusion protein of GST with 2BC, the precursor of 2C (not shown). Expression of GST-2BC was less efficient compared with GST-2C, most likely due to the toxic nature of the 2B moiety (17). Nevertheless, GST-2BC hydrolyzed ATP with an efficiency comparable to that of GST-2C.4

Motifs A and B belong to the purine NTP-binding motif originally described by Walker and colleagues (27). This motif appears in a variety of NTP-binding and NTP-hydrolyzing proteins. Mutational changes of conserved residues within motifs A and B of poliovirus 2C have been shown to severely impair virus viability (Table I) (38, 39). Replacement of the conserved Lys residue with Gln in motif A abolished ATPase activity of purified 2C (29). GST-2C mA3, carrying the K135Q mutation (29), was used in this study. Confirming previous results, GST-2C mA3 was devoid of ATPase activity (Fig. 4). We have extended the analysis of purified mutant protein 2C to motifs B and C. Replacement of Asp177 to Leu in B (GST-2C mB4) resulted in a loss of ATPase activity of GST-2C. This was expected if 2C is a genuine ATPase since motif B has been reported to be essential for the function of many NTPases (56). The function of motif C, unique for superfamily III helicases and present in protein 2C of all picornaviruses (28), has not been genetically dissected before. We replaced the conserved Asn223 in motif C with either Asp (GST-2C mC1) or Gly (GST-2C mC2). The ATPase activities of both mutants were inhibited to undetectable levels. Thus, motif C of protein 2C is required for the hydrolysis of ATP. Replacement of the corresponding Asn523 by Ile in papillomavirus protein E1 did not impair ATP binding but the mutant protein was no longer able to support DNA replication (57), which was possibly due to the loss of E1's helicase activity. Mutation of motif C in E1 may have abolished NTPase activity and, as a consequence, helicase activity, since helicase activity of E1 depends on NTP hydrolysis (58). Mutation of motif C in NS1 of minute virus of mice, a parvovirus, reduced the ability of NS1 to hydrolyze ATP by 60% (59). Whether motif C of all superfamily III helicases is functionally related, i.e. involved in NTP hydrolysis, remains to be proven.

ATP was the preferred substrate among NTPs and dNTPs for the NTPase activity of GST-2C although hydrolysis of some non-ATP nucleotides was detectable. However, their rate of hydrolysis was at most 3% of that of ATP. This result is contradictory to an earlier study, in which GTP was hydrolyzed as efficiently as ATP (30). The reason for the discrepancy may reflect different reaction conditions. Mainly, a pH of 7.5 and a low concentration of ATP of 0.2 mM/µg of protein (here 6.8 and 0.5 mM/µg) may have contributed to an inefficient ATP hydrolysis in the Rodríguez and Carrasco study (30), leading to an overestimation of the associated GTPase activity. Alternatively, the protein described by these authors may have carried a cellular contaminating GTPase. On the other hand, the reaction conditions described in our study are based on optimal ATP hydrolysis and may not be optimal for GTP hydrolysis. However, our optimized reaction conditions agree well with those found for other NTPases with less substrate specificity (56). When included in ATPase reactions, non-ATP rNTPs inhibited to various degrees the ATPase activity of 2C. As was the case for GTPgamma S, inhibition was purely competitive indicating that GTP and most likely other rNTPs as well, bind at the same site as ATP does. Among all the rNTPs tested, ATPgamma S was the most effective inhibitor, an observation that indicated a preference for ATP at the NTP-binding site of protein 2C. The substrate specificity of 2C is unique among superfamily III helicases. Large T antigen as well as E1 accept a variety of rNTPs and dNTPs as substrates for hydrolysis (56).

It has been known for nearly four decades that guanidine inhibits poliovirus replication at concentrations of <= 2 mM (31, 60). Indeed, guanidine was one of the first drugs discovered that specifically interfered with the replication of a large number of animal and plant RNA viruses (for references, see Refs. 36 and 61). At low concentrations (<= 2 mM), guanidine has no apparent inhibitory effect on the proliferation of HeLa cells, the preferred host for poliovirus growth in tissue culture (61). Numerous studies have led to the conclusion that the main target of guanidine is poliovirus RNA replication yet all attempts to correlate inhibition with a specific biochemical event have failed (1). Poliovirus mutants resistant to guanidine (gr) are easily selected and they map to 2C (35-37). Interestingly, the mutations conferring resistance to 2 mM GdnHCl map to the vicinity of motif B (Fig. 1, changes of Asn179 to Ala or Gly; Refs. 35 and 36). Therefore, it was appealing to test the ATPase activity of GST-2C in the presence of 2 mM GdnHCl. Remarkably, the enzymatic activity of the enzyme was indeed abolished in the presence of the drug (Fig. 7A).

The genetic response of poliovirus to the presence of GdnHCl is complex. Mutations in 2C of gr or gd viral phenotypes are spread between amino acid residues 72 and 318 (36, 37). Most of the mutants harbor one of two "hallmark" mutations and can be classified accordingly as class N and class M mutants (37) (Fig. 1). In class N mutants, Asn179 is always replaced by either Gly or Ala, while in class M mutants, Met187 is always replaced by Leu. Both classes contain gr and gd phenotypes, depending on additional mutations within 2C. However, N179G/A or M187L alone are sufficient to confer gr or gd phenotypes, respectively (52, 62). At low concentrations of GdnHCl (<= 1 mM) there is a tendency toward selection of class M mutants, whereas at higher concentrations class N mutants are selected (37, 62). Accordingly, class N mutants generally tolerate higher concentrations of GdnHCl than class M mutants (62).

We considered it prudent to generate GST-2C preparations harboring class N and class M mutations, and to test these ATPases for their response to different concentrations of GdnHCl. It was satisfying to observe that the ATPase activity of GST-2C GR1, an enzyme belonging to the class N mutants, was 100-fold more tolerant to the drug than wt GST-2C (Fig. 7A). These results identify for the first time a biochemical function of a poliovirus gene product that is targeted by guanidine. GST-2C GD1 ATPase, an N class mutant selected at 2 mM GdnHCl, exhibited the same tolerance to GdnHCl as the GST-2C GR1 ATPase. It should be noted that the GST-2C GD1 ATPase originated from a gd mutant of poliovirus (36). The GST-2C R111 ATPase, on the other hand, harbors a class M mutation, and was 10-fold less resistant to GdnHCl when compared with the class N mutants (Fig. 7A). This we expected because the poliovirus variant R111 was selected at 0.5 mM GdnHCl; it expressed a gd phenotype (52). It therefore appears that poliovirus mutants selected at various GdnHCl concentrations harbor mutations in their 2C ATPase that confer GdnHCl resistance in assays of ATP hydrolysis at drug concentrations roughly corresponding to the genetic selection conditions.

The inhibition of the ATPase activity by GdnHCl appeared to be non-competitive. GdnHCl may be an allosteric inhibitor that mimicks a natural regulator of the ATPase activity of 2C by binding to a specific site within the protein inducing conformational changes. It is possible that these changes affect other (unknown) functions of 2C as well. Such a function of 2C from gd polioviruses (GD1 and R111) may depend on GdnHCl, because their ATPase function does not require GdnHCl. Perhaps, a function of 2C distinct from ATPase activity became dependent on guanidine-induced (structural?) changes while concomitantly the ATPase activity became tolerant to the drug.

RNA binding capabilities of protein 2C of poliovirus (16, 22, 23, 30) and hepatitis A virus (63) have been reported. We tested the effects of several kinds of RNAs (poly(U), single-stranded, heteropolymeric RNA, tRNA) on the ATPase activity of GST-2C. Unexpectedly, RNA inhibited the ATPase activity up to 90% (not shown). The mechanism behind this observation and its biological significance is under investigation.

Is 2C a helicase? This question has been entertained in many laboratories but no answer can be given today. Despite the motifs A, B, and C, all of which are essential for ATP hydrolysis, no further sequence similarity is apparent between 2C and other known helicases. Experiments performed with GST-2C4 or with MBP-2C (30) did not reveal helicase activity. In contrast, bacterially expressed MBP-E1 of papillomavirus (58) and the MBP-CI protein of plum pox potyvirus (64) are helicases, an observation suggesting that protein fusion is not necessarily detrimental to helicase function. Considering a strand-displacement activity of the poliovirus potymerase 3Dpol during RNA chain elongation (65), poliovirus may not need an additional helicase. Strand displacement in the absence of a helicase has been demonstrated for adenovirus, and other DNA viruses with a linear double-stranded genome (66-68). Many of these viruses have been shown to initiate DNA synthesis by using a viral protein as a primer (protein priming) (69). This is also the mechanism by which poliovirus RNA synthesis is initiated (70), a similarity that may support the notion that poliovirus may replicate its genome without a virus-encoded helicase. Yet 2C may express helicase in the presence of other viral or even cellular proteins, a possibility that has been tested but so far proved inconclusive.4

Even if poliovirus 2C is not a helicase, there are numerous possibilities of the ATPase activity to function in biochemical events during poliovirus replication. Whether these events relate to the organization of the membranous replication complex (9, 16), in the rearrangement of cellular organelles (10, 13, 18, 71), to chaperone function in the formation of protein complexes (9, 10, 44), or to processes of viral uncoating (42) or assembly (43) remains to be seen. There are many known biological processes in which NTPases regulate formation and function of proteinaceous complexes. The NTPase activity of both actin and tubulin enables them to control the state of polymerization (72). ATP hydrolysis by N-ethylmaleimide-sensitive fusion protein provokes the disassembly of the 20 S docking complex that mediates vesicle fusion (73). GTP-bound ADP-ribosylation factor directs the assembly of coatomer on membranes allowing vesicle budding (74). Hydrolysis of the bound GTP in ADP-ribosylation factor induces the disassembly of the coatomer, a prerequisite for vesicle fusion. E. coli DnaK, one of the best studied molecular chaperones, is able to bind and release proteins concomitant with ATP binding and hydrolysis (75). SV40 large T antigen participates in a complex containing the mammalian DnaK homologue Hsc70, a chaperone of the heat-shock protein family (76). Hepatitis B virus assembly and reverse transcription depends on a ribonucleoprotein complex, the formation of which requires ATP hydrolysis mediated by Hsp70 (77). It appears that many processes regulated by NTPases are associated with membrane traffic, protein folding, or nucleic acid replication. The involvement of protein 2C in similar events during poliovirus replication is likely.

The high degree of conservation of protein 2C among picornaviruses in general and of the NTP binding motif in particular (1) argues for a key role of protein 2C, and its responses to ATP, during picornavirus replication. Identification and characterization of the mechanisms that regulate the ATPase activity of 2C will shed light on 2C functioning in virus replication. We suggest that GST-2C is a valuable tool to study these mechanisms.

Human picornaviruses (enteroviruses, rhinoviruses, parechoviruses, and hepatoviruses), a very large group of pathogens, cause a bewildering array of different diseases (78). No effective chemotherapy for any of these diseases has been developed. It is sensible to consider the ATPase activity of picornavirus 2C as a target for drug intervention of picornavirus diseases. This may even be valuable for poliovirus. Poliovirus, the causative agent of poliomyelitis, has been targeted for global eradication by the year 2000 or shortly thereafter. Eventually, all vaccination against poliomyelitis will cease. There remains the problem of possible human carriers of poliovirus whose shedding of the infectious agent may jeopardize local eradication. In those cases, however, rare, an effective anti-poliovirus drug may be of great importance.

    ACKNOWLEDGEMENTS

We thank Drs. Aniko Paul and Sandy Simon for suggestions in experimental design, Drs. Rohit Duggal and Michael Shepley for carefully reading the manuscript, and Dr. Jorge Galán for the anti-GST antibody.

    FOOTNOTES

* This work was supported in part by the, NIAID, National Institutes of Health Grants AI1512225 and AI3210007.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger Recipient of stipends from the Swiss National Science Foundation, the Freie Akademische Gesellschaft Basel, and the Theodor Engelmann-Stiftung Basel, Switzerland.

§ To whom correspondence should be addressed. Tel.: 516-632-8787; Fax: 516-632-8891; E-mail: wimmer{at}asterix.bio.sunysb.edu.

2 Q. Liu and E. Wimmer, unpublished data.

3 T. Pfister and E. Wimmer, unpublished observation.

4 T. Pfister and E. Wimmer, unpublished results.

    ABBREVIATIONS

The abbreviations used are: RC, replication complex; MBP, maltose-binding protein; NTP, nucleoside triphosphate; GdnHCl, guanidine hydrochloride; gr, guanidine-resistant; gd, guanidine-dependent; GST, glutathione S-transferase; PAGE, polyacrylamide gel electrophoresis; NTPase, nucleoside triphosphatase; GTPgamma S, guanosine 5'-3-O-(thio)triphosphate; ATPgamma S, adenosine 5'-O-(thiotriphosphate).

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Abstract
Introduction
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