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J Biol Chem, Vol. 274, Issue 11, 6992-7001, March 12, 1999
From the Department of Molecular Genetics and Microbiology, State
University of New York at Stony Brook,
Stony Brook, New York 11794-5222
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.
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).
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 ( 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 DH5
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- 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 [ 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.
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.
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.
It has been shown that poliovirus 2C catalyzes the reaction of ATP 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 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.
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 ATP
Inhibition of ATP hydrolysis by ATP 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).
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.
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 GTP It has been known for nearly four decades that guanidine inhibits
poliovirus replication at concentrations of 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 ( 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.
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.
*
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.
§
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.
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;
GTP
Characterization of the Nucleoside Triphosphatase Activity of
Poliovirus Protein 2C Reveals a Mechanism by Which Guanidine Inhibits
Poliovirus Replication*
and
ABSTRACT
Top
Abstract
Introduction
References
INTRODUCTION
Top
Abstract
Introduction
References
View larger version (28K):
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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).
2 mM) of
GdnHCl on poliovirus replication and that of many other picornaviruses
by inference.
EXPERIMENTAL PROCEDURES
. The 2C coding sequence was confirmed by sequencing
(Sequenase, U. S. Biochemical Corp., Cleveland, OH).
-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.
-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).
RESULTS
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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- -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.
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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 ( 
) or mangesese acetate (
) 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.
ADP + Pi (29, 30). The colorimetric assay used does not
distinguish between Pi and pyrophosphate (PPi).
Therefore, we used -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 [-33P]ADP
indicating that the unlabeled 
-phosphate has been cleaved off.
[-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 [ -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.
Genetic variants of poliovirus protein 2C used in this study
ATPase activity of GST-2C mutants
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.
Substrates of GST-2C
S and GTPS (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. GTPS 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 ATPS,
which presumably indicates that ATPS competes with ATP for binding
to the NTP-binding site of 2C.
Inhibitory effect of rNTPs on ATP hydrolysis by GST-2C
S and GTPS 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 ATPS, 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 ATPS. Inhibition of ATP hydrolysis by GTPS was also
competitive (Fig. 6B), indicating that GTPS 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 ATPS
(Fig. 6C) and 0.25 mM for GTPS (Fig.
6D). Since Ki represents the dissociation
constant of the inhibitor-enzyme complex, the results suggest that
ATPS binds 2-fold stronger than GTPS 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 ATP S (A
and C) and GTPS
(B and D). A and
B, Lineweaver-Burk plots of reactions containing 0.1 mM () or 0.2 mM (
) inhibitor and variable
concentrations of substrate (ATP). Reaction conditions were as for Fig.
5E. The curve of Fig. 5E () is superimposed
for comparison with an uninhibited reaction. C and
D, Dixon plots of reactions containing 1 mM
(
) or 2 mM (
) 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.
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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 (), GST-2C GR1 (
), 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 () or 0.085 mM () GdnHCl. Reaction conditions were as for Fig.
5E. The curve of Fig. 5E () is superimposed
for comparison with an uninhibited reaction. Each data point represents
the average of duplicate reactions.
DISCUSSION
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, ATPS
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).
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).
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).
ACKNOWLEDGEMENTS
FOOTNOTES
Recipient of stipends from the Swiss National Science Foundation,
the Freie Akademische Gesellschaft Basel, and the Theodor Engelmann-Stiftung Basel, Switzerland.
ABBREVIATIONS
S, guanosine
5'-3-O-(thio)triphosphate;
ATPS, adenosine
5'-O-(thiotriphosphate).
REFERENCES
Top
Abstract
Introduction
References
Copyright © 1999 by The American Society for Biochemistry and Molecular Biology, Inc.
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R. C. Rust, L. Landmann, R. Gosert, B. L. Tang, W. Hong, H.-P. Hauri, D. Egger, and K. Bienz Cellular COPII Proteins Are Involved in Production of the Vesicles That Form the Poliovirus Replication Complex J. Virol., October 15, 2001; 75(20): 9808 - 9818. [Abstract] [Full Text] [PDF]
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W. D. Zhao and E. Wimmer Genetic Analysis of a Poliovirus/Hepatitis C Virus Chimera: New Structure for Domain II of the Internal Ribosomal Entry Site of Hepatitis C Virus J. Virol., April 15, 2001; 75(8): 3719 - 3730. [Abstract] [Full Text]
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T. Pfister and E. Wimmer Polypeptide p41 of a Norwalk-Like Virus Is a Nucleic Acid-Independent Nucleoside Triphosphatase J. Virol., February 15, 2001; 75(4): 1611 - 1619. [Abstract] [Full Text]
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R. Banerjee and A. Dasgupta Specific Interaction of Hepatitis C Virus Protease/Helicase NS3 with the 3'-Terminal Sequences of Viral Positive- and Negative-Strand RNA J. Virol., February 15, 2001; 75(4): 1708 - 1721. [Abstract] [Full Text]
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A. V. Paul, E. Rieder, D. W. Kim, J. H. van Boom, and E. Wimmer Identification of an RNA Hairpin in Poliovirus RNA That Serves as the Primary Template in the In Vitro Uridylylation of VPg J. Virol., November 15, 2000; 74(22): 10359 - 10370. [Abstract] [Full Text]
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E. Rieder, A. V. Paul, D. W. Kim, J. H. van Boom, and E. Wimmer Genetic and Biochemical Studies of Poliovirus cis-Acting Replication Element cre in Relation to VPg Uridylylation J. Virol., November 15, 2000; 74(22): 10371 - 10380. [Abstract] [Full Text]
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M. S. Marín, R. Casais, J. M. M. Alonso, and F. Parra ATP Binding and ATPase Activities Associated with Recombinant Rabbit Hemorrhagic Disease Virus 2C-Like Polypeptide J. Virol., November 15, 2000; 74(22): 10846 - 10851. [Abstract] [Full Text]
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T. Ahola, J. A. den Boon, and P. Ahlquist Helicase and Capping Enzyme Active Site Mutations in Brome Mosaic Virus Protein 1a Cause Defects in Template Recruitment, Negative-Strand RNA Synthesis, and Viral RNA Capping J. Virol., October 1, 2000; 74(19): 8803 - 8811. [Abstract] [Full Text]
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D. Egger, N. Teterina, E. Ehrenfeld, and K. Bienz Formation of the Poliovirus Replication Complex Requires Coupled Viral Translation, Vesicle Production, and Viral RNA Synthesis J. Virol., July 15, 2000; 74(14): 6570 - 6580. [Abstract] [Full Text]
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H. Shimizu, M. Agoh, Y. Agoh, H. Yoshida, K. Yoshii, T. Yoneyama, A. Hagiwara, and T. Miyamura Mutations in the 2C Region of Poliovirus Responsible for Altered Sensitivity to Benzimidazole Derivatives J. Virol., May 1, 2000; 74(9): 4146 - 4154. [Abstract] [Full Text]
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M. Klein, D. Hadaschik, H. Zimmermann, H. J. Eggers, and B. Nelsen-Salz The picornavirus replication inhibitors HBB and guanidine in the echovirus-9 system: the significance of viral protein 2C J. Gen. Virol., April 1, 2000; 81(4): 895 - 901. [Abstract] [Full Text]
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T. Pfister, K. W. Jones, and E. Wimmer A Cysteine-Rich Motif in Poliovirus Protein 2CATPase Is Involved in RNA Replication and Binds Zinc In Vitro J. Virol., January 1, 2000; 74(1): 334 - 343. [Abstract] [Full Text]
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