Published Ahead of Print 10 August 2011.
2011, 85(20):10764. DOI: 10.1128/JVI.05062-11.
Gorbalenya, Heléne Norder, Bruno Canard and Miquel Coll
Robert Janowski, Bruno Coutard, Maria Solà, Alexander E.
Lionel Costenaro, Zuzanna Kaczmarska, Carme Arnan,
the Main Protease, 3C, from Human
Structural Basis for Antiviral Inhibition of
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JOURNAL OF VIROLOGY, Oct. 2011, p. 10764–10773
Copyright © 2011, American Society for Microbiology. All Rights Reserved.
Vol. 85, No. 20
Structural Basis for Antiviral Inhibition of the Main Protease,
3C, from Human Enterovirus 93?
Lionel Costenaro,1,2† Zuzanna Kaczmarska,1,2Carme Arnan,1,2‡ Robert Janowski,1,2
Bruno Coutard,3Maria Sola `,2Alexander E. Gorbalenya,4Hele ´ne Norder,5
Bruno Canard,3and Miquel Coll1,2*
Institute for Research in Biomedicine, Barcelona, Spain1; Institut de Biologia Molecular de Barcelona (CSIC), Barcelona,
Spain2; Architecture et Fonction des Macromole ´cules Biologiques (UMR 6098 CNRS), Marseille, France3;
Molecular Virology Laboratory, Leiden University Medical Center, Leiden, The Netherlands4; and
Swedish Institute for Disease Control, Solna, Sweden5
Received 11 May 2011/Accepted 2 August 2011
Members of the Enterovirus genus of the Picornaviridae family are abundant, with common human pathogens
that belong to the rhinovirus (HRV) and enterovirus (EV) species, including diverse echo-, coxsackie- and
polioviruses. They cause a wide spectrum of clinical manifestations ranging from asymptomatic to severe
diseases with neurological and/or cardiac manifestations. Pandemic outbreaks of EVs may be accompanied by
meningitis and/or paralysis and can be fatal. However, no effective prophylaxis or antiviral treatment against
most EVs is available. The EV RNA genome directs the synthesis of a single polyprotein that is autocatalytically
processed into mature proteins at Gln2Gly cleavage sites by the 3C protease (3Cpro), which has narrow,
conserved substrate specificity. These cleavages are essential for virus replication, making 3Cproan excellent
target for antivirus drug development. In this study, we report the first determination of the crystal structure
of 3Cprofrom an enterovirus B, EV-93, a recently identified pathogen, alone and in complex with the anti-HRV
molecules compound 1 (AG7404) and rupintrivir (AG7088) at resolutions of 1.9, 1.3, and 1.5 Å, respectively.
The EV-93 3Cproadopts a chymotrypsin-like fold with a canonically configured oxyanion hole and a substrate
binding pocket similar to that of rhino-, coxsackie- and poliovirus 3C proteases. We show that compound 1 and
rupintrivir are both active against EV-93 in infected cells and inhibit the proteolytic activity of EV-93 3Cproin
vitro. These results provide a framework for further structure-guided optimization of the tested compounds to
produce antiviral drugs against a broad range of EV species.
Enteroviruses (EVs) are small, nonenveloped, icosahedral,
positive-sense, single-stranded RNA viruses classified into a
genus of the Picornaviridae family, one of the largest and most
important families of viral pathogens of vertebrates, including
humans (36). The Enterovirus genus encompasses 234 human
pathogens that form 7 species spread worldwide: human en-
teroviruses A through D (HEV-A, HEV-B, HEV-C, and
HEV-D) and human rhinoviruses A through C (HRV-A,
HRV-B, and HRV-C) (23). Echoviruses and coxsackievirus B
(CV-B) are classified within the HEV-B species, and poliovi-
ruses (PVs) are classified within HEV-C. There are also EVs
that infect nonhuman primates, cattle, and swine that may play
roles in zoonotic spread and the emergence of new human
pathogens. In humans, infections range from asymptomatic to
more severe illnesses that are manifested as aseptic meningitis,
encephalitis, gastroenteritis, myocarditis, paralysis, and polio-
myelitis, with high mortality rates in infected newborn infants.
Outbreaks of different EVs are frequently reported. For ex-
ample, EV-71 of HEV-A caused serious complications—en-
cephalitis and myocarditis—and death during epidemics of
hand, foot, and mouth disease in Asia in 1997, 1998, 2000, and
2008 (44). The disease manifestation of acute flaccid paralysis
is also associated with nonpoliovirus EVs within the HEV-B
species, including newly discovered viruses like EV-93, which is
the subject of this study (20). Despite the enormous health care
impact of EV infections, no antiviral drugs have been approved
to control these infections (for a recent review, see reference 11).
The EV genome is a positive-sense, single-stranded RNA of
between 7.4 and 7.5 kb with a single open reading frame
translated into a large polyprotein of approximately 2,200
amino acids (?250 kDa) (13, 38). This polyprotein is rapidly
processed by co- and posttranslational cleavages into three
precursor molecules, P1, P2, and P3, and then into mature viral
proteins: the structural proteins VP4 to VP1 from P1 and the
nonstructural proteins associated with replication, 2A to 2C
and 3A to 3D from P2 and P3, respectively, from the N to the
C terminus (22). Most of these cleavages are mediated by the
3C protease (3Cpro) either alone or as a domain of 3CDpro. In
addition to its key role in processing the polyprotein, 3Cpro
cleaves a number of host proteins to remodel the cellular
environment for virus reproduction (12, 43). Due to its central
role in the control of genome expression, 3Cprocould be con-
sidered the “main” protease (49). Crystal structures have been
determined for EV 3Cprofrom CV-B3, HRV-2, HRV-14,
PV-1, and EV-71 (7, 10, 27, 30, 31, 34). The protease adopts a
* Corresponding author. Mailing address: Institute for Research in
Biomedicine, Barcelona Science Park, Baldiri Reixac 10, 08028 Bar-
celona, Spain. Phone: 34 93 4034951. Fax: 34 93 4034979. E-mail:
† Present address: Institute of Biotechnology and Biomedicine, Uni-
versitat Auto `noma de Barcelona, 08193 Bellaterra, Spain.
‡ Present address: Centre de Regulacio ´ Geno `mica, Dr. Aiguader 88,
08003 Barcelona, Spain.
?Published ahead of print on 10 August 2011.
on November 20, 2013 by guest
chymotrypsin-like fold with the Cys-His-Glu catalytic triad
present in a shallow groove between two topologically equiv-
alent six-stranded ? barrels. It cleaves sites that have predom-
inantly Gln2Gly at the P12P1? positions and a restricted
evolutionary variation at the P4 position (17, 45).
Because of their essential role in virus replication and a
narrow substrate specificity, EV 3Cpros are excellent targets for
antiviral therapy and have been the focus of extensive struc-
ture/activity studies to develop inhibitor compounds, mainly
against HRVs (for a recent review, see reference 11). Two
compounds against the common cold, rupintrivir (AG7088)
and its orally bioavailable analogue compound 1 (AG7404),
progressed to phase-II/I clinical trials (19, 41). Both com-
pounds (Fig. 1) are peptidomimetic inhibitors and imitate the
P4 to P1 peptide substrate, with an ?,?-unsaturated ester at
P1? as a Michael acceptor to form an irreversible covalent
bond with the active-site Cys residue. Rupintrivir was shown to
have low toxicity and potent antiviral activity against all HRV
serotypes tested (48), with a mean 50% effective concentration
(EC50) of 23 nM, and also against four related EVs (40).
Similar results were later obtained against HRV clinical iso-
lates and four additional EVs, as well as with compound 1 (5,
Here we present the crystal structure of the main protease of
human enterovirus B EV-93 (EV-93 3Cpro) alone and in com-
plex with compound 1 and rupintrivir at resolutions of 1.9 Å,
1.3 Å, and 1.5 Å, respectively. This 20.2-kDa cysteine protease
adopts a chymotrypsin-like fold with a catalytic triad, Cys147-
His40-Glu71, located in the cleft between the two six-stranded
? barrels. The residues that form the inhibitor binding pockets
are highly conserved among EV 3Cpros, which explains the
broad activity of compounds developed initially against a spe-
cific virus. To corroborate this finding, we showed that both
compound 1 and rupintrivir are active against EV-93 in in-
fected cells and inhibit the proteolytic activity of EV-93 3Cpro
MATERIALS AND METHODS
Protein purification. EV-93 3Cprowas expressed in Escherichia coli cells and
purified at high yields (?25 mg per liter of cell culture). For this process, the 3C
protease gene from HEV-B EV-93 strain 38-03 (20) was cloned into a pDEST14
expression vector (Invitrogen) with an N-terminal MK, as the additional Lys can
improve protein expression (8), and a C-terminal His6tag. Four E. coli strains
(BL21 pLysS, Rosetta, Origami pLysS, and C41) were tested to identify the
optimal conditions for protein overexpression. Soluble protein was detected by
dot blotting for three media and temperatures for a total of 36 conditions (4).
The best results were obtained using E. coli BL21(DE3)-pLysS grown in Superior
Broth medium (Athena) at 17°C. Cells were grown in this medium (3 liters),
which contained 100 ?g ml?1ampicillin and 34 ?g ml?1chloramphenicol, at
37°C until an optical density at 600 nm (OD600) of 0.6 was reached. They were
then induced with 0.5 mM isopropyl-?-D-thiogalactopyranoside and further in-
cubated overnight at 17°C. Cells were harvested by centrifugation, resuspended
in extraction buffer (50 mM Tris-HCl [pH 8], 300 mM NaCl, 10 mM imidazole,
0.1% [vol/vol] Triton X-100, 5% [vol/vol] glycerol, 0.25 mg/ml lysozyme, and
Complete EDTA-free antiproteases [Roche]), and then stored at ?80°C.
Thawed cells were lysed by sonication on ice and centrifuged at 30,000 ? g for
1 h at 4°C. The supernatant was loaded into a 5-ml Ni-affinity HisTrap HP
column (GE Healthcare), and recombinant EV-93 3Cprowas eluted with 0.5 M
imidazole in 50 mM Tris-HCl (pH 8)–300 mM NaCl. EV-93 3Cprowas further
purified through a gel filtration column (HiLoad 16/60 Superdex 200; GE
Healthcare) in 10 mM bicine (pH 8.5)–300 mM NaCl and concentrated for
crystallization to 2 mg/ml, as determined spectrophotometrically using a theo-
retical extinction coefficient of 0.677 ml mg?1cm?1at 280 nm. EV-93 3Cprowas
purified without dithiothreitol (DTT) or with 1 mM DTT in all buffers except the
extraction buffer (5 mM). The C147A mutant of EV-93 3Cprowas obtained by
site-directed mutagenesis (Stratagene), checked by DNA sequencing, and puri-
fied as the native protein. The EV-93 3Cpro-compound 1 and EV-93 3Cpro-
rupintrivir complexes were purified by gel filtration after overnight incubation at
20°C with a 2-fold molar excess of the inhibitor. The antiviral agents rupintrivir
(AG7088) and compound 1 (AG7404) were both generously provided by Pfizer.
Selected fractions were concentrated to 3 mg/ml for the EV-93 3Cpro-compound
1 complex and to 5 mg/ml for the EV-93 3Cpro-rupintrivir complex.
Crystallization and structure determination. Crystallization trials were set up
at 20°C with a NanoDrop dispenser in 96-well sitting drop plates using commer-
cial screens. Drops were prepared by mixing 100 nl of protein solution with 100
nl of reservoir solutions. Crystal optimization was performed manually with 1?1
?l sitting drops. Needle-like crystals of EV-93 3Cprogrew in 18% (vol/vol) PEG
8000, 0.2 M Mg acetate, and 0.1 M cacodylate (pH 7). Crystals were briefly
soaked in a cryo-protectant solution comprising mother liquor with an additional
20% (vol/vol) glycerol and then flash cooled in liquid nitrogen. EV-93 3Cpro-
compound 1 crystals were grown in 1.8 M (NH4)2HPO4–0.1 M Tris (pH 8.3) and
were briefly soaked in 2.3 M (NH4)2HPO4–0.1 M Tris (pH 8.3) supplemented
with 13.4% ethylene glycol prior to being flash cooled. EV-93 3Cpro-rupintrivir
crystals were obtained in 0.25 M MgCl2, 0.1 M Tris (pH 8.5), and 25% PEG 8000
and soaked in mother liquor with an additional 25% PEG 400 before being flash
Diffraction intensities were recorded on microfocus beamline ID23-2 at the
European Synchrotron Radiation Facility (ESRF; Grenoble, France) at a wave-
length of 0.8726 Å and a beam diameter of 10 ?m. Data for native protein and
the compound 1 complex were indexed, integrated, and scaled using the
MOSFLM and SCALA programs (9, 46), while the DENZO and SCALEPACK
programs (37) were used for the rupintrivir complex data. Statistics for the
best-measured data sets are reported in Table 1. EV-93 3Cprocrystals belong to
the monoclinic space group P21and have unit cell parameters that are similar for
native protein and the compound 1 and rupintrivir complexes. The calculated
Matthews coefficients (VM? 1.9, 2.0, and 1.9 Å3Da?1) indicate the presence of
two molecules of EV-93 3Cproin the asymmetric units with corresponding sol-
vent contents of 37.5%, 38.9%, and 36.6%, respectively (29).
The initial phases were obtained by molecular replacement using the program
PHASER (32) with the PV-1 3Cprostructure as a search model (PDB code,
1L1N) (34). The EV-93 3Cprostructure was built initially with ARP/wARP 6.1.1
(42) and then by iterative cycles of restrained refinement with REFMAC5 (9, 35)
and model building/solvent addition with COOT (15). The EV-93 3Cpro-com-
pound 1 structure was refined with PHENIX (1), while REFMAC5 was used to
refine the EV-93 3Cpro-rupintrivir complex. Geometry restraint information for
compound 1 was generated with eLBOW from the SMILES description of the
FIG. 1. Chemical structure of rupintrivir (AG7088) and its orally
bioavailable analogue compound 1 (AG7404). Asterisks indicate car-
bon atoms that make an irreversible covalent bond with the active-site
cysteine residue of 3Cpro.
VOL. 85, 2011 ANTIVIRAL INHIBITION OF EV-93 MAIN PROTEASE10765
on November 20, 2013 by guest
antiviral method and using the semiempirical quantum-mechanical optimization
method AM1 (33). Geometry restraint information for rupintrivir was calculated
with SKETCHER within CCP4i (9). In the first two structures (EV-93 3Cpro
alone and in complex with compound 1), hydrogen atoms were added in their
riding positions, but they were not added for the rupintrivir complex structure.
Refinement statistics are reported in Table 1. Structural figures were made with
PyMOL (www.pymol.org). The sequence alignment was obtained from Muscle-
mediated (14), polyprotein-wide EV alignment built using the Viralis software
platform (16). The sequence alignment figure was generated with ESPript (18).
Amino acid conservation scores were calculated and mapped onto the protein
structure with the ConSurf server (25).
Antiviral activity in infected cells. The antiviral activities of rupintrivir
(AG7088) and compound 1 (AG7404) were determined by infecting monolayers
of human rhabdomyosarcoma cells (RD) with 100 or 1,000 50% cell culture
infective doses (CCID50) of EV-93 in two 96-well plates. After an adsorption
period of 2 h at 37°C, the virus was removed and serial dilutions of one com-
pound per plate were added. The cultures were further incubated for 7 days, until
the complete cytopathogenic effect (CPE) was observed in the wells with infected
and untreated virus controls. The wells were read visually. The compounds were
diluted from 1 to 0.0016 ?g/ml. After 7 days of culture, the supernatant was
collected from wells that exhibited the full CPE in the presence of the lowest
concentration of the compounds used. This virus was used for successive rounds
of infection, a procedure that was repeated 15 times in order to generate drug-
In vitro proteolytic activity assay. A peptide representing the predicted
2C23A cleavage site of EV-93 (Ac-RHSVGATLEALFQ2GPPVYREIKIS-
NH2; Genepep) was used to test EV-93 3Cproproteolytic activity in vitro and its
inhibition by two antiviral agents, rupintrivir (AG7088) and compound 1
(AG7404). The lyophilized peptide and antiviral agents were dissolved in 100%
dimethyl sulfoxide (DMSO) at a concentration of 0.5 mM. Cleavage reaction
mixtures containing 30 ?M peptide, 3 ?M EV-93 3Cpro, 50 mM HEPES (pH
7.2), and 150 mM NaCl in a total volume of 100 ?l were incubated at 30°C. The
reactions were stopped after 20 h by the addition of 0.5% (final concentration)
trifluoroacetic acid (TFA) or by freezing at ?20°C. Samples were analyzed by
reverse-phase high-performance liquid chromatography (HPLC) on a Source
5RPC ST 4.6/150 column (GE Healthcare) using a 2% to 90% linear gradient of
acetonitrile in 0.1% TFA. To keep them independent of the initial amount of the
substrate, trans cleavage efficiencies (E) are reported as the fraction of the
substrate converted to products, based on the integrated peak areas at 215 nm
corresponding to the remaining substrate (r) and the products (pi), as follows:
E ? ? pi/(r ? ? pi). The substrate and products were analyzed by matrix-assisted
laser desorption ionization–time of flight mass spectrometry (MALDI-TOF) and
comparison with the reference samples (the synthesized full peptides, Ac-RHS
VGATLEALFQ-OH and H-GPPVYREIKIS-NH2). The presence of the protein
was confirmed by tandem mass spectrometry (MS-MS) fragmentation and
Protein structure accession numbers. Atomic coordinates and structure fac-
tors have been deposited in the Protein Data Bank under accession codes 3Q3X,
3Q3Y, and 3RUO for EV-93 3Cpro, EV-93 3Cpro-compound 1, and EV-93
RESULTS AND DISCUSSION
Structure determination and model quality. EV-93 3Cpro
was expressed in E. coli cells and purified at a high yield (?25
mg per liter of cell culture). Needle-like crystals, up to approx-
imately 10 by 10 by 170 ?m, were obtained in a few weeks from
a solution containing PEG 8000, magnesium acetate, and
cacodylate. Further optimizations of the crystallization condi-
tions, including additive screen and macro- or microseeding,
did not yield larger crystals of the native protein. Taking ad-
vantage of the highly focused beam of the ID23-2 line at the
European Synchrotron Radiation Facility (ESRF; Grenoble,
France), we collected two data sets from a single crystal, irra-
diating each time a fresh crystal portion along its long dimen-
sion, with a shifted but overlapping ?-angle range. Merging the
best parts of these data sets allowed us to obtain a complete
data set at high resolution (Table 1). The stability of the crystal
TABLE 1. Crystallographic data and refinement statistics
Values for indicated protease or compounda
EV-93 3Cpro-compound 1
Resolution range (Å)
Cell dimensions (Å, degrees)
a ? 39.07, b ? 65.22, c ? 66.36,
? ? 90.67
2 Mg2?ions, 6 glycerol
a ? 39.04, b ? 64.45, c ? 68.74,
? ? 90.81
2 compound 1 molecules, 8 ethylene
glycol molecules, 3 HPO4
a ? 39.00, b ? 63.91, c ? 66.36,
? ? 90.43
2 rupintrivir molecules, 2 Mg2?
No. of observed reflections
No. of unique reflections
No. of protein atoms (non-H)
No. of water molecules
No. of hetero compounds
RMSD for bond length (Å);
RMSD for bond angles (°)
Mean B value (Å2)
Mean B value for inhibitor
allowed region (%)
0.012; 1.400.008; 1.41
aValues in parentheses refer to the high-resolution shell.
bRmerge? ?h?i?Ii,h? ?Ih??/?h?iIi,h, where Ii,his the ith-intensity measurement of reflection h and ?Ih? is the average intensity for multiple measurements.
cRworkand Rfree? ???Fo? ? ?Fc??/??Fo?. Rfreewas calculated for 5% of the reflections not used for refinement.
dRamachandran analysis was done with PROCHECK.
10766COSTENARO ET AL. J. VIROL.
on November 20, 2013 by guest
during irradiation was most likely due to its low solvent con-
tent, 37.5%, corresponding to dense crystal packing with two
molecules in the asymmetric unit.
We solved the structure of EV-93 3Cproby molecular re-
placement, using the structure of the homologous PV-1 3Cpro
as a search model, the two proteins sharing 61% of identical
amino acids. The structure of EV-93 3Cprowas refined at 1.9 Å
to a final Rworkof 14.8% (Rfree? 21%; see Table 1 for refine-
ment statistics). We modeled all residues except the His6tag
and the last two and three C-terminal residues for chains A and
B, respectively, which we did not model because of poor elec-
tron density. Figure 2 shows the electron density in a repre-
sentative region of the structure. The Ramachandran plot
shows 88.4% of the residues to be in the most favored regions,
10.3% and 1.3% in the additionally and generously allowed
regions, respectively (26). The two molecules of the asymmet-
ric unit are related by a noncrystallographic 2-fold axis. The
refined structure contains 339 water molecules, 2 magnesium
ions, and 6 glycerol molecules.
The structure of EV-93 3Cproin complex with compound 1
was solved by molecular replacement using the native structure
and was refined at 1.32 Å to a final Rworkof 12.5% (Rfree?
17%; Table 1). The final model encompasses all EV-93 3Cpro
amino acids except the first N-terminal and the last three
C-terminal (and His6tag) residues. One molecule of com-
pound 1 per protein chain was unequivocally and precisely
defined (see Fig. 5a), the ethoxycarbonyl group corresponding
to the P1? position (Fig. 1) being more mobile. The structure
also contains 543 water, 8 ethylene glycol, 3 HPO4
between the two structures are 0.465 Å (for 267 superimposed
C? atoms) for both chains and 0.262 Å (for 137 superimposed
C? atoms) for one protein chain, with an angular shift of about
2° between the second chains of the two structures.
2?, and 2
?molecules. The root mean square deviations (RMSDs)
The structure of EV-93 3Cproin complex with rupintrivir
was solved by molecular replacement using the native structure
as a starting model. One rupintrivir molecule per protein
chain, including molecules from the 1-fluorobenzene-4-yl
group, which is different from the corresponding compound 1
group (Fig. 1, P2 position), was clearly defined in both 2Fo?Fc
and Fo?Fc electron density maps. Given the higher resolution
of the EV-93 3Cpro-compound 1 complex structure, the mo-
lecular replacement calculation was repeated with protein mol-
ecule A of the complex structure (without ligand and solvent
atoms) as the initial model. The electron density maps showed
a position for the rupintrivir molecule identical to that shown
when using the native structure as the starting model, so the
molecule was added. The structure was refined at 1.50 Å to a
final Rworkof 16.6% (Rfree? 19.8%; Table 1). The structure
also contains 375 water molecules, 2 Mg2?ions, and 1 Cl?ion.
Overall structure and active site of EV-93 3Cpro. EV-93
3Cprofolds into two antiparallel ? barrels (residues 15 to 77
and 97 to 173, respectively) that are oriented 90° apart, linked
by a 20-amino-acid loop with a short ?-helix in its middle, and
flanked by two other ?-helixes at the N and C termini, 14 and
6 amino acids long, respectively (Fig. 3a). The two barrels are
topologically equivalent and are formed by six antiparallel ?
strands with the first four (A to D) organized into a Greek key
motif. Our structure confirms that EV-93 3Cproadopts a chy-
motrypsin-like fold similar to that of other picornavirus 3Cpros.
The RMSDs between 3Cpros from EV-93 and other EVs are
0.28 Å (160 C?) for CV-B3, 0.70 Å (152 C?) for PV-1, 0.77 Å
(150 C?) for HRV-2, 1.02 Å (150 C?) for HRV-14, and 1.16 Å
(170 C?) for EV-71 (7, 10, 27, 30, 34). These figures underline
the high conservation of the 3Cprostructure in viruses of the
Enterovirus genus. Analysis of EV-93 3Cprointerfaces with
the PISA server (24) suggests that the homodimer formed by
the two chains of the asymmetric unit (Fig. 3a) might be stable
FIG. 2. Typical ?A-weighted 2Fo?Fc electron density map of the crystal structure of EV-93 3Cpro-compound 1. The electron density, contoured
at 1.3?, is shown as a blue mesh with the residues depicted in ball-and-stick format (stereo view).
VOL. 85, 2011 ANTIVIRAL INHIBITION OF EV-93 MAIN PROTEASE10767
on November 20, 2013 by guest
in solution with an interface area of 1,710 Å2mainly involving
parts of the ?1-helix and B2 strand (residues 1, 5, 107, 113, and
143 from both chains). However, a gel filtration chromatogram
showed only one peak corresponding to a monomer of EV-93
3Cpro, and there is no further evidence that dimerization is
required for the proteolytic activity of EV-93 3Cpro.
The active-site residues are located in the cleft between the
two barrels with the nucleophilic Cys147from the C-terminal
barrel and the general acid base pair His40-Glu71from the
N-terminal barrel (Fig. 3). Early in the refinement of the native
structure of EV-93 3Cpro, residual-difference density close to
the sulfur of the active-site cysteine in both copies indicated
that Cys147residues were oxidized at least to the stage of
sulfinic acid, ?SO2
case, with partial occupations for the O?atom (Fig. 3b). Since
the purified protein is active (see below), such oxidation must
have occurred during crystallization or X-ray data collection.
Oxidized active-site Cys residues were also observed in other
picornaviral 3Cpros (2, 3). As a result, the side chains of His40
adopt two conformations, one corresponding to the canonical
orientation, as seen in other EV 3Cpros, and making hydrogen
bonds with the two other active-site residues, and the second
corresponding to the oxidized-cysteine state, being rotated
about 120° out of the active site.
Substrate hydrolysis by cysteine proteases occurs through a
covalent tetrahedral intermediate between the active-site nu-
cleophile and the carbonyl carbon of the scissile bond. The
resulting oxyanion is stabilized by strong hydrogen bonds with
amide groups of the protease, which are collectively called the
oxyanion hole. In the EV-93 3Cprostructure (Fig. 3b), the
amide groups of Cys147, Gln146, and Gly145form this oxyanion
?, and partially to that of sulfonic acid,
?. We therefore modeled both residues as the latter
hole with a conformation similar to that of other EV 3Cpros,
which is adequate to stabilize the tetrahedral intermediate.
3C proteases were shown to recognize amino acid residues
around the cleavage site, mostly at the P4…P1 2 P1? positions
that fit into corresponding specific binding subsites (S4…S1,
S1?) of the protease. Based on comparison of the structure of
covalently bound to a peptide (acetyl-
LEALFQ-ethyl propionate) inhibitor, including P6 to P1 sub-
strate residues (7), with the EV-93 3Cprostructure, we propose
that the substrate binding pocket of EV-93 3Cprois formed by
residues belonging to the ? strands B2, E2, F2 and to the C2 to
D2 loop head of the oxyanion hole (Fig. 3b). In particular,
mutations in the B2 to C2 loop were shown to have a signifi-
cant impact on the proteolytic activity of 3Cprofrom foot-and-
mouth disease virus, which belongs to another genus of the
Picornaviridae family (47).
Rupintrivir and compound 1 inhibit EV-93 replication in
infected cells. Since compound 1 and rupintrivir, developed as
irreversible inhibitors of HRV 3Cpros, inhibit 3Cpros encoded
by viruses of HEV species (40, 41), we reasoned that they could
also be active against EV-93. Indeed, rupintrivir inhibited
EV-93 replication in infected RD cells, a prototype cell line for
enterovirus growth (48), with a mean EC50of 33 nM (range, 17
to 50 nM). The EC50for compound 1 was 93 nM (range, 37 to
112 nM). These results are consistent with mean EC50s ob-
tained for related HEVs: 88 nM (range, 7 to 183 nM) and 75
nM (range, 7 to 249 nM) for rupintrivir and compound 1,
respectively (40, 41). We were unable to isolate a drug-resis-
tant virus after 15 successive passages of the virus in the pres-
ence of either of the compounds at concentrations that allowed
observation of the full cytopathogenic effect caused by the
virus. For HRVs, most mutations conferring resistance to
FIG. 3. Crystal structure of EV-93 3Cpro. (a) Ribbon representation of the two molecules present in the asymmetric unit with the noncrystal-
lographic 2-fold axis (}) perpendicular to the plane. The protease folds into two antiparallel ? barrels (in green and orange tones from the N to
the C terminus), forming the chymotrypsin-like fold. The catalytic triad is highlighted as stick representations. (b) Active site of EV-93 3Cpro. Key
residues are highlighted as ball-and-stick representations (stereo view). Main-chain amides forming the oxyanion hole are indicated by “n.”
10768COSTENARO ET AL.J. VIROL.
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rupintrivir were obtained after only a few serial passages, 3 for
HRV-14, 4 for HRV-2 and HRV-39, and 6 for HRV-Hanks
(6), and thus 15 passages were considered sufficient. As mu-
tating residues in HRVs are mostly conserved in sequence and
structure compared to those in EV-93, we cannot rule out that
the difference between EV-93 and HRVs in obtaining drug-
resistant viruses is due to different passaging procedures.
Rupintrivir and compound 1 inhibit EV-93 3Cproprotease in
vitro. In order to verify that compound 1 and rupintrivir could
also target the EV-93 3C protease, we tested the effect of each
antiviral agent on the in vitro proteolytic activity of EV-93
3Cproon a cognate peptide substrate (P13 to P11?) that mimics
the 2C23A cleavage site. As illustrated in Fig. 4A, reverse-
phase HPLC analyses of overnight incubations of the peptide
with EV-93 3Cproprovide clear evidence of the expected pro-
teolytic cleavage. The identities of the substrate and its cleav-
age products were verified by MALDI-TOF and comparison
with the reference samples, while the presence of the protein
was confirmed by MS-MS fragmentation and SDS-PAGE. The
trans cleavage efficiency (E) of EV-93 3Cprowas estimated to
be more than 95% for the native enzyme, regardless of the
presence or absence of DTT (Fig. 4A and Table 2). This
finding suggests that the oxidation of the active-site Cys147, as
seen in the EV-93 3Cprostructure (and thus the inactivation of
the enzyme), occurred after its purification, during its crystal-
lization or during X-ray data collection. The proteolytic assay
performed with the C147A mutant of EV-93 3Cproshowed no
effect on the substrate and did not render detectable cleavage
products (Fig. 4D), thereby confirming that Cys147is the active
nucleophilic residue. Proteolytic assays performed in the pres-
FIG. 4. Inhibition of the in vitro proteolytic activity of EV-93 3Cproby rupintrivir. Reverse-phase chromatograms show in red the products of
the digestion of the EV-93 2C23A peptide (Ac-RHSVGATLEALFQ2GPPVYREIKIS-NH2) by EV-93 3Cpro(p) without rupintrivir (r) (A) or
with rupintrivir at two inhibitor-to-protein molar ratios, 1:3 (B) and 30:3 (C). (D) C147A mutant protein. Chromatograms of the substrate peptide
alone are shown in blue in all panels.
TABLE 2. trans-cleavage efficiencies of 2C23A peptide
by EV-93 3Cpro
molar ratio (?M)
a???, efficiency of ?90%; ??, efficiency of ?20%; ?, efficiency of ?10%;
0, no visible product peaks.
VOL. 85, 2011ANTIVIRAL INHIBITION OF EV-93 MAIN PROTEASE 10769
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ence of an inhibitor with three different inhibitor-to-protease
molar ratios indicated that both rupintrivir and compound 1
efficiently inhibit native EV-93 3Cproin vitro (Fig. 4B and C
and Table 2). No cleavage product was detected with a 10-fold
excess of inhibitors over the enzyme, and cleavage efficiencies
of less than 10% were observed with equimolar amounts of the
two antivirals relative to the 3Cpro(Table 2). These results are
consistent with prior results for potency and irreversible inhi-
FIG. 5. Stereo views of compound 1 and rupintrivir bound to the EV-93 3Cproactive-site pocket. (a and b) Refined structures of EV-93 3Cpro
in complex with compound 1 (a) and rupintrivir (b). Both compounds are represented as sticks, with their 2Fo?Fc-weighted electron density
contoured at 1.5 ? and represented as a blue mesh and their Fo?Fc-weighted-difference electron densities contoured at ?3 ? and ?3 ? and shown
in red and green, respectively. EV-93 3Cproresidues interacting with the compounds are shown as sticks and are labeled. (c) Conservation of the
compound 1 and rupintrivir binding pocket. The rupintrivir structure (yellow carbon atoms) is overlaid on the EV-93 3Cpro-compound 1 structure
(green carbon atoms). The EV-93 3Cpromolecular surface is colored from cyan to magenta for variable to conserved residues, respectively, based
on the multiple-sequence alignment presented in Fig. 6. EV-93 3Cproresidues interacting with the antiviral compounds are shown as sticks and
are labeled. Positions P4 to P1? are labeled in all figures.
10770COSTENARO ET AL. J. VIROL.
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bition by these compounds of 3Cprofrom HRVs and EVs (40,
41) and corroborate their strong antiviral effect against a broad
spectrum of picornaviruses.
Rupintrivir and compound 1 as potential antivirals against
all EVs. To characterize the molecular interactions of com-
pound 1 and rupintrivir with EV-93 3Cpro, we cocrystallized
their complexes and solved their crystallographic structures at
very high resolutions. The structure of EV-93 3Cpro-compound
1 is the first known structure of a protease in complex with this
antiviral agent. The electron density allowed us to unequivo-
cally and precisely build one molecule of compound 1 or rupin-
trivir per protein (Fig. 5). In both cases, the inhibitor electro-
philic ? carbon (Fig. 1, asterisk) is covalently bound to the
active-site Cys147after its Michael addition, forming a stable
tetrahedral adduct and resulting in the irreversible inactivation
of the protease. Compound 1 binds to EV-93 3Cproin a par-
tially extended conformation with its peptidomimetic back-
bone making antiparallel ?-sheet-type hydrogen bonds with
part of the solvent-exposed ? strand, E2, of the protein (resi-
dues 162 to 164). The inhibitor’s P4 part (Fig. 5a) lies in the
deep groove formed by the ? strands E2, F2, and B2 and
interacts with protein residues 125 to 128, 164 to 165, 168, and
170. The P3 part makes two main-chain hydrogen bonds with
Gly163–164, and the hetero ring is mainly solvent exposed, in-
teracting with Gly128only on one side. P2 2-propynyl stacks
against His40, and residues 71 and 127 further constrain its
conformation. The P1 part is deeply inserted between ?
strands E2 (residues 162 to 164) and loop 142 to 144, making
this pocket wider by about 1 Å than that of the native structure.
A P1 glutamine-like side chain makes hydrogen bonds with
Thr142and His161, most probably mimicking the recognition of
the natural substrate P1 Gln, which is highly conserved in 3C
cleavage sequences. The P1? carbonyl oxygen of the ethyl ester
is positioned above the oxyanion hole formed by the amide
groups of Cys147, Gln146, and Gly145but makes a hydrogen
bond only with the latter. The ethoxycarbonyl group is more
mobile and is either solvent exposed or interacts with residues
23 to 25.
FIG. 6. Multiple-sequence alignment of 3Cpros for 11 EVs representing the currently known species diversity of the Enterovirus genus. The
sequence names indicate the names and accession IDs of the viruses, and the corresponding species are indicated at the ends of the sequences.
Identical and similar residues are boxed in black and gray, respectively. Residues making side chain interactions or only main-chain interactions
between EV-93 3Cproand compound 1 are highlighted in red and blue, respectively. Additional residues of EV-93 3Cprothat interact with
rupintrivir are highlighted in magenta. Catalytic residues Cys174, His40, and Glu71are indicated by red asterisks. Secondary structural elements
correspond to EV-93 3Cpro, for which the sequence (top line) has been modified with N- and C-terminal extensions in this study. BEV, bovine
enterovirus; PEV, porcine enterovirus; SEV, simian enterovirus.
VOL. 85, 2011 ANTIVIRAL INHIBITION OF EV-93 MAIN PROTEASE10771
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The binding mode of rupintrivir 1 to EV-93 3Cprois very
similar to that of compound 1. The differences between the two
antivirals lie in the least-conserved P3 and P2 positions (Fig. 1
and 5b), where a 2-pyridon-1,3-diyl group that cycles with the
following amine is replaced by a valine amino acid (4-methyl-
pentan-2-one-1,3-diyl) and the ethynyl group is replaced by a
1-fluorobenzen-4-yl group in rupintrivir. Variations at the P3
position imply the loss of van der Waals contacts with Gly128in
the case of rupintrivir, since its valine side chain is exposed to
the solvent and does not interact with the protein. In contrast,
compared with that in compound 1 the P2 substitution in
rupintrivir results in additional stacking interactions of the
1-flurobenzen-4-yl ring with His40and Glu71side chains and
new interactions of the fluorine atom with Thr130and Arg39.
The stacking interactions of the inhibitor ring with His40and
Glu71were also observed in the HRV-2 3Cpro-rupintrivir com-
plex (30), as was the H bond of the fluorine atom with Thr130,
which is an Asn in HRV-2 3Cpro. The contact with Arg39is not
present in the HRV-2 3Cpro-rupintrivir complex, since this
residue is a Thr in the HRV-2 protein and has a much shorter
side chain. The enhanced interactions of rupintrivir with the
proteases at site S2 could explain why EC50s for this antiviral
are almost systematically lower than those obtained for com-
pound 1 against EV-93 and HRVs (see above and references
40 and 41). Interactions of rupintrivir with EV-93 and HRV-2
3Cpros also changed for the following residues: Leu125and
G128, which correspond to Ile and Ser in the HRV-2 protein,
respectively, and Asn165and Thr142, which have different side
chain conformations in the two complexes.
A noticeable feature of the binding pocket is the conserva-
tion of EV-93 3Cproresidues interacting with compound 1
within all EVs. For 11 EV species representing the entire
genetic diversity of this genus, most of the 3Cproresidues
making side chain interactions (71%; in red in Fig. 6) or only
main-chain interactions (62%; in blue) with compound 1 are
identical or physico-chemically similar. The four not strictly
conserved residues involved in side chain interactions (Leu125,
Gly128, Thr130, and Phe170) make steric interactions with com-
pound 1 or rupintrivir that are compatible with the amino acid
diversity observed. Thr130interacts only with the fluorine end
of the 1-fluorobenzen-4-yl group of rupintrivir (Fig. 1, P2), as
was discussed above. A higher level of conservation of the 3C
inhibitor binding pocket is observed in HRV serotypes (5).
In studies to control natural rhinovirus infection by 3Cpro
inhibitors, compound 1 or rupintrivir showed unsatisfactory
performance and was therefore excluded from further clinical
development (39). Our results with EV-93 indicate that these
compounds could be valuable antivirals against other EV spe-
cies. The high level of conservation among EVs of the residues
forming the 3Cprobinding pockets for compound 1 and rupin-
trivir and the broad-spectrum antiviral activity of these com-
pounds in vitro reinforce their potential as excellent candidates
for developing potent antivirals against all EVs (28). These
results also suggest that the level of conservation of the resi-
dues forming the substrate binding pocket could be useful in
the process of designing antiviral compounds against new,
Conclusion. In summary, we report the first determination
of the crystallographic structure of the main protease from a
human enterovirus B (EV-93 3Cpro) alone and in complex with
the HRV antiviral molecules compound 1 and rupintrivir at
resolutions of 1.9, 1.3, and 1.5 Å, respectively. The chymotryp-
sin-like fold of the protease presents the catalytic triad Cys-
His-Glu in the cleft between the two six-stranded ? barrels,
adjacent to a canonically configured oxyanion hole. We showed
that compound 1 and rupintrivir inhibit the proteolytic activity
of EV-93 3Cproin vitro and are active against EV-93 in infected
cells. The primary and tertiary structures of the 3Cprobinding
pockets for these two compounds are highly conserved among
EVs, which explains their broad-spectrum antiviral capacity.
These results reinforce the structural framework for designing
antiviral drugs against the 3Cproto control enterovirus infec-
This work was supported by the EC (Integrated Project VIZIER,
contract LSHG-CT-2004-511960, and Cooperation Project SILVER,
GA 260644), the Spanish Ministerio de Ciencia e Innovacio ´n
(BFU2008-02372/BMC to M.C.), and the Generalitat de Catalunya
(2009SGR-1309 to M.C.).
We thank Violaine Lantez and Karen Dalle for technical assistance
in protein production. We also thank the personnel of the Automated
Crystallography Platform (Barcelona Science Park, Spain) for techni-
cal assistance with crystallization and preliminary X-ray data collec-
tion; Chris Lauber for help with alignment; and Igor Sidorov, Alexan-
der Kravchenko, and Dmitry Samborskiy for administering the Viralis
software platform. We acknowledge the European Synchrotron Radi-
ation Facility for providing access to synchrotron radiation facilities.
We thank Pfizer for generously providing rupintrivir and compound 1.
1. Adams, P. D., et al. 2010. PHENIX: a comprehensive Python-based system
for macromolecular structure solution. Acta Crystallogr. D Biol. Crystallogr.
2. Anand, K., et al. 2002. Structure of coronavirus main proteinase reveals
combination of a chymotrypsin fold with an extra alpha-helical domain.
EMBO J. 21:3213–3224.
3. Bergmann, E. M., S. C. Mosimann, M. M. Chernaia, B. A. Malcolm, and
M. N. James. 1997. The refined crystal structure of the 3C gene product from
hepatitis A virus: specific proteinase activity and RNA recognition. J. Virol.
4. Berrow, N. S., et al. 2006. Recombinant protein expression and solubility
screening in Escherichia coli: a comparative study. Acta Crystallogr. D Biol.
5. Binford, S. L., et al. 2005. Conservation of amino acids in human rhinovirus
3C protease correlates with broad-spectrum antiviral activity of rupintrivir, a
novel human rhinovirus 3C protease inhibitor. Antimicrob. Agents Che-
6. Binford, S. L., et al. 2007. In vitro resistance study of rupintrivir, a novel
inhibitor of human rhinovirus 3C protease. Antimicrob. Agents Chemother.
7. Bjorndahl, T. C., L. C. Andrew, V. Semenchenko, and D. S. Wishart. 2007.
NMR solution structures of the apo and peptide-inhibited human rhinovirus
3C protease (serotype 14): structural and dynamic comparison. Biochemistry
8. Care, S., et al. 2008. The translation of recombinant proteins in E. coli can
be improved by in silico generating and screening random libraries of a
?70/?96 mRNA region with respect to the translation initiation codon.
Nucleic Acids Res. 36:e6.
9. Collaborative Computational Project Number 4. 1994. The CCP4 suite:
programs for protein crystallography. Acta Crystallogr. D Biol. Crystallogr.
10. Cui, S., et al. 2011. Crystal structure of human enterovirus 71 3C protease.
J. Mol. Biol. 408:449–461.
11. De Palma, A. M., I. Vliegen, E. De Clercq, and J. Neyts. 2008. Selective
inhibitors of picornavirus replication. Med. Res. Rev. 28:823–884.
12. Dougherty, J. D., N. Park, K. E. Gustin, and R. E. Lloyd. 2010. Interference
with cellular gene expression, p. 165–180. In E. Ehrenfeld, E. Domingo, and
R. P. Roos (ed.), The picornaviruses. ASM Press, Washington, DC.
13. Dougherty, W. G., and B. L. Semler. 1993. Expression of virus-encoded
proteinases: functional and structural similarities with cellular enzymes. Mi-
crobiol. Rev. 57:781–822.
14. Edgar, R. C. 2004. MUSCLE: multiple sequence alignment with high accu-
racy and high throughput. Nucleic Acids Res. 32:1792–1797.
10772 COSTENARO ET AL.J. VIROL.
on November 20, 2013 by guest
15. Emsley, P., and K. Cowtan. 2004. Coot: model-building tools for molecular Download full-text
graphics. Acta Crystallogr. D Biol. Crystallogr. 60:2126–2132.
16. Gorbalenya, A. E., et al. 2010. Practical application of bioinformatics by the
multidisciplinary VIZIER consortium. Antiviral Res. 87:95–110.
17. Gorbalenya, A. E., and E. J. Snijder. 1996. Viral cysteine proteinases. Per-
spect. Drug Discov. Des. 6:64–86.
18. Gouet, P., E. Courcelle, D. I. Stuart, and F. Metoz. 1999. ESPript: analysis of
multiple sequence alignments in PostScript. Bioinformatics 15:305–308.
19. Hayden, F. G., et al. 2003. Phase II, randomized, double-blind, placebo-
controlled studies of rupintrivir nasal spray 2-percent suspension for preven-
tion and treatment of experimentally induced rhinovirus colds in healthy
volunteers. Antimicrob. Agents Chemother. 47:3907–3916.
20. Junttila, N., et al. 2007. New enteroviruses, EV-93 and EV-94, associated
with acute flaccid paralysis in the Democratic Republic of the Congo. J. Med.
21. Kaiser, L., C. E. Crump, and F. G. Hayden. 2000. In vitro activity of pleco-
naril and AG7088 against selected serotypes and clinical isolates of human
rhinoviruses. Antiviral Res. 47:215–220.
22. Kitamura, N., et al. 1981. Primary structure, gene organization and polypep-
tide expression of poliovirus RNA. Nature 291:547–553.
23. Knowles, N. J., T. Hovi, A. M. Q. King, and G. Stanway. 2010. Overview of
taxonomy, p. 19–32. In E. Ehrenfeld, E. Domingo, and R. P. Roos (ed.), The
picornaviruses. ASM Press, Washington, DC.
24. Krissinel, E., and K. Henrick. 2007. Inference of macromolecular assemblies
from crystalline state. J. Mol. Biol. 372:774–797.
25. Landau, M., et al. 2005. ConSurf 2005: the projection of evolutionary con-
servation scores of residues on protein structures. Nucleic Acids Res. 33:
26. Laskowski, R. A., M. W. MacArthur, D. S. Moss, and J. M. Thornton. 1993.
PROCHECK: a program to check the stereochemical quality of protein
structures. J. Appl. Cryst. 26:283–291.
27. Lee, C.-C., et al. 2009. Structural basis of inhibition specificities of 3C and
3C-like proteases by zinc-coordinating and peptidomimetic compounds.
J. Biol. Chem. 284:7646–7655.
28. Lee, E. S., et al. 2007. Development of potent inhibitors of the coxsackievirus
3C protease. Biochem. Biophys. Res. Commun. 358:7–11.
29. Matthews, B. W. 1968. Solvent content of protein crystals. J. Mol. Biol.
30. Matthews, D. A., et al. 1999. Structure-assisted design of mechanism-based
irreversible inhibitors of human rhinovirus 3C protease with potent antiviral
activity against multiple rhinovirus serotypes. Proc. Natl. Acad. Sci. U. S. A.
31. Matthews, D. A., et al. 1994. Structure of human rhinovirus 3C protease
reveals a trypsin-like polypeptide fold, RNA-binding site, and means for
cleaving precursor polyprotein. Cell 77:761–771.
32. McCoy, A. J., R. W. Grosse-Kunstleve, L. C. Storoni, and R. J. Read. 2005.
Likelihood-enhanced fast translation functions. Acta Crystallogr. D Biol.
33. Moriarty, N. W., R. W. Grosse-Kunstleve, and P. D. Adams. 2009. Electronic
ligand builder and optimization workbench (eLBOW): a tool for ligand
coordinate and restraint generation. Acta Crystallogr. D Biol. Crystallogr.
34. Mosimann, S. C., M. M. Cherney, S. Sia, S. Plotch, and M. N. James. 1997.
Refined X-ray crystallographic structure of the poliovirus 3C gene product.
J. Mol. Biol. 273:1032–1047.
35. Murshudov, G. N., A. A. Vagin, and E. J. Dodson. 1997. Refinement of
macromolecular structures by the maximum-likelihood method. Acta Crys-
tallogr. D Biol. Crystallogr. 53:240–255.
36. Norder, H., et al. 2011. Picornavirus non-structural proteins as targets for
new anti-virals with broad activity. Antiviral Res. 89:204–218.
37. Otwinowski, Z., and W. Minor. 1997. Processing of X-ray diffraction data
collected in oscillation mode, p. 307–326. In C. W. J. Carter and R. M. Sweet
(ed.), Methods in enzymology, vol. 276, part A. Academic Press, New York,
38. Palmenberg, A. C. 1990. Proteolytic processing of picornaviral polyprotein.
Annu. Rev. Microbiol. 44:603–623.
39. Patick, A. K. 2006. Rhinovirus chemotherapy. Antiviral Res. 71:391–396.
40. Patick, A. K., et al. 1999. In vitro antiviral activity of AG7088, a potent
inhibitor of human rhinovirus 3C protease. Antimicrob. Agents Chemother.
41. Patick, A. K., et al. 2005. In vitro antiviral activity and single-dose pharma-
cokinetics in humans of a novel, orally bioavailable inhibitor of human
rhinovirus 3C protease. Antimicrob. Agents Chemother. 49:2267–2275.
42. Perrakis, A., R. Morris, and V. S. Lamzin. 1999. Automated protein model
building combined with iterative structure refinement. Nat. Struct. Biol.
43. Porter, A. G. 1993. Picornavirus nonstructural proteins: emerging roles in
virus replication and inhibition of host cell functions. J. Virol. 67:6917–6921.
44. Qiu, J. 2008. Enterovirus 71 infection: a new threat to global public health?
Lancet Neurol. 7:868–869.
45. Skern, T., et al. 2002. Structure and function of picornavirus proteinases, p.
199–212. In B. L. Semler and E. Wimmer (ed.), Molecular biology of picor-
naviruses. American Society for Microbiology, Washington, DC.
46. Steller, I., R. Bolotovsky, and M. G. Rossmann. 1997. An algorithm for
automatic indexing of oscillation images using Fourier analysis. J. Appl.
47. Sweeney, T. R., N. Roque-Rosell, J. R. Birtley, R. J. Leatherbarrow, and S.
Curry. 2007. Structural and mutagenic analysis of foot-and-mouth disease
virus 3C protease reveals the role of the beta-ribbon in proteolysis. J. Virol.
48. Wecker, I., and V. ter Meulen. 1977. RD cells in the laboratory diagnosis of
enteroviruses. Med. Microbiol. Immunol. 163:233–240.
49. Ziebuhr, J., E. J. Snijder, and A. E. Gorbalenya. 2000. Virus-encoded pro-
teinases and proteolytic processing in the Nidovirales. J. Gen. Virol. 81:853–
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