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J Virol. Oct 2011; 85(19): 10319–10331.
PMCID: PMC3196414

Enterovirus 71 and Coxsackievirus A16 3C Proteases: Binding to Rupintrivir and Their Substrates and Anti-Hand, Foot, and Mouth Disease Virus Drug Design [down-pointing small open triangle]


Enterovirus 71 (EV71) and coxsackievirus A16 (CVA16) are the major causative agents of hand, foot, and mouth disease (HFMD), which is prevalent in Asia. Thus far, there are no prophylactic or therapeutic measures against HFMD. The 3C proteases from EV71 and CVA16 play important roles in viral replication and are therefore ideal drug targets. By using biochemical, mutational, and structural approaches, we broadly characterized both proteases. A series of high-resolution structures of the free or substrate-bound enzymes were solved. These structures, together with our cleavage specificity assay, well explain the marked substrate preferences of both proteases for particular P4, P1, and P1′ residue types, as well as the relative malleability of the P2 amino acid. More importantly, the complex structures of EV71 and CVA16 3Cs with rupintrivir, a specific human rhinovirus (HRV) 3C protease inhibitor, were solved. These structures reveal a half-closed S2 subsite and a size-reduced S1′ subsite that limit the access of the P1′ group of rupintrivir to both enzymes, explaining the reported low inhibition activity of the compound toward EV71 and CVA16. In conclusion, the detailed characterization of both proteases in this study could direct us to a proposal for rational design of EV71/CVA16 3C inhibitors.


Hand, foot, and mouth disease (HFMD) is a common viral illness among infants and young children, with clinical characterizations of prodromal fever followed by pharyngitis, mouth ulcers, and a rash on the hands and feet (7, 8). Human enterovirus 71 (EV71) and coxsackievirus A16 (CVA16) are the two major causative agents of HFMD. Clinically, infections with the two viruses manifesting as HFMD are indistinguishable. However, EV71 can also result in severe neurological diseases, such as aseptic meningitis and acute flaccid paralysis (AFP), and even death (7, 8, 38). Since the first reported case of HFMD in New Zealand in 1957 (31), it has continued to spread globally and is a continuing threat to global public health (2, 5, 14, 16, 17, 29). In the last decade, regularly reoccurring outbreaks of HFMD have been relatively centralized in the Asia-Pacific region (9, 10, 33, 40). In 2008, an unexpected HFMD outbreak hit mainland China, resulting in >480,000 cases nationwide, >120 fatal cases, and great economic losses (41). Thus far, no prophylactic or therapeutic method is available to treat HFMD (43). These urgent issues and the potential for an HFMD pandemic in the future prompted us to exploit a more effective approach to combat these highly pathogenic viruses.

Both EV71 and CVA16 belong to the genus Enterovirus in the family Picornaviridae (30). Like other members of the family, both viruses contain a genome of single-stranded, positive-sense RNA with a single open reading frame (ORF) encoding a large polyprotein precursor. In infected cells, this polyprotein is further cleaved into four structural (Vp1 to Vp4) and seven nonstructural (2A to 3D) proteins via the virus-encoded 2A and 3C proteases. Upon translation of the polyprotein, the 2A protease automatically cleaves the joining sequence between Vp1 and 2A. However, 3C is the main protease, because it is responsible for the cleavage of the other eight junction sites within the remainder of the polyprotein (30). In addition, the 3C protease also acts as a constituent of the replication complex via its binding to the 5′ untranslated region (UTR) of the viral genomic RNA (32). There are also reports demonstrating that the EV71 3C facilitates progeny virus production and helps the virus evade host antiviral immunity by interaction with or cleavage of host factors (22, 37). The pivotal roles of the 3C protease in the life cycles of EV71 and CVA16 make it an ideal target for anti-HFMD drug design.

Rupintrivir (also referred to as AG7088) is a drug that was initially designed as a specific inhibitor of the human rhinovirus (HRV) 3C protease but was later found to exhibit broad-spectrum antiviral activity against other members of the family Picornaviridae (6, 23, 27). Compared to its extremely high potency against HRV 3C, the compound exhibits nearly 2 orders of magnitude lower inhibition activity toward 3C proteases from EV71 and CVA16 (21, 36). Therefore, structure-based modifications of rupintrivir are urgently required to generate more specific and effective inhibitors of EV71/CVA16 3C, which necessitates assistance from high-resolution structures of the free and/or the substrate-bound and/or the inhibitor-bound enzymes. However, with the atomic structures of EV71 proteins (such as 3C and 3D RdRp) starting to be unveiled recently (12, 39), to date, only a 3-Å structure of EV71 3C is available (12).

Here, we thoroughly characterize the 3C proteases from EV71 and CVA16 by defining their substrate specificities and reporting a series of high-resolution structures of both enzymes in free-, peptide-bound, or inhibitor-bound form. These data enabled us to explain the substrate preferences of EV71 and CVA16 3Cs for particular P4, P1, and P1′ residue types and their relative malleability for P2 amino acids. Furthermore, a half-closed S2 subsite and a size-reduced S1′ subsite are revealed by the enzyme-rupintrivir complexes, which we believe are unique to the 3C proteases of members of human enterovirus group A. These structural features cause rupintrivir to tilt the P1′ ester group away from the enzyme after binding to EV71/CVA16 3C, demonstrating that the inhibitor is not as well accommodated as in HRV 3C. This explains why rupintrivir exhibits dramatically lower efficacy for EV71 and CVA16 than for HRV. We also propose a strategy for rupintrivir modification to more effectively inhibit EV71 and CVA16.


Expression and purification of the 3C proteases from EV71 and CVA16.

The original viral strains selected in this study are Anhui1-09-China (GenBank accession no. GQ994988) for EV71 and Beijing0907 (GenBank accession no., ACV33372) for CVA16. The DNA fragment encoding the full-length 3C protease from either virus was synthesized such that the protease gene was immediately followed by a hexahistidine tag coding sequence (Takara Corporation). The gene was then subcloned into pET-21a via NdeI and XhoI restriction sites.

For site-directed mutagenesis, the plasmid encoding the wild-type EV71 (or CVA16) 3C protease was used as a template to generate the construct coding for the C147A mutant enzyme. The following primer pairs were used: CVA16-3C-C147A-mutant-forward, 5′-GCAGGACAGGCTGGAGGTGTG-3′; CVA16-3C-C147A-mutant-reverse, 5′-CACACCTCCAGCCTGTCCTGC-3′; EV71-3C-C147A-mutant-forward, 5′-GCAGGACAGGCTGGGGGAGTG-3′; EV71-3C-C147A-mutant-reverse, 5′-CACTCCCCCAGCCTGTCCTGC-3′.

Both mutant constructs were successfully obtained using the Phusion Site Mutagenesis Kit (NEB) according to the manufacturer's instructions. All the constructs made in this study were verified by direct DNA sequencing.

Each of the four proteases was expressed and purified using the following protocol. One microliter of the plasmid was transformed into Escherichia coli BL-21(DE3) competent cells, and cultures were grown to an optical density at 600 nm (OD600) of 0.8 in LB medium (supplemented with ampicillin) and induced with 0.1 mM IPTG (isopropyl-β-d-thiogalactopyranoside) for 5 h at 37°C. The cells were harvested and resuspended in cold lysis buffer (50 mM HEPES [pH 6.5] and 150 mM NaCl) and homogenized by sonication. Cell debris was removed by centrifugation at 16,000 rpm for 30 min. The resultant supernatant was added to Ni-nitrilotriacetic acid (NTA) resin (GE) and gently mixed at 4°C for 1 h. The resin was then collected and treated with 2.5 M NaCl in 50 mM HEPES (pH 6.5) for another 5 h. This extra step disrupted the nonspecific interaction between the 3C protease and RNA contaminants. The nonspecific contaminants were then removed by washing the resin with 20 column volumes of lysis buffer. The 3C protease was subsequently eluted with 200 mM imidazole in lysis buffer and concentrated to an appropriate volume. The eluate was further purified by gel filtration chromatography using a Superdex 75 Hiload 16/60 column (GE). The enzyme fractions were pooled and concentrated to approximately 10 to 12 mg/ml in buffer containing 50 mM HEPES (pH 6.5), 150 mM NaCl, 1 mM EDTA, and 5 mM dithiothreitol (DTT).

Preparation of the peptide-enzyme, as well as rupintrivir-enzyme, complexes.

The peptides used in this study were purchased from ChinaPeptides Co., Ltd., with purity of over 95%. Each peptide was dissolved in 100% dimethyl sulfoxide (DMSO) to a final concentration of 50 mM and stored in small aliquots at −80°C before use in the cleavage assay or the cocrystallization trials. Rupintrivir was purchased from Toronto Research Chemicals Inc. and dissolved at 30 mM in DMSO.

The noncovalent peptide-enzyme complex was prepared at 1:5 molar ratio of enzyme to peptide, since this has been shown to work well for related proteases (28, 45). Typically 150 μl of the C147A mutant 3C protease at 12 mg/ml was mixed with 30 μl of the 50 mM peptide and incubated with rotation at 4°C overnight. This yields a complex with a 10-mg/ml protein concentration that is ready for subsequent crystallization screenings.

A similar method was used to obtain the complex of rupintrivir bound to EV71/CVA16 3C, except that the enzyme: inhibitor molar ratio was set to 1:3. After an overnight incubation of 150 μl wild-type enzyme (12 mg/ml) with 30 μl rupintrivir solution (30 mM), the complex preparation still has a protein concentration of 10 mg/ml and is immediately screened for crystallizing conditions.

Crystallization, data collection, and structure determination.

All the crystals were obtained by the hanging-drop vapor diffusion method at 18°C. For the peptide-enzyme complexes, we screened a series of 16-mer peptides (corresponding to the substrate-peptides in this study) or 10-mer peptides (corresponding to the “P” portion of the respective substrate-peptide) to cocrystallize with the C147A mutant proteases from either EV71 or CVA16. Finally, only peptide FAGLRQAVTQ with the CVA16 enzyme and peptide KPVLRTATVQGPSLDF with the EV71 enzyme yielded crystals. The final conditions used to crystallize each protein preparation were as follows: (i) CVA16 3C, 0.1 M Bis-Tris (pH 5.5), 0.1 M ammonium acetate, and 17% (wt/vol) polyethylene glycol (PEG) 10000; (ii) FAGLRQAVTQ plus CVA16-C147A 3C, 0.1 M HEPES, pH 7.5, 0.2 M lithium sulfate, and 25% (wt/vol) PEG 3350; (iii) rupintrivir plus CVA16 3C, 0.1 M sodium acetate (pH 4.6), 0.1 M magnesium chloride, and 25% (wt/vol) PEG 4000; (iv) KPVLRTATVQGPSLDF plus EV71-C147A 3C, 0.1 M Tris-HCl (pH 8.5), 20 mM lithium sulfate, and 25% (wt/vol) PEG 5000 monomethyl ether; and (v) rupintrivir plus EV71 3C, 0.1 M sodium acetate (pH 4.6), 0.2 M ammonium sulfate, and 25% (wt/vol) PEG 4000.

Seventeen percent (vol/vol) glycerol in mother liquor worked well as a cryoprotectant for all of the crystal species in this study. For data collection, a single crystal was mounted on a nylon loop and flash cooled with a nitrogen gas stream at 100 K. Diffraction data for the rupintrivir plus EV71 3C crystal were collected at beam line NE3A (wavelength, 1.0000 Å) of KEK, Japan, while data sets for the rest of the crystal species were obtained with an in-house Rigaku MicroMax007 rotating-anode X-ray generator equipped with an image plate detector. Raw data were processed and scaled using HKL2000 (26).

All crystal structures were determined by the molecular-replacement method. We first solved the structure of CVA16 3C using the CVB3 3C structure (Protein Data Bank [PDB] code 2ZTY) as the search model. Then, with the newly obtained CVA16 3C structure as the input model, the remaining structures were solved. In each case, the initial model was obtained by MOLREP and subsequently refined in Refmac5 (CCP4 suite) (11) using rigid-body refinement and maximum-likelihood procedures. Then, a series of iterative cycles of manual rebuilding were performed in COOT (13) and followed by refinement with Phenix.refine (1). During the course of model building and refinement, the stereochemistry of the structure was monitored by PROCHECK (19). The detailed statistics are summarized in Table 1. All figures were generated using PyMOL (http://pymol.sourceforge.net) and ESPript (15).

Table 1.
Data collection and refinement statistics

In vitro cleavage assay.

For each peptide (natural or mutated), the cleavage assay was performed in a reaction volume of 200 μl, using 2.1 μl enzyme (10 mg/ml) and 2 μl peptide (50 mM) in the reaction buffer (50 mM HEPES, pH 6.5, 150 mM NaCl, 1 mM EDTA, 2 mM DTT, 10% glycerol) to yield a final concentration of 5 μM enzyme and 500 μM peptide. Cleavage reactions were routinely incubated at 25°C for 5 min and then terminated by the addition of an equal volume of 2% trifluoroacetic acid. The samples were analyzed by reverse-phase high-performance liquid chromatography (HPLC) on a C18 column (4.6 by 250 mm) using a 20-min, 5 to 45% linear gradient of acetonitrile in 0.1% trifluoroacetic acid. The absorbance was monitored at 215 nm. The area of the product peak was calculated and transformed into a product-peptide concentration, based on which the efficiency of each peptide being processed by EV71/CVA16 3C could be determined. The cleavage efficiency was defined as the average rate within 5 min of a selected peptide being cleaved by 5 μM either protease with an initial peptide concentration of 500 μM.

Enzyme inhibition assay.

A fluorescent peptide with the sequence Dabcyl-KIGNTIEALFQGPPKFRE-Edans (purchased from GL BioChem [Shanghai] Ltd., with a purity of 90%) was used as the substrate for the inhibition assay. This peptide contains the 2C/3A junction site. In our in vitro cleavage assay, the site was demonstrated to be most efficiently processed by both 3C proteases. With excitation at 340 nm, enhanced fluorescence could be observed at 490 nm after the cleavage of the peptide. This enabled us to monitor the peptide cleavage in real time.

To determine the 50% inhibitory concentrations (IC50s) of rupintrivir against EV71/CVA16 3C, 1 μM protease, 20 μM fluorescent peptide, and gradient concentrations of inhibitor were mixed in a buffer containing 50 mM HEPES (pH 6.5), 150 mM NaCl, 1 mM EDTA, 2 mM DTT, and 10% glycerol. The initial velocities of the enzymatic reactions (within the first 5 min) were determined and fitted to a sigmoidal dose-response equation with nonlinear regression analysis using the program GraphPad Prism. The data from three independent assays were used as input for Prism to calculate the IC50 and 95% confidence interval values.

Protein structure accession numbers.

The atomic coordinates and structure factors of all the structures solved in this study have been deposited in the Protein Data Bank (Table 1).


Cleavage specificities of EV71 and CVA16 3C proteases.

We first determined the cleavage efficiencies of EV71 and CVA16 3C proteases for the junctions derived from both viruses. The two 3Cs we characterized in this study were from strain Anhui1-09-China (for EV71) and Beijing0907 (for CVA16). The whole-genome sequence for isolate Beijing0907 is not available; therefore, the junction sequence information for the coxsackievirus was obtained from strain shzh01. The two CVA16 isolates encode 3C proteases that differ from each other at only one position (V103I).

As with other picornaviruses, the 3C proteases from both EV71 and CVA16 are responsible for the cleavage of eight junction sites within the respective viral polyproteins. All these sites contain a Q/G or Q/S scissor bond (Fig. 1). According to the nomenclature of Berger and Schechter (4), the amino acids within each junction site are designated “P” or “P′” residues. The newly generated C terminus after the cleavage of the scissor bond is denoted P1, preceded by the P2, P3, etc., residues, and the N terminus yielded by cleavage is denoted P1′, followed by the P2′, P3′, etc., residues. Accordingly, those sites within the 3C protease that accommodate substrate “P” or “P′” residues are designated “S” or “S′” subsites. Overall, conservation in sequence at each site between the EV71 and CVA16 viruses was observed for the junctions within 2A to 2C and 3A to 3C of the viral polyprotein, whereas the joining sequences are relatively discrepant for the Vp2/Vp3, Vp3/Vp1, and 3C/3D junctions. Therefore, 11 peptides (P10 to P6′) covering all eight cleavage sites within the polyprotein precursors of both viruses were synthesized and investigated for their susceptibilities to 3C cleavage (Table 2).

Fig. 1.
Overview of the domain organization within the viral polyproteins of EV71 and CVA16. The single ORF of EV71 or CVA16 could be further divided into three regions (P1, P2, and P3). The four structural subunits encoded by the P1 region and the seven nonstructural ...
Table 2.
Cleavage efficiencies of EV71 and CVA16 3C proteases toward SPs or MPsa

On the whole, for each substrate peptide (SP) tested, the proteases exhibit similar cleavage efficiencies, which is consistent with the high sequence identity between the two enzymes (94%). Of the 11 SPs, the one that could be most efficiently processed was SP-7, which corresponds to the 2C/3A junction site, indicating fast and effective separation of the P3 region from the P2 region of the viral polyprotein. Both enzymes also show high enzymatic activities toward peptides SP-1, -9, -10, and -11, corresponding to Vp2/Vp3 (EV71), 3B/3C (CVA16), 3C/3D (EV71), and 3C/3D (CVA16) junctions, respectively. Moderate proteolytic efficiency was observed for peptide SP-8, representing the sequence joining coxsackievirus 3A and 3B subunits. Finally, the substrates to be least processed are those peptides representing the junction sites within the P2 region of the polyprotein precursor, including SP-5 (2A/2B of CVA16) and SP-6 (2B/2C of CVA16), as well as SP-2; the sequence links the Vp2 and Vp3 subunits in CVA16 (Table 2). We failed to determine the cleavage efficiencies of both proteases toward peptides SP-3 and SP-4 due to severe dissolving problems for the two peptides. Still, it was quite unexpected that the Vp2/Vp3 site of EV71 was much more (about 17-fold) efficiently cleaved than that of CVA16.

To further elucidate the substrate specificities of the EV71 and CVA16 3Cs, a series of mutational peptides (MPs) with substitutions for amino acids in the P6-P1′ region of SP-1 were tested in vitro. The “P-side” portion of SP-1 was also successfully cocrystallized with the CVA16 enzyme; therefore, the cleavage result for these MPs is presented alongside a description of the substrate-enzyme complex structure. Overall, it is notable that both EV71 and CVA16 3Cs exhibit marked preference for P4, P1, and P1′ residue types, whereas there is little sequence conservation at the rest of the P and P′ positions in the natural cleavage sites of both proteases (Table 2). Accordingly, within the context of SP-1 sequence, mutations at the P4 and P1′ positions could dramatically reduce the rate of peptide cleavage by EV71/CVA16 3C. Variations at the P6, P5, and P2 positions only moderately affect its ability to be processed by both proteases (Table 2).

Overall structures of CVA16 3C and EV71-C147A 3C.

As expected, the CVA16 3C protease also forms a chymotrypsin-like fold, which is a typical feature of all reported structures of 3C/3C-like proteases (3, 20, 23, 24, 35, 42). The CVA16 3C structure contains 179 amino acids from residues L4 to E182, forming two domains. The first domain is largely composed of a 7-stranded β-barrel structure (aI to gI), surrounded by surface loops. The second domain also contains a compact barrel core, which is composed of seven β-strands (aII to cII and fII to iII) arranged in an antiparallel manner. This core structure is further flanked by the most N-terminal α-helix (helix A) and a strand-loop-strand β-ribbon structure (dII-eII), which (according to the previous reports on other 3C proteases) should play an important role in substrate recognition by the enzyme (34, 45). Overall, the two domains are connected via a long loop (amino acids 78 to 100) over the “rear” surface of the molecule. On the “face” side, three closely positioned residues (H40, E71, and C147) comprise the canonical catalytic triad residing in the open cleft formed by the two domains (Fig. 2 A).

Fig. 2.
Overall structures of the 3C proteases from CVA16 and EV71. (A) Cartoon representation of the structure of CVA16 3C. Domain I (strands aI to gI) and domain II (strands aII to iII) are colored green and magenta, respectively. The α-helices are ...

Despite great effort, we failed to obtain any crystals of the wild-type EV71 3C protease. However, a catalytically mutated protease, EV71-C147A 3C, was successfully crystallized in the presence of peptide KPVLRTATVQGPSLDF (SP-9). Our initial intention was to have a complex structure with the peptide docking into the substrate binding groove of EV71 3C. Nevertheless, in the solved structure, the peptide unexpectedly formed an extended β-strand conformation, located next to strand fI of the protease molecule (Fig. 2B). In addition, a cleavage for unknown reasons between Ala7 and Thr8 must have occurred during the process of crystallization (as demonstrated by the observance of clear electron densities for the carboxyl group of Ala7), leaving only 7 residues present in the structure. These residues could form 6 H bonds with the parental protease molecule and 5 H bonds with the neighboring enzyme molecule simultaneously, thereby functioning like “glue” in crystal packing (Fig. 2C).

Overall, EV71-C147A 3C also folds into two domains and exhibits extremely high structural similarity to its coxsackievirus counterpart. A structural alignment of the two proteases yields a root mean square deviation (RMSD) of 0.365 Å for all equivalent Cα pairs (Fig. 2D). For the EV71 3C protease, all of the skeletal units (e.g., the core barrel of each domain) and structural elements involved in catalysis (including the catalytic triad and the β-ribbon related to substrate recognition) are sterically arranged exactly the same as in the CVA16 3C structure.

Unexpectedly, both structures of the free enzymes solved in this study were shown to be more structurally discrepant from a recently reported 3-Å unliganded structure of EV71 3C (12) than from other picornaviral 3C structures, such as the HRV, coxsackievirus B3 (CVB3), and poliovirus 3C proteases (20, 23, 24). The major difference lies in the substrate-recognizing β-ribbon structure connecting strands cII and fII, which displays an unusual “open” conformation in the structure reported by Cui et al. (12). Nevertheless, both proteases in this study retain this β-ribbon in a close state (Fig. 2E). We further demonstrated that this ribbon structure remains close after the binding of substrate or inhibitor molecule to the enzymes (see below).

The complex structure of a substrate-peptide with CVA16 3C.

We then sought to elucidate the substrate specificities of EV71 and CVA16 3Cs. A peptide corresponding to the P portion of the SP-1 peptide (sequence, FAGLRQAVTQ) was cocrystallized with CVA16-C147A 3C. As a whole, the peptide-bound protease has the same structure as the substrate-free enzyme and the substrate-recognizing β-ribbon of the protease maintains its close conformation. The peptide, with clear electron density for 8 amino acids, binds largely within a deep surface groove that intersects the interdomain cleft (Fig. 3 A). In common with other 3C proteases (28, 45), by forming a β-strand conformation, the peptide makes intimate contacts with the protease via an extensive network of H-bond and apolar interactions (Fig. 3B).

Fig. 3.
Noncovalent complex structure of CVA16-C147A 3C with peptide SP-1 (“P” portion). (A) Surface representation to highlight the substrate binding groove of the protease. The peptide, shown in stick form is contoured for an electron density ...

In the natural cleavage sites within the EV71 and CVA16 polyprotein precursors, the P1 position is invariably occupied by Gln. In our structure, this residue is accommodated by the S1 subsite containing H161 at the bottom and T142 located on one side wall of the pocket. These 2 residues in total form three strong hydrogen bonds (2.8 to 2.9Å) with the side chain of P1-Gln in the substrate (Fig. 3B). Therefore, the S1 pocket displays a character of extremely good chemical complementarity to the glutamate residue.

In contrast to the absolute conservatism for P1-Gln, the P2 residues of the 3C substrates from both viruses are rather variable (Table 2). Our cleavage assay with peptides containing mutations at P2 showed that amino acids with various side chains (acidic, basic, aliphatic, and polar) are acceptable. Consistent with this, a pocket with enough size to accommodate various residue side chains is observed in our structure (Fig. 3A). The P2-Thr, located at the entry of the S2 subsite, leaves most of the specificity pocket unoccupied and mainly hydrophobically interacts with L127 and I162 of the enzyme (Fig. 3B). Despite the malleability of the P2 residue, both proteases favor aliphatic or aromatic side chains at this specific position. Replacement of P2-Thr by Phe can increase the rate of cleavage by about 3-fold, whereas introduction of Glu or Lys at P2 results in a reduction of the cleavage rate. It is notable that a single mutation of P2-Thr to Lys could lead to an approximately 5-fold decrease in the processing rate. This should partially account for the observed lower processing efficiency of both proteases for the CVA16 Vp2/Vp3 junction than for that of EV71.

The side chain of P3-Val is oriented toward the bulk solvent region. The residue contacts G164 of the protease via only two backbone H bonds (Fig. 3B). This explains why, in common with other picornaviral proteases, there is no specificity for particular residues at this position in natural 3C cleavage sites. P4-Ala, in contrast, is accommodated in a highly apolar depression that is formed by the side chains of L125, L127, and F170. In EV71 and CVA16, apart from amino acids at positions P1 and P1′, the P4 residue is the only one exhibiting conservatism in the natural joining sequences (Table 2). The favored amino acid is Ala, and Gly and Val are also acceptable. This phenomenon could be well explained by the feature of the S4 subsite whose size and hydrophobic characters well suit the methyl group of alanine. The limited S4 subsite is also a clue that residues any larger than valine could not be tolerated by the proteases, which was confirmed in our in vitro cleavage assay. Both enzymes failed to cleave the MP-3 peptide, which contains a leucine at the P4 position. Extra interactions with the protease at this position include two backbone H bonds formed between the alanine and the main-chain groups of N126 and S128 (Fig. 3B).

There are no apparent pockets in the protease for the P5 to P10 residues of the substrate. Therefore, these amino acids provide only optional interactions with the protease and show great variance in sequence (Table 2). For the complex structure in this study, the Gln residue at the P5 position is located at the entry of the substrate binding groove (Fig. 3A). With its side chain oriented toward the bulk solvent, P5-Gln interacts with the enzyme only by forming an H bond with the side chain of N126. P6-Arg is the most N-terminal residue that makes intimate contact with the protease. It is arranged such that its side chain spreads over the surface of the enzyme molecule. This enables the guanidine group to interact extensively with Y122 and F124 of the enzyme (Fig. 3B). The contributions of P5-Gln and P6-Arg to the peptide-enzyme interaction were also confirmed by our cleavage assay using peptides MP-1 and MP-2. Mutation of these 2 residues to alanine, thereby eliminating the side chain-mediated H-bond contacts, could apparently reduce the rate of peptide cleavage by both proteases (1.2-fold and 1.9-fold, respectively).

P7-Leu and P8-Gly are totally solvent exposed and do not make any direct contacts with the enzyme, and P9-Ala and P10-Phe are not visible in the structure due to flexibility.

Both EV71 and CVA16 3Cs strongly favor Gly at the substrate's P1′ position. Despite the fact that serine also occurs in the natural cleavage sites, replacement of P1′-Gly by Ser could lead to more than a 20-fold decrease in the cleavage rate (Table 2). This result implies a small S1′ subsite in both proteases. Consistently, we demonstrated, via the rupintrivir-enzyme complexes, that the S1′ subsite of EV71/CV16 3C is indeed significantly smaller than those of other picornaviral 3Cs, such as HRV (see results below).

Rupintrivir exhibits structural and chemical complementarities to the CVA16/EV71 3C.

Rupintrivir is a specific HRV 3C inhibitor (23). The compound also exhibits moderate antiviral activity against EV71 and CVA16 (36). A report by Tsai et al. showed that the 50% effective concentrations (EC50) of rupintrivir for EV71 and CVA16 are 0.781 μM and 0.331 μM, respectively, based on a fluorescence resonance energy transfer (FRET) assay (36), which are almost 2 orders of magnitude higher than that of the compound for HRV (6, 27). In this study, we also experimentally determined the IC50s of rupintrivir against both EV71 and CVA16 3C proteases at the protein level using a fluorescent substrate peptide (for details, see Materials and Methods). These enzyme-based values were measured at 1.65 and 2.06 μM for EV71 3C and CVA16 3C, respectively (Table 3). The enzyme-based data obtained in this study and the cell-based results of Tsai et al. all indicate potent inhibition by rupintrivir of EV71/CVA16 3C, although not as efficient as the compound for HRV 3C. To explore the specific binding mode of rupintrivir to EV71/CVA16 3C and, thereby, to provide a structural basis for the development of EV71/CVA16 3C-specific inhibitors, we also determined the structures of both proteases in complex with rupintrivir.

Table 3.
IC50s of rupintrivir against EV71 3C and CVA16 3C

Overall, rupintrivir can be described as a peptide mimic inhibitor containing a lactam at P1, a fluorophenylalanine at P2, a Val at P3, a 5-methyl-3-isoxazole at P4, and an α,β-unsaturated ester at P1′ (Fig. 4) (23). In both proteases, the compound is accommodated in the substrate binding groove with a covalent linkage to the nucleophilic Cys147 (Fig. 5 A and B). Consistent with the extremely high structural similarities between the EV71 and CVA16 3Cs, rupintrivir exhibits the same binding mode in both structures. Superimposition of the two complex structures yields an RMSD of 0.221 Å for the equivalent Cα atoms of the protein molecules and 0.236 Å for all atoms of the inhibitor moiety (Fig. 5C). In light of these observed similarities, the structure of rupintrivir bound to CVA16 3C was used as a representative for subsequent analyses.

Fig. 4.
Chemical structure of rupintrivir. Overall, rupintrivir could be described as a peptide-mimic inhibitor containing a lactam at P1, a fluorophenylalanine at P2, a Val at P3, a 5-methyl-3-isoxazole at P4, and an α,β-unsaturated ester at ...
Fig. 5.
Rupintrivir exhibits the same mode of binding to the EV71 and CVA16 3C proteases. (A and B) Overall structure of the enzyme-inhibitor complex. The structures of rupintrivir bound to the 3C proteases from CVA16 and EV71 are shown in panels A and B, respectively. ...

As shown in Fig. 6, rupintrivir binds to CVA16 3C via a network of extensive H bonds and hydrophobic interactions. These include three hydrogen bonds provided by the P4 isoxazole group, two by the P3-Ala, four at the P1 position, and one by the terminal P1′ ester group. Conversely, the P2 group and the aliphatic portion of P4 mainly contribute to the enzyme-inhibitor interaction by contacting the hydrophobic S2 and S4 subsites of the protease. In addition, the fluoride at the tip of P2 fluorophenylalanine could also have polar interaction with R39 of the protein (see results below).

Fig. 6.
Detailed interactions between rupintrivir and CVA16 3C. H-bond interactions are shown as dashed lines. The five groups of the inhibitor are labeled P1′ to P4 and colored orange. The residues in the enzyme that interact with rupintrivir are shown ...

The detailed interactions, including H bonds and hydrophobic contacts, between rupintrivir and EV71 3C are exactly the same as those observed in the CVA16 3C with compound structure. Therefore, overall, rupintrivir exhibits structural and chemical complementarities to both CVA16 and EV71 3Cs.

Rupintrivir is not as well accommodated in CVA16/EV71 3C as in HRV 3C.

Compared to a previously reported structure of rupintrivir in HRV 3C (23), the compound binds to CVA16 and HRV 3Cs with similar modes. Superimposition of the two complex structures reveals well-aligned enzyme entities, as well as inhibitor molecules at the P4, P3, and P1 positions, where most (9 out of 10) of the H-bond contacts with the enzyme are located. However, great discrepancies were observed for the other two groups of the compound in the CVA16 3C structure: (i) the P2 group makes a rotary movement (around atom C-5 as the rotating axis) of ~1.7 Å out of the S2 pocket and (ii) the P1′ group adopts a tilted conformation, exposing its ester chain to the solvent (Fig. 7 A). The latter difference is extremely unexpected, because the P1′ group could be well accommodated in the S1′ subsite of HRV 3C. By “lying down,” rupintrivir's P1′ group forms two H bonds with the oxyanion hole (main-chain amide of G145 and C147) of HRV 3C. However, in the context of the 3C protease from CVA16, the tilted P1′ group of rupintrivir supports only one H-bond interaction with the enzyme (Fig. 7B). Therefore, despite the complementarity of rupintrivir to CVA16/EV71 3C, the compound binds the 3C protease of HRV more stably, well explaining the observed lower efficacy of rupintrivir for EV71/CVA16 than for HRV (23). Next, we set out to explore the structural basis for these observed differences by comparison of the S2 and S1′ subsites in the HRV and CVA16/EV71 3C proteases.

Fig. 7.
Rupintrivir is not as well accommodated in CVA16 3C as in HRV 3C. (A) Comparison of rupintrivir in CVA16 (magenta) and HRV (cyan) 3C proteases. The 1.7-Å uplift of the P2 group and the tilted P1′ ester chain of the inhibitor in CVA16 3C ...

Structural basis for the modification of rupintrivir.

Within the CVA16-3C-rupintrivir structure, it is noteworthy that a residue with a long side chain (K130) is located at the distal end of the S2 subsite. With its side chain atoms half closing the pocket, the P2 group of rupintrivir could not be deeply buried in the S2 subsite of CVA16 3C and thus adopted a relatively tilted conformation. This tilted P2-fluorophenylalanine was further stabilized via extensive polar interactions between the group and R39 of CVA16 3C, a residue located at the top right corner of the S2 subsite. These include a direct polar attraction of the P2 group from the R39 residue (~3.6 Å) and an indirect interaction between the two entities via a water-mediated H-bond network (Fig. 8 A). However, in the rhinoviral protease, the R39 and K130 residues are replaced by T39 and N130, respectively. Not only do these substitutions leave the S2 subsite fully open to the solvent, but they also disrupt the potential contact of T39 with the rupintrivir fluoride. With a pocket fully open to the solvent, the P2 group of rupintrivir is easily buried deep in the S2 subsite of HRV 3C. This lying-down conformation for the inhibitor's P2 group was further stabilized via a weak H bond (3.5 Å) contributed by a bottom-residing glutamic acid (E71) (Fig. 8B).

Fig. 8.
The half-closed S2 subsite and an S1′ pocket with reduced size in CVA16 3C. (A and B) Surface representation of the S2 subsites in the CVA16 (A) and HRV (B) 3C proteases. The residues referred to in the text are presented as thin sticks and are ...

For the S1′ subsite in both CVA16 and HRV 3Cs, the specificity pocket is formed by the backbone atoms of the oxyanion hole residues (G145 to C147), the main-chain atoms of H24/K24, and the side chain of F25. Superimposition of the subsites reveals that these residues are well aligned, except for the H24/K24 pair, where the carbonyl group of H24 in CVA16 3C is moved ~0.9 Å into the pocket relative to that of K24 in HRV 3C (Fig. 8C). This results in a smaller S1′ pocket in CVA16 3C than in the rhinoviral enzyme. Further analysis shows that residues Q19, E107, and H108 are positioned in steric proximity to H24 in CVA16 3C. By surrounding the side chain of H24 like a cap, these residues confine the histidine residue within a limited space. They also form three hydrogen bonds with H24 and thereby help stabilize its observed conformation. However, in HRV 3C, these 3 residues are replaced by T19, N107, and Q108 (respectively), and due to a different orientation of the aII-bII loop (the loop connecting the aII and bII strands), residue K24 is easily exposed to solvent (Fig. 8D). The confinement arising from residues Q19, E107, and H108 in CVA16 3C might cause a 0.9-Å shift for the main-chain carbonyl group of residue 24 relative to that of the HRV enzyme.

It should be noted that the inhibitor's P2 group is uplifted ~1.7 Å compared to that in HRV 3C. Taking the rigidity of the drug into account, this shift should be transferred to the opposite side of the inhibitor, moving the P1 and P1′ groups toward the body of the enzyme. In support of this, the compound indeed inserts its P1 lactam deeper into the S1 subsite of CVA16 3C. For the S1′ subsite, which is smaller and lacks malleability, the ester chain at the P1′ position would have conflicted with CVA16 3C if it was retained in the conformation observed in the HRV 3C-rupintrivir complex. Together, these factors force the P1′ chain of the inhibitor to adopt an alternative (in this case tilted) conformation when it binds to CVA16 3C.

Identical rupintrivir-enzyme interaction modes at the S2 and S1′ subsites were observed in the structure of the inhibitor in EV71 3C. Therefore, the half-closed S2 and size-reduced S1′ subsites are also typical features of the EV71 enzyme. We believe that the special characters of the S2 and S1′ subsites within the EV71/CVA16 enzyme together cause rupintrivir to tilt both its P2 and P1′ groups and that the less avid binding of rupintrivir to EV71/CVA16 3C is the result of the subpar fit of the compound to the protease at both the S2 and S1′ subsites.


EV71 and CVA16 are phylogenetically closely related (25). Overall, the 3C proteases from different EV71 and CVA16 strains display 90 to 98% sequence identity. Accordingly, the two proteases in this study exhibited similar in vitro cleavage capabilities and did not show a preference for peptides derived from their respective viruses. It was unexpected that the Vp2/Vp3 junction of EV71 is much more efficiently processed than that of CVA16, especially in the context where the nonstructural junctions (2A-2C and 3A-3D) of both viruses are each cleaved in parallel by EV71/CVA16 3C (Table 2). Despite the fact that we failed to determine the cleavage rates of the two proteases for peptides representing Vp3/Vp1 junctions of EV71 and CVA16, a more effective in vivo processing of the capsid region for EV71 than for CVA16 could still be expected. This could lead to fast assembly and production of EV71 progeny virus in infected individuals. Supportive evidence comes from a report by Zhang et al. (44), who demonstrated that CVA16 might experience a lower evolution rate than EV71. Further experiments are needed to confirm this inference.

It is also noteworthy that a single mutation of P2-Thr to P2-Lys within the SP-1 peptide could lead to a decrease in cleavage efficiency of about 6-fold (Table 2). In terms of the 17-fold difference in the 3C processing rates between peptides SP-1 and SP-2, which differ from each other at only two positions (P2 and P2′), it is a natural inference that an F-to-I mutation at the P2′ position should also affect the cleavage rate of the peptide by about 3-fold.

We also reported the rupintrivir-enzyme complex structures in this study. Compared to rupintrivir in HRV 3C, the P1′ group of the inhibitor is oriented toward the bulk solvent after it is bound to CVA16/EV71 3C. We hypothesize that this tilted ester group orientation will decrease the binding affinity of rupintrivir to CVA16/EV71 3C. Although the ester group can interact with the enzyme molecule by forming one H bond, the switch from a Z conformation (as observed in the HRV 3C-rupintrivir complex) to the tilted conformation (as observed in our structures) requires energy. In support of this, a recent report compared the abilities of two compounds (differing only at the P1′ position) to inhibit the EV71 3C protease and demonstrated that substitution of the ester chain by aldehyde improves the potency of the compound by ~100-fold (18).

Based on the findings in this study, we propose that modifications of rupintrivir at the P2 and P1′ positions will allow the compound to bind more tightly to EV71/CVA16 3C. An option for P1′ modification is to replace the entire ester chain with aldehyde. For the P2 group, the fluorophenylalanine chain is too large to be accommodated by the half-closed S2 subsite. Therefore, an unmodified benzyl ring would likely fit the S2 pocket better.

It is also noteworthy that the residues forming the featured S2 and S1′ subsites in our enzyme structures are highly conserved (with either no variations or one variation at H108) among the main proteases of viruses from human enterovirus group A but not those of other groups (e.g., human enterovirus groups B, C, and D) (Fig. 9). A reasonable inference is that the half-closed S2 subsite and an S1′ pocket with a reduced size are common characteristics of group A enteroviral 3C proteases. Moreover, apart from EV71 and CVA16, many other group A enteroviruses, such as coxsackieviruses A4, A5, A8, and A10, also clinically cause HFMD. Consequently, we believe that the structures presented in this study provide a solid basis for the development of 3C-based anti-HFMD virus drugs.

Fig. 9.
Multiple-sequence alignment of the 3C proteases from human enteroviruses A to D compared to that of HRV 3C. The spiral lines indicate α-helices, and the horizontal arrows represent β-strands. Residue numbers for CVA16 3C are given above ...


G.F.G. is a leading principal investigator of the Innovative Research Group of the National Natural Science Foundation of China (NSFC) (grant no. 81021003).


[down-pointing small open triangle]Published ahead of print on 27 July 2011.


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