Crystal structures of the 3C proteases from Coxsackievirus B3 and B4

Enteroviruses cause a wide range of disorders with varying presentations and severities, and some enteroviruses have emerged as serious public health concerns. These include Coxsackievirus B3 (CVB3), an active causative agent of viral myocarditis, and Coxsackievirus B4 (CVB4), which may accelerate the progression of type 1 diabetes. The 3C proteases from CVB3 and CVB4 play important roles in the propagation of these viruses. In this study, the 3C proteases from CVB3 and CVB4 were expressed in Escherichia coli and purified by affinity chromatography and gel-filtration chromatography. The crystals of the CVB3 and CVB4 3C proteases diffracted to 2.10 and 2.01 Å resolution, respectively. The crystal structures were solved by the molecular-replacement method and contained a typical chymotrypsin-like fold and a conserved His40–Glu71–Cys147 catalytic triad. Comparison with the structures of 3C proteases from other enteroviruses revealed high similarity with minor differences, which will guide the design of 3C-targeting inhibitors with broad-spectrum properties.

1. Introduction

Enteroviruses are a group of small, non-enveloped RNA viruses belonging to the Enterovirus genus within the Picorna­viridae family (Zell et al., 2017). They are icosahedral in shape and have a diameter of around 25–30 nm. Poliovirus, which attacks the central nervous system and causes poliomyelitis, is one of the best-known enteroviruses and therefore has been studied thoroughly since it was discovered nearly a century ago (Nomoto, 2007). Decades of investigations not only led to the development of vaccines against poliovirus, but have also significantly advanced the field of molecular virology (Racaniello & Baltimore, 1981; Mbani et al., 2023). With the global ambition of the worldwide eradication of poliomyelitis, poliovirus is currently under control and is rare in most parts of the world. Other non-polio enteroviruses include group A coxsackieviruses (types 1–22 and 24), group B coxsackie­viruses (types 1–6), echoviruses (types 1–7, 9, 11–21, 24–27 and 29–33), many types of numbered enteroviruses and rhinoviruses (Zell et al., 2017; Baggen et al., 2018; Simmonds et al., 2020). They cause unnoticed infections in most cases, but can also result in serious disease and even death, especially in infants, young children and immunocompromised individuals, thus necessitating further attention and investigation.

Several specific non-polio enteroviruses have emerged as serious public health threats, such as Coxsackievirus A16 (CVA16), Enterovirus A71 (EV-A71), Coxsackievirus A6 (CVA6), Enterovirus D68 (EV-D68), Coxsackievirus B3 (CVB3) and Coxsackievirus B4 (CVB4). EV-A71, CVA16 and CVA6 are the three most common pathogens of hand, foot and mouth disease (HFMD), a widespread disease generally characterized by a brief fever and rashes or blisters around the hands, feet and mouth (Kimmis et al., 2018; Saguil et al., 2019). EV-D68 is an important causative agent of acute respiratory illness and acute flaccid myelitis (Sooksawasdi Na Ayudhya et al., 2021). CVB3 is a well known cause of myocarditis, and infected individuals may develop dilated cardiomyopathy (DCM; Zhang et al., 2023), while CVB4 infection can promote the development of type 1 diabetes (T1D; Alhazmi et al., 2021). There are currently no approved drugs for the treatment of the diseases caused by these emerging enteroviruses. Therefore, it is important to develop antiviral drugs to control these infections in preparation for a possible outbreak.

Despite their diversity, all enteroviruses comprise a single-stranded, positive-sense RNA genome that has a single open reading frame (ORF) and encodes a large polyprotein precursor. In infected cells, this viral polyprotein is further processed by viral proteases into four structural proteins, namely VP1, VP2, VP3 and VP4, and seven nonstructural proteins, 2A–2C and 3A–3D (Laitinen et al., 2016). During maturation of the polyprotein, the 3C protease is responsible for most of the cleavages (Sun et al., 2016). In addition, the 3C protease can bind to the 5′-untranslated region (UTR) of the viral genomic RNA and exert essential functions in viral replication (Blair et al., 1998; Sun et al., 2016). Moreover, enteroviral 3C protease is able to promote viral replication and mediate immune evasion by cleaving host factors (Barral et al., 2009; Sun et al., 2016; Jin et al., 2018). These pivotal roles of enteroviral 3C protease make it an excellent target for three-dimensional structure analysis, which could further direct the structure-based design of antiviral drugs.

Currently, the crystal structures of 3C proteases from several enteroviruses, including poliovirus (PV), Human rhinovirus C15 (HRV-C15), CVB3, EV-B93, EV-D68, CVA16 and EV-A71, have been solved (Lu et al., 2011; Mosimann et al., 1997; Lee et al., 2009; Yuan et al., 2020; Cui et al., 2011; Costenaro et al., 2011; Tan et al., 2013). However, structural data for enteroviral 3C proteases in the Protein Data Bank (PDB) are still limited, and further structural information about entero­viral 3C proteases at the atomic level would greatly enhance our understanding of this viral enzyme.

In this study, we determined the crystal structure of the 3C protease from CVB3 at 2.10 Å resolution and reported the crystal structure of the 3C protease from CVB4 for the first time at 2.01 Å resolution. The structures reveal the presence of a typical chymotrypsin-like fold and a conserved catalytic triad, which are highly similar to those observed in 3C proteases from other enteroviruses. Our structures of the 3C proteases from CVB3 and CVB4 could guide the design of 3C-targeting inhibitors with broad-spectrum properties to facilitate the development of anti-enteroviral drugs.

2. Materials and methods

2.1. Macromolecule production

The full-length cDNAs encoding the CVB3 and CVB4 3C proteases were codon-optimized, commercially synthesized (Tsingke, People's Republic of China) and cloned into pET-21a vectors between NdeI and XhoI restriction sites. To produce the target proteins, the recombinant plasmids were transformed into competent Escherichia coli BL21 (DE3) cells. The bacteria were then grown at 37°C in lysogeny broth (LB) medium and induced by the addition of isopropyl β-D-1-thiogalactopyranoside (IPTG) to a final concentration of 1 mM when the optical density at 600 nm reached about 0.6. After this, the culture temperature was set to 18°C and the cells were cultured for a further 16 h. Subsequently, the cells were harvested by centrifugation, resuspended in lysis buffer and lysed by ultrasonication. The cell lysates were centrifuged to remove cell debris, and the supernatant was loaded onto a gravity column packed with Ni–NTA resin (GE Healthcare). After extensive washing with lysis buffer to remove non­specific proteins bound to the column, the His-tagged 3C proteases were eluted using an elution buffer containing an increasing concentration gradient of imidazole. The eluted fractions were collected, concentrated and further purified by size-exclusion chromatography on a Superdex 200 Increase 10/300 GL column (GE Healthcare). The molecular weight and purity of the target proteins were evaluated by SDS–PAGE. Macromolecule-production information is summarized in Table 1.

Table 1
Macromolecule-production information

Source organism	Coxsackievirus B3	Coxsackievirus B4
DNA source	Commercially synthesized	Commercially synthesized
Cloning sites	NdeI–XhoI	NdeI–XhoI
Cloning vector	pET-21a	pET-21a
Expression vector	pET-21a	pET-21a
Expression host	Escherichia coli strain BL21 (DE3)	Escherichia coli strain BL21 (DE3)
Complete amino-acid sequence of the construct produced	MGPAFEFAVAMMKRNSSTVKTEYGEFTMLGIYDRWAVLPRHAKPGPTILMNDQEVGVLDAKELVDKDGTNLELTLLKLNRNEKFRDIRGFLAKEEVEVNEAVLAINTSKFPNMYIPVGQVTEYGFLNLGGTPTKRMLMYNFPTRAGQCGGVLMSTGKVLGIHVGGNGHQGFSAALLKHYFNDEQLEHHHHHH	MGPAFEFAVAMMKRNASTVKTEYGEFTMLGIYDRWAVLPRHAKPGPTILMNDQEVDVVDAKELVDKDGTNLELTLLKLNRNEKFRDIRGFLTREEAEVNEAVLAINTSKFPNMYIPVGQVTDYGFLNLGGTPTKRMLMYNFPTRAGQCGGVLMSTGKVLGIHVGGNGHQGFSAALLRHYFNDEQLEHHHHHH

2.2. Crystallization

The purified CVB3 and CVB4 3C proteases were concentrated to 10 mg ml⁻¹ using a concentration tube. Crystallization screening was performed manually with commercial kits (Hampton Research) in a 48-well plate using the sitting-drop vapor-diffusion method at 293 K. As summarized in Table 2, the best-diffracting crystals of the CVB3 3C protease were grown in a condition composed of 0.2 M calcium acetate hydrate, 0.1 M sodium cacodylate trihydrate pH 6.5, 18% polyethylene glycol 8000, while high-quality crystals of the CVB4 3C protease were grown in a condition composed of 0.16 M magnesium chloride hexahydrate, 0.08 M Tris–HCl pH 8.5, 24%(w/v) polyethylene glycol 4000, 20%(v/v) glycerol. The obtained crystals were mounted on a cryo-loop (Hampton Research) and then quickly cooled in liquid nitrogen for better collection of X-ray diffraction data.

Table 2
Crystallization

Protein	CVB3 3C	CVB4 3C
Method	Sitting-drop vapor diffusion	Sitting-drop vapor diffusion
Plate type	48-well plate	48-well plate
Temperature (K)	293	293
Protein concentration (mg ml^−1)	10	10
Buffer composition of protein solution	300 mM NaCl, 25 mM HEPES pH 7.5, 5 mM DTT, 5% glycerol	300 mM NaCl, 25 mM HEPES pH 7.5, 5 mM DTT, 5% glycerol
Composition of reservoir solution	0.2 M calcium acetate hydrate, 0.1 M sodium cacodylate trihydrate pH 6.5, 18% polyethylene glycol 8000	0.16 M magnesium chloride hexahydrate, 0.08 M Tris–HCl pH 8.5, 24%(w/v) polyethylene glycol 4000, 20%(v/v) glycerol
Volume and ratio of drop	1 ml protein plus 1 ml reservoir (1:1)	1 ml protein plus 1 ml reservoir (1:1)
Volume of reservoir (µl)	60	60
Cryoprotectant	20% ethylene glycol	20% ethylene glycol

2.3. Data collection and processing

Diffraction data collection was completed at 100 K at a wavelength of 0.97918 Å on macromolecular crystallography beamline 10U2 (BL10U2) at Shanghai Synchrotron Radiation Facility (SSRF), Shanghai, People's Republic of China. The diffraction data were processed using the HKL-2000 software package (Otwinowski & Minor, 1997). The space groups of the crystals of the CVB3 and CVB4 3C proteases were identified to be C2 and P2₁, respectively. Data-collection and processing details are summarized in Table 3.

Table 3
Data collection and processing

Values in parentheses are for the outer shell.

	CVB3 3C	CVB4 3C
PDB code	8y2t	8y2u
Diffraction source	BL10U2, SSRF	BL10U2, SSRF
Wavelength (Å)	0.97918	0.97918
Temperature (K)	100	100
Detector	Dectris EIGER S 9M	Dectris EIGER S 9M
Crystal-to-detector distance (mm)	242	324
Rotation range per image (°)	1	1
Total rotation range (°)	360	360
Exposure time per image (s)	0.1	0.1
Space group	C2	P21
a, b, c (Å)	78.22, 64.57, 39.74	39.10, 64.43, 67.99
α, β, γ (°)	90.00, 115.27, 90.00	90.00, 92.47, 90.00
Mosaicity (°)	0.23	0.18
Resolution (Å)	2.10 (2.21–2.10)	2.01 (2.06–2.01)
Total No. of reflections	31251	132282
No. of unique reflections	9502	22518
Completeness (%)	90.6 (98.3)	99.7 (99.0)
Multiplicity	3.3 (2.9)	5.9 (4.4)
〈I/σ(I)〉	11.4 (3.2)	4.9 (1.6)
Rmerge (%)	4.1 (26.3)	29.4 (163.9)
Rr.i.m.	0.027	0.136
Overall B factor from Wilson plot (Å^2)	43.6	15.08

2.4. Structure solution and refinement

The crystal structures of the 3C proteases from CVB3 and CVB4 were solved by molecular replacement in Phaser (McCoy et al., 2007). A previously determined structure of the CVB3 3C protease (PDB entry 2ztz; Lee et al., 2009) was used as the search model. The original model was built using Coot (Emsley et al., 2010) and refined using Phenix (Liebschner et al., 2019). The quality of the final model was verified using MolProbity (Chen et al., 2010). Structure-refinement statistics are summarized in Table 4. PyMOL (https://www.pymol.org) was used to perform structural analysis and produce structural figures. The coordinates and structure factors for the 3C proteases from CVB3 and CVB4 have been deposited in the PDB as entries 8y2t and 8y2u, respectively.

Table 4
Structure refinement

Values in parentheses are for the outer shell.

	CVB3 3C	CVB4 3C
PDB code	8y2t	8y2u
Resolution range (Å)	33.75–2.10	39.06–2.01
Completeness (%)	90.15	99.52
No. of reflections, working set	9498	22483
No. of reflections, test set	9502	22518
Final Rcryst	0.2066	0.1980
Final Rfree	0.2553	0.2404
No. of non-H atoms
Total	1414	2957
Protein	1397	2804
Water	17	153
R.m.s.d., bond lengths (Å)	0.008	0.007
R.m.s.d., bond angles (°)	1.045	0.924
Average B factors (Å^2)
Protein	48.3	17.22
Water	48.2	22.8
Ramachandran plot
Favored regions (%)	97.75	97.51
Additionally allowed (%)	2.25	2.49
Outliers (%)	0	0

3. Results and discussion

3.1. Crystal structure of the 3C protease from CVB3

The obtained crystals of the CVB3 3C protease belonged to space group C2, and diffracted to 2.10 Å resolution (Table 3). The crystal structure was solved by the molecular-replacement method and contains one monomer per asymmetric unit. CVB3 3C protease forms a chymotrypsin-like fold and can be divided into two β-barrel domains (Fig. 1a), which can also be seen in the structures of other enteroviral 3C proteases (Lu et al., 2011; Mosimann et al., 1997; Lee et al., 2009; Yuan et al., 2020; Cui et al., 2011; Costenaro et al., 2011; Tan et al., 2013). Domain I is composed of seven β-strands (βaI–βgI), a short α-helix (αB) connecting βcI and βdI, and a C-terminal α-helix (αD) (Fig. 1a). Domain II mainly consists of seven β-strands (βaII–βgII) and an N-terminal α-helix (αA) (Fig. 1a). The nomenclature of secondary-structural elements is with reference to the 3C protease structures from other enteroviruses (Lu et al., 2011; Mosimann et al., 1997; Lee et al., 2009; Yuan et al., 2020; Cui et al., 2011; Costenaro et al., 2011; Tan et al., 2013). The two main domains are connected by a long loop which contains a short α-helix (αC). His40, Glu71 and Cys147, which are located in the cleft between the two domains, act as the catalytic triad and play vital roles in enzymatic activity (Fig. 1b). However, the electron density for the side chain of Cys147 is missing in the high-resolution structure, which may reflect the different conformation of this catalytic residue in the apo state. Two motifs (KFRDI and VGK) have previously been identified as RNA-binding sites in picornaviral 3C proteases (Mosimann et al., 1997; Shih et al., 2004; Matthews et al., 1994; Leong et al., 1993; Walker et al., 1995). In the structure of the CVB3 3C protease, the KFRDI motif is located in the connecting loop between domain I and II just after the αC helix, while the VGK motif is located in the loop between βeII and βfII (Fig. 1b).

Figure 1 Crystal structure of the 3C protease from CVB3. (a) Overall structure of the CVB3 3C protease. The structure is colored from blue at the N-terminus to red at the C-terminus. The α-helices in the two domains (I and II) are labeled αA–αD. The β-strands are labeled βaI–βgI in domain I and βaII–βgII in domain II according to their occurrence along the primary structure. (b) Catalytic triad and RNA-binding sites of the CVB3 3C protease. The 2Fo − Fc electron densities for the catalytic residues (His40, Glu71 and Cys147) are contoured at 1σ. The RNA-binding motifs, KFRDI and TGK, are colored green. (c) Comparison of structures of the CVB3 3C protease. The structure solved in this study is colored magenta. The structures solved previously, named form I (PDB entry 2zty) and form II (PDB entry 2ztz), are colored cyan and orange, respectively. The loop consisting of residues 143–146 (the 143–146 loop) is indicated by a green arrow. (d) An enlarged view of the comparison of catalytic residues. The catalytic residues are shown as sticks. Electron density for Cys147 is missing in our structure.

Previously, two crystal structures of the CVB3 3C protease have been deposited in the PDB as entries 2zty (form I) and 2ztz (form II) (Lee et al., 2009). Form I belongs to space group P2₁ and contains two molecules in the asymmetric unit, while form II belongs to space group C2 and contains only one molecule in the asymmetric unit, like our structure. Superposition of our structure onto the two previously determined structures revealed a root-mean-square deviation (r.m.s.d.) for C^α atoms of 0.453 and 0.268 Å, respectively, indicating that these three structures are very similar (Fig. 1c). Despite their high similarity, subtle differences can be found in residues 143–146 (the 143–146 loop) and the orientations of the side chains of Cys147 and His40 (Fig. 1d). These variations may be related to substrate-binding regulation.

3.2. Crystal structure of the 3C protease from CVB4

The crystal structure of CVB4 3C protease was solved at 2.01 Å resolution and contains two molecules in the asymmetric unit, named molecule A and molecule B (Tables 3 and 4 and Fig. 2a). Molecule A is tightly packed on molecule B by means of a network of hydrogen-bonding and hydrophobic interactions (Fig. 2b). Specifically, Gly1, Glu5, Tyr113, Arg143 and Ser107 in molecule A form one or two hydrogen-bonding inter­actions with Glu5, Gly1, Tyr13, Ser107 and Arg143, respectively. Additional hydrogen-bonding interactions were found between Pro110 in molecule A and Gln146 and Pro141 via a bridging water molecule. A hydrophobic interaction was found between the Phe4 residues in molecules A and B.

Figure 2 Crystal structure of the 3C protease from CVB4. (a) Overall structure of the CVB4 3C protease. The two molecules (molecule A and molecule B) in the asymmetric unit of the CVB4 3C protease structure are shown in cartoon representation. Molecule B (cyan) is packed against the surface of molecule A (green). (b) A magnified view showing detailed crystal-packing interactions. The residues involved in crystal packing are shown as sticks. Hydrogen-bonding interactions are indicated as black dashed lines. W represents the water molecule that mediates hydrogen-bonding interactions. (c) Comparison of molecule A and molecule B in the CVB4 3C protease structure. The catalytic residues (His40, Glu71 and Cys147) are shown as sticks. (d) Overall structure of molecule A. The structure is colored from blue at the N-terminus to green at the C-terminus. Four α-helices in two domains (I and II) are marked αA–αD. A total of 16 β-strands are labeled (βaI–βgI in domain I and βaII–βgII in domain II) according to their occurrence along the primary structure. (e) Catalytic triad and RNA-binding sites of the CVB4 3C protease. The 2Fo − Fc electron densities for the catalytic residues are contoured at 1σ. The RNA-binding motifs, KFRDI and TGK, are colored magenta.

Both molecules A and B in the structure of the CVB4 3C protease form a chymotrypsin-like fold (Fig. 2c), which possesses a catalytic triad and key RNA-binding sites, and can be further divided into 16 β-strands (βaI–βgI in domain I and βaII–βiII in domain II), four α-helices (αA–αD) and several connecting loops (Figs. 2d and 2e). The electron density of all of the catalytic residues (His40, Glu71 and Cys147) of the CVB4 3C protease can be traced in our structure (Fig. 2e). However, the side-chain orientation of Cys147 in molecule A shows a small difference from that in molecule B (Fig. 2c).

3.3. Comparison with other 3C protease structures

At present, the structures of 3C proteases from several viruses in the family Picornaviridae have been reported, including those from Senecavirus A (SVA) in the genus Senecavirus, Foot-and-mouth disease virus (FMDV) in the genus Aphthovirus, Hepatitis A virus (HAV) in the genus Hepatovirus and PV, EV-B93, EV-D68, CVA16, HRV-C15 and EV-A71 in the genus Enterovirus (Lu et al., 2011; Mosimann et al., 1997; Yuan et al., 2020; Cui et al., 2011; Costenaro et al., 2011; Tan et al., 2013; Meng et al., 2022; Bergmann et al., 1997; Birtley et al., 2005). Although the 3C structures from HAV, FDMV and SVA show several large deviations from the enteroviral 3C structures (Meng et al., 2022; Bergmann et al., 1997; Birtley et al., 2005), they possess a chymotrypsin fold similar to those from enteroviruses. The crystal structures of the CVB3 and CVB4 3C proteases solved in this study further confirm this typical structural feature of picornaviral 3C proteases. Structural superposition revealed high similarity between the CVB3 and CVB4 3C proteases, with an r.m.s.d. of 0.370 Å for equivalent C^α atoms. The overall r.m.s.d.s between the 3C protease from CVB3 and those from other entero­viruses are 0.624 Å (165 C^α atoms) for PV, 0.353 Å (152 C^α atoms) for EV-B93, 0.608 Å (164 C^α atoms) for EV-D68, 0.575 Å (165 C^α atoms) for CVA16, 0.802 Å (171 C^α atoms) for HRV-C15 and 0.926 Å (164 C^α atoms) for EV-A71 (Fig. 3a). The overall r.m.s.d.s between the 3C protease from CVB4 and those from other enteroviruses are 0.624 Å (165 C^α atoms) for PV, 0.353 Å (152 C^α atoms) for EV-B93, 0.608 Å (164 C^α atoms) for EV-D68, 0.575 Å (165 C^α atoms) for CVA16, 0.802 Å (171 C^α atoms) for HRV-C15 and 0.926 Å (164 C^α atoms) for EV-A71 (Fig. 3a). The most obvious difference in these structures is that a surface loop adopts an open conformation in EV-A71 3C protease, while it adopts a closed conformation in the other 3C proteases (Fig. 3a). These data underline the high conservation of the 3C proteases in enteroviruses (Figs. 3a and 3b). Thus, inhibitors targeting a certain 3C protease should have broad-spectrum activities.

Figure 3 Comparison of enteroviral 3C proteases with reported structures. (a) Structural alignment of 3C proteases. 3C proteases from poliovirus (PV, cyan, PDB entry 1l1n; Mosimann et al., 1997), EV-B93 (orange, PDB entry 3q3x; Costenaro et al., 2011), EV-D68 (lemon, PDB entry 3zv8; Tan et al., 2013), CVA16 (slate, PDB entry 3sj8; Lu et al., 2011), HRV-C15 (yellow, PDB entry 6ku7; Yuan et al., 2020), EV-A71 (wheat, PDB entry 3osy; Cui et al., 2011), CVB3 (gray, this study) and CVB4 (magenta, this study) are included in the comparison. Alignments of these 3C proteases yield r.m.s.d. values that demonstrate the similarity between these enzymes. (b) Multiple-sequence alignment of 3C proteases from different enteroviruses. The MEGA software (version 10.2.5) was used to perform the alignment. The sequence-alignment visualization was generated using ESPript 3.0. (c) Structural alignment of the CVB3 (gray, this study), CVB4 (magenta, this study) and EV-B93 (orange, PDB entry 3q3x) 3C proteases. Obvious local differences are labeled with green lines or dashed boxes. (d) An enlarged view of reigions 1–3 with obvious local differences. (e) An enlarged view of the catalytic triad of 3C proteases.

As the EV-B93 3C protease has the lowest r.m.s.d. with both the CVB3 and CVB4 3C proteases, we superimposed these three enzymes to reveal local differences. As shown in Fig. 3(c), several regions with differences were labeled. Region 1 is located in domain I of these 3C proteases. Both the CVB3 and CVB4 3C proteases have two β-strands (βeI and βfI) in region 1, while EV-B93 has only one β-strand (βeI) (Fig. 3d). Region 2 and region 3 are located in domain II and EV-B93 has one β-strand in these two regions (Fig. 3d). The CVB4 3C protease has three β-strands (βbII, βcII and βdII) in region 2 and two β-strands (βeII and βfII) in region 3, while the CVB3 3C protease has two β-strands (βbII and βcII) in region 2 and one β-strand (βdII) in region 3 (Fig. 3d). Regions 4–6 are also located in domain II, where the loops of the CVB3 3C protease show an obvious deviation from those of the CVB4 and EV-B93 3C proteases (Fig. 3c). Moreover, minor changes are observed in the orientation of catalytic residues of the three 3C proteases (Fig. 3e). These observations indicate several local differences among these highly similar 3C proteases, which may modestly affect the substrate recognition of these 3C proteases.

Despite sharing very little sequence homology with enteroviral 3C proteases, the main proteases from coronaviruses also harbor a chymotrypsin-like fold (Jin et al., 2020; Zhou et al., 2021). Moreover, the main protease possesses a domain III that participates in protease dimerization in the chymotrypsin-like fold. Due to this structural similarity, inhibitors that are effective against enteroviral 3C protease can be repurposed to inhibit the activities of coronaviral main proteases (Lockbaum et al., 2021).

4. Conclusion

In conclusion, the crystal structures of the 3C proteases from CVB3 and CVB4 were solved and reveal the presence of a conserved His40–Glu71–Cys147 catalytic triad and a chymotrypsin-like fold. We believe that these data will add to the design of broad-spectrum inhibitors targeting 3C proteases to block the replication of enteroviruses.