Autoprocessing: an essential step for expression and purification of enterovirus 71 3Cpro in Escherichia coli

A gene encoding the 3BC of human enterovirus 71 (EV71) was cloned and inserted into a derivative of plasmid pET-32a(+) driven by T7 promoter. The expressed 3C protease (3C^pro) autocatalytically cleaved itself from the recombinant protein Trx-3BC and the mature 3C^pro partitioned in the soluble fraction of bacterial lysate. The 13-amino-acid peptide substrates with the junction of 3B/3C were used to verify the proteolysis activity of the purified 3C^pro. The EV71 3C^pro had a K_m value of 63 μM (measured by a continuous fluorescence assay). The other solid-phase activity assay of the EV71 3C^pro was developed using HPLC to analyze the proteolytic products. The combination of two activity assays contributes to promote the identification of the specific inhibitors targeted to the EV71 3C^pro.     

Optimize the interactions at S4 with efficient inhibitors targeting 3C proteinase from enterovirus 71

Enterovirus 71 (EV71) is the causative agent of hand, foot and mouth disease and can spread its infections to the central nervous and other systems with severe consequences. The replication of EV71 depends on its 3C proteinase (3C^pro), a significant drug target. By X‐ray crystallography and functional assays, the interactions between inhibitors and EV71 3C^pro were evaluated. It was shown that improved interactions at S4 for the substrate binding could significantly enhance the potency. A new series of potent inhibitors with high ligand efficiency was generated for developing antivirals to treat and control the EV71‐associated diseases. Copyright © 2016 John Wiley & Sons, Ltd.     

Enterovirus 71 3C Protease Cleaves a Novel Target CstF-64 and Inhibits Cellular Polyadenylation

Identification of novel cellular proteins as substrates to viral proteases would provide a new insight into the mechanism of cell–virus interplay. Eight nuclear proteins as potential targets for enterovirus 71 (EV71) 3C protease (3C^pro) cleavages were identified by 2D electrophoresis and MALDI-TOF analysis. Of these proteins, CstF-64, which is a critical factor for 3′ pre-mRNA processing in a cell nucleus, was selected for further study. A time-course study to monitor the expression levels of CstF-64 in EV71-infected cells also revealed that the reduction of CstF-64 during virus infection was correlated with the production of viral 3C^pro. CstF-64 was cleaved in vitro by 3C^pro but neither by mutant 3C^pro (in which the catalytic site was inactivated) nor by another EV71 protease 2A^pro. Serial mutagenesis was performed in CstF-64, revealing that the 3C^pro cleavage sites are located at position 251 in the N-terminal P/G-rich domain and at multiple positions close to the C-terminus of CstF-64 (around position 500). An accumulation of unprocessed pre-mRNA and the depression of mature mRNA were observed in EV71-infected cells. An in vitro assay revealed the inhibition of the 3′-end pre-mRNA processing and polyadenylation in 3C^pro-treated nuclear extract, and this impairment was rescued by adding purified recombinant CstF-64 protein. In summing up the above results, we suggest that 3C^pro cleavage inactivates CstF-64 and impairs the host cell polyadenylation in vitro, as well as in virus-infected cells. This finding is, to our knowledge, the first to demonstrate that a picornavirus protein affects the polyadenylation of host mRNA.     

Crystal structure of human enterovirus 71 3C protease

Human enterovirus 71 (EV71) is the major pathogen that causes hand, foot and mouth disease that particularly affects young children. Growing hand, foot and mouth disease outbreaks were observed worldwide in recent years and caused devastating losses both economically and politically. However, vaccines or effective drugs are unavailable to date. The genome of EV71 consists of a positive sense, single-stranded RNA of ∼ 7400 bp, encoding a large precursor polyprotein that requires proteolytic processing to generate mature viral proteins. The proteolytic processing mainly depends on EV71 3C protease (3C^pro) that possesses both proteolysis and RNA binding activities, which enable the protease to perform multiple tasks in viral replication and pathogen-host interactions. The central roles played by EV71 3C^pro make it an appealing target for antiviral drug development. We determined the first crystal structure of EV71 3C^pro and analyzed its enzymatic activity. The crystal structure shows that EV71 3C^pro has a typical chymotrypsin-like fold that is common in picornaviral 3C^pro. Strikingly, we found an important surface loop, also denoted as β-ribbon, which adopts a novel open conformation in EV71 3C^pro. We identified two important residues located at the base of the β-ribbon, Gly123 and His133, which form hinges that govern the intrinsic flexibility of the ribbon. Structure-guided mutagenesis studies revealed that the hinge residues are important to EV71 3C^pro proteolytic activities. In summary, our work provides the first structural insight into EV71 3C^pro, including a mobile β-ribbon, which is relevant to the proteolytic mechanism. Our data also provides a framework for structure-guided inhibitor design against EV71 3C^pro.     

以EV71 3Cpro为靶标的抗病毒药物筛选模型的建立及抗3Cpro化合物筛选

建立一种以EV71 3C蛋白酶为靶标的抗肠病毒药物筛选模型,并应用于小分子化合物库筛选具有抗EV71活性的化合物.从临床手足口病例标本中分离肠道病毒进行PCR鉴定及基因组测序.通过插入突变在黄色荧光YFP编码框合适位点处引入EV71 3C酶切位点,构建对3C蛋白酶敏感的报告质粒pc DNA3-m YFP,然后将其与表达3C的质粒共转293A细胞,在3C抑制剂Rupintrivir存在与否的情况下通过荧光显微镜和酶标仪检测Ex(500nm)/Em(535nm)荧光信号的变化,判断建模是否成功;利用建好的筛选模型在高通量药物筛选平台对小分子化合物库进行初筛和复筛;再利用空斑分析检测筛选出的活性化合物是否对临床分离的EV71毒株具有抑制作用.m YFP在293A细胞中表达良好,3C的表达使荧光信号下降80%,Rupintrivir的存在则几乎不影响荧光表达,说明以3C为靶位的筛选模型构建成功.经过高通量初筛和复筛从26 000多种小分子化合物中获得26种能够显著回复m YFP表达的活性化合物;空斑分析显示其中2种化合物具有较为明显的抑制EV71复制的活性.因此,我们所构建的3C-m YFP共表达系统是一种简便有效的、可用于高通量筛选抗EV71 3C~(pro)药物的筛选模型.     

EV71 3C protease cleaves host anti-viral factor OAS3 and enhances virus replication

The global spread of enteroviruses (EVs) has become more frequent, severe and life-threatening. Intereron (IFN) I has been proved to control EVs by regulating IFN-stimulated genes (ISG) expression. 20-50-oligoadenylate synthetases 3 (OAS3) is an important ISG in the OAS/RNase L antiviral system. The relationship between OAS3 and EVs is still unclear. Here, we reveal that OAS3, superior to OAS1 and OAS2, significantly inhibited EV71 replication in vitro. However, EV71 utilized autologous 3C protease (3C^pro) to cleave intracellular OAS3 and enhance viral replication. Rupintrivir, a human rhinovirus 3C protease inhibitor, completely abolished the cleavage of EV71 3C^pro on OAS3. And the proteolytically deficient mutants H40G, E71A, and C147G of EV71 3C^pro also lost the ability of OAS3 cleavage. Mechanistically, the Q982-G983 motif in C-terminal of OAS3 was identified as a crucial 3C^pro cutting site. Further investigation indicated that OAS3 inhibited not only EV71 but also Coxsackievirus B3 (CVB3), Coxsackievirus A16 (CA16), Enterovirus D68 (EVD68), and Coxsackievirus A6 (CA6) subtypes. Notably, unlike other four subtypes, CA16 3C^pro could not cleave OAS3. Two key amino acids variation Ile36 and Val86 in CA16 3C^pro might result in weak and delayed virus replication of CA16 because of failure of OAS and 3AB cleavage. Our works elucidate the broad anti-EVs function of OAS3, and illuminate a novel mechanism by which EV71 use 3C^pro to escape the antiviral effect of OAS3. These findings can be an important entry point for developing novel therapeutic strategies for multiple EVs infection.     

Mutations at KFRDI and VGK domains of enterovirus 71 3C protease affect its RNA binding and proteolytic activities

The 3C proteases (3C^pro) of enterovirus 71 (EV71) is a good molecular target for drug discovery. Notably, this protease was found to possess RNA-binding activity. The regions responsible for RNA binding were classified as KFRDI (positions 82–86) and VGK (positions 154–156) in 3C^pro by mutagenesis study. Although the RNA-binding regions are structurally distinct from the catalytic site of EV71 3C^pro, mutations in the RNA-binding regions influenced 3C^pro proteolytic activity. In contrast, mutations at the catalytic site had almost no influence on RNA binding ability. We identified certain mutations within 3C^pro which abrogated both the RNA-binding activity of the expressed, recombinant, protease and the ability to rescue virus from an infectious full-length clone of EV71 (pEV71). Interestingly, mutation at position 84 from Arg(R) to Lys(K) was found to retain good RNA binding and proteolytic activity for the recombinant 3C^pro; however, no virus could be rescued when pEV71 with the R84K mutation was introduced into the infectious copy. Together, these results may provide useful information for using 3C^pro as the molecular target to develop anti-EV71 agents.The second and the third authors contributed equally to this work.     

Insights into Cleavage Specificity from the Crystal Structure of Foot-and-Mouth Disease Virus 3C Protease Complexed with a Peptide Substrate

Picornavirus replication is critically dependent on the correct processing of a polyprotein precursor by 3C protease(s) (3C^pro) at multiple specific sites with related but non-identical sequences. To investigate the structural basis of its cleavage specificity, we performed the first crystallographic structural analysis of non-covalent complexes of a picornavirus 3C^pro with peptide substrates. The X-ray crystal structure of the foot-and-mouth disease virus 3C^pro, mutated to replace the catalytic Cys by Ala and bound to a peptide (APAKQ|LLNFD) corresponding to the P5-P5′ region of the VP1-2A cleavage junction in the viral polyprotein, was determined up to 2.5 Å resolution. Comparison with free enzyme reveals significant conformational changes in 3C^pro on substrate binding that lead to the formation of an extended interface of contact primarily involving the P4-P2′ positions of the peptide. Strikingly, the deep S1′ specificity pocket needed to accommodate P1′-Leu only forms when the peptide binds. Substrate specificity was investigated using peptide cleavage assays to show the impact of amino acid substitutions within the P5-P4′ region of synthetic substrates. The structure of the enzyme-peptide complex explains the marked substrate preferences for particular P4, P2 and P1 residue types, as well as the relative promiscuity at P3 and on the P′ side of the scissile bond. Furthermore, crystallographic analysis of the complex with a modified VP1-2A peptide (APAKE|LLNFD) containing a Gln-to-Glu substitution reveals an identical mode of peptide binding and explains the ability of foot-and-mouth disease virus 3C^pro to cleave sequences containing either P1-Gln or P1-Glu. Structure-based mutagenesis was used to probe interactions within the S1′ specificity pocket and to provide direct evidence of the important contribution made by Asp84 of the Cys-His-Asp catalytic triad to proteolytic activity. Our results provide a new level of detail in our understanding of the structural basis of polyprotein cleavage by 3C^pro.     

Enterovirus 71 Protease 2Apro Targets MAVS to Inhibit Anti-Viral Type I Interferon Responses

Enterovirus 71 (EV71) is the major causative pathogen of hand, foot, and mouth disease (HFMD). Its pathogenicity is not fully understood, but innate immune evasion is likely a key factor. Strategies to circumvent the initiation and effector phases of anti-viral innate immunity are well known; less well known is whether EV71 evades the signal transduction phase regulated by a sophisticated interplay of cellular and viral proteins. Here, we show that EV71 inhibits anti-viral type I interferon (IFN) responses by targeting the mitochondrial anti-viral signaling (MAVS) protein—a unique adaptor molecule activated upon retinoic acid induced gene-I (RIG-I) and melanoma differentiation associated gene (MDA-5) viral recognition receptor signaling—upstream of type I interferon production. MAVS was cleaved and released from mitochondria during EV71 infection. An in vitro cleavage assay demonstrated that the viral 2A protease (2A^pro), but not the mutant 2A^pro (2A^pro-110) containing an inactivated catalytic site, cleaved MAVS. The Protease-Glo assay revealed that MAVS was cleaved at 3 residues between the proline-rich and transmembrane domains, and the resulting fragmentation effectively inactivated downstream signaling. In addition to MAVS cleavage, we found that EV71 infection also induced morphologic and functional changes to the mitochondria. The EV71 structural protein VP1 was detected on purified mitochondria, suggesting not only a novel role for mitochondria in the EV71 replication cycle but also an explanation of how EV71-derived 2A^pro could approach MAVS. Taken together, our findings reveal a novel strategy employed by EV71 to escape host anti-viral innate immunity that complements the known EV71-mediated immune-evasion mechanisms.     

Seneca Valley Virus 3Cpro Substrate Optimization Yields Efficient Substrates for Use in Peptide-Prodrug Therapy

The oncolytic picornavirus Seneca Valley Virus (SVV-001) demonstrates anti-tumor activity in models of small cell lung cancer (SCLC), but may ultimately need to be combined with cytotoxic therapies to improve responses observed in patients. Combining SVV-001 virotherapy with a peptide prodrug activated by the viral protease 3C^pro is a novel strategy that may increase the therapeutic potential of SVV-001. Using recombinant SVV-001 3C^pro, we measured cleavage kinetics of predicted SVV-001 3C^pro substrates. An efficient substrate, L/VP4 (k_cat/K_M = 1932 ± 183 M^-1s^-1), was further optimized by a P2’ N→P substitution yielding L/VP4.1 (k_cat/K_M = 17446 ± 2203 M^-1s^-1). We also determined essential substrate amino acids by sequential N-terminal deletion and substitution of amino acids found in other picornavirus genera. A peptide corresponding to the L/VP4.1 substrate was selectively cleaved by SVV-001 3C^pro in vitro and was stable in human plasma. These data define an optimized peptide substrate for SVV-001 3C^pro, with direct implications for anti-cancer therapeutic development.     

Structural basis for antiviral inhibition of the main protease, 3C, from human enterovirus 93

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 Gln↓Gly cleavage sites by the 3C protease (3C^pro), which has narrow, conserved substrate specificity. These cleavages are essential for virus replication, making 3C^pro an excellent target for antivirus drug development. In this study, we report the first determination of the crystal structure of 3C^pro from 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 3C^pro adopts 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 3C^pro in 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.     

Maturation Mechanism of Severe Acute Respiratory Syndrome (SARS) Coronavirus 3C-like Proteinase

The 3C-like proteinase (3CL^pro) of the severe acute respiratory syndrome (SARS) coronavirus plays a vital role in virus maturation and is proposed to be a key target for drug design against SARS. Various in vitro studies revealed that only the dimer of the matured 3CL^pro is active. However, as the internally encoded 3CL^pro gets matured from the replicase polyprotein by autolytic cleavage at both the N-terminal and the C-terminal flanking sites, it is unclear whether the polyprotein also needs to dimerize first for its autocleavage reaction. We constructed a large protein containing the cyan fluorescent protein (C), the N-terminal flanking substrate peptide of SARS 3CL^pro (XX), SARS 3CL^pro (3CLP), and the yellow fluorescent protein (Y) to study the autoprocessing of 3CL^pro using fluorescence resonance energy transfer. In contrast to the matured 3CL^pro, the polyprotein, as well as the one-step digested product, 3CLP-Y-His, were shown to be monomeric in gel filtration and analytic ultracentrifuge analysis. However, dimers can still be induced and detected when incubating these large proteins with a substrate analog compound in both chemical cross-linking experiments and analytic ultracentrifuge analysis. We also measured enzyme activity under different enzyme concentrations and found a clear tendency of substrate-induced dimer formation. Based on these discoveries, we conclude that substrate-induced dimerization is essential for the activity of SARS-3CL^pro in the polyprotein, and a modified model for the 3CL^pro maturation process was proposed. As many viral proteases undergo a similar maturation process, this model might be generally applicable.     

Molecular dynamic simulations analysis of ritonavir and lopinavir as SARS-CoV 3CL(pro) inhibitors

Since the emergence of the severe acute respiratory syndrome (SARS) to date, neither an effective antiviral drug nor a vaccine against SARS is available. However, it was found that a mixture of two HIV-1 proteinase inhibitors, lopinavir and ritonavir, exhibited some signs of effectiveness against the SARS virus. To understand the fine details of the molecular interactions between these proteinase inhibitors and the SARS virus via complexation, molecular dynamics simulations were carried out for the SARS-CoV 3CL^pro free enzyme (free SARS) and its complexes with lopinavir (SARS-LPV) and ritonavir (SARS-RTV). The results show that flap closing was clearly observed when the inhibitors bind to the active site of SARS-CoV 3CL^pro. The binding affinities of LPV and RTV to SARS-CoV 3CL^pro do not show any significant difference. In addition, six hydrogen bonds were detected in the SARS-LPV system, while seven hydrogen bonds were found in SARS-RTV complex.     

Structure–activity relationships of heteroaromatic esters as human rhinovirus 3C protease inhibitors

Human rhinovirus 3C protease (HRV 3C^pro) is known to be a promising target for development of therapeutic agents against the common cold because of the importance of the protease in viral replication as well as its expression in a large number of serotypes. To explore non-peptidic inhibitors of HRV 3C^pro, a series of novel heteroaromatic esters was synthesized and evaluated for inhibitory activity against HRV 3C^pro, to determine the structure–activity relationships. The most potent inhibitor, 7, with a 5-bromopyridinyl group, had an IC₅₀ value of 80 nM. In addition, the binding mode of a novel analog, 19, with the 4-hydroxyquinolinone moiety, was explored by molecular docking, suggesting a new interaction in the S1 pocket.     

Conformational Plasticity of the 2A Proteinase from Enterovirus 71

The 2A proteinase (2A^pro) is an enterovirally encoded cysteine protease that plays essential roles in both the processing of viral precursor polyprotein and the hijacking of host cell translation and other processes in the virus life cycle. Crystallographic studies of 2A^pro from enterovirus 71 (EV71) and its interaction with the substrate are reported here. EV71 2A^pro was comprised of an N-terminal domain of a four-stranded antiparallel β sheet and a C-terminal domain of a six-stranded antiparallel β barrel with a tightly bound zinc atom. Unlike in other 2A^pro structures, there is an open cleft across the surface of the protein in an open conformation. As demonstrated by the crystallographic studies and modeling of the complex structure, the open cleft could be fitted with the substrate. On comparison 2A^pro of EV71 to those of the human rhinovirus 2 and coxsackievirus B4, the open conformation could be closed with a hinge motion in the bII2 and cII β strands. This was supported by molecular dynamic simulation. The structural variation among different 2A^pro structures indicates a conformational flexibility in the substrate-binding cleft. The open structure provides an accessible framework for the design and development of therapeutics against the viral target.     

Differential Regulation of TLR Signaling on the Induction of Antiviral Interferons in Human Intestinal Epithelial Cells Infected with Enterovirus 71

Enterovirus 71 (EV71) causes hand-foot-and-mouth disease, which can lead to fatal neurological complications in young children and infants. Few gastrointestinal symptoms are observed clinically, suggesting the presence of a unique immunity to EV71 in the gut. We reported a robust induction of interferons (IFNs) in human intestinal epithelial cells (HT-29), which was suppressed in other types such as RD and HeLa cells. The underlying mechanism for the apparent difference remains obscure. In this study we report that in EV71-infected HT-29 cells, TLR/TRIF signaling was essential to IFN induction; viral replication increased and the induction of IFN-α, -β, -ω, -κ, and -ε decreased markedly in TRIF-silenced HT-29 cells. Importantly, TRIF was degraded by viral 3C^pro in RD cells, but resisted cleavage, and IRF3 was activated and translocated into the nucleus in HT-29 cells. Taken together, our data suggest that IFNs were induced differentially in human HT-29 cells through an intact TLR/TRIF signaling, which differs from other cell types and may be implicated in viral pathogenesis in EV71 infection.     

Identification of quinone analogues as potential inhibitors of picornavirus 3C protease in vitro

Picornaviruses are non-enveloped viruses that represent a large family of positive-sense single-stranded RNA viruses including a number of causative agents of many human and animal diseases such as coxsackievirus B3 (CVB3) and rhinoviruses (HRV). In this study, we performed a high-throughput screening of a compound library composed of ～6000 small molecules in search of potential picornavirus 3C protease (3C^pro) inhibitors. As results, we identified quinone analogues that effectively inhibited both CVB3 3C^pro and HRV 3C^pro with IC₅₀ values in low micromolar range. Together with predicted binding modes of these compounds to the active site of the viral protease, it is implied that structural features of these non-peptidic inhibitors may act as useful scaffold for further anti-picornavirus drug design and development.     

siRNA Targeting the 2Apro Genomic Region Prevents Enterovirus 71 Replication In Vitro

Enterovirus 71 (EV71) is the most important etiological agent of hand, foot, and mouth disease (HFMD) in young children, which is associated with severe neurological complications and has caused significant mortalities in recent HFMD outbreaks in Asia. However, there is no effective antiviral therapy against EV71. In this study, RNA interference (RNAi) was used as an antiviral strategy to inhibit EV71 replication. Three small interfering RNAs (siRNAs) targeting the 2A^pro region of the EV71 genome were designed and synthesized. All the siRNAs were transfected individually into rhabdomyosarcoma (RD) cells, which were then infected with strain EV71-2006-52-9. The cytopathic effects (CPEs) in the infected RD cells, cell viability, viral titer, and viral RNA and protein expression were examined to evaluate the specific viral inhibition by the siRNAs. The results of cytopathogenicity and MTT tests indicated that the RD cells transfected with the three siRNAs showed slight CPEs and significantly high viability. The 50% tissue culture infective dose (TCID₅₀) values demonstrated that the viral titer of the groups treated with three siRNAs were lower than those of the control groups. qRT–PCR and western blotting revealed that the levels of viral RNA and protein in the RD cells treated with the three siRNAs were lower than those in the controls. When RD cells transfected with siRNAs were also infected with strain EV71-2008-43-16, the expression of the VP1 protein was significantly inhibited. The levels of interferon α (IFN-α) and IFN-β did not differ significantly in any group. These results suggest that siRNAs targeting the 2A^pro region of the EV71 genome exerted antiviral effects in vitro.     

Profiling of substrate specificities of 3C-like proteases from group 1, 2a, 2b, and 3 coronaviruses

Background

Coronaviruses (CoVs) can be classified into alphacoronavirus (group 1), betacoronavirus (group 2), and gammacoronavirus (group 3) based on diversity of the protein sequences. Their 3C-like protease (3CL^pro), which catalyzes the proteolytic processing of the polyproteins for viral replication, is a potential target for anti-coronaviral infection.

Methodology/Principal Findings

Here, we profiled the substrate specificities of 3CL^pro from human CoV NL63 (group 1), human CoV OC43 (group 2a), severe acute respiratory syndrome coronavirus (SARS-CoV) (group 2b) and infectious bronchitis virus (IBV) (group 3), by measuring their activity against a substrate library of 19×8 of variants with single substitutions at P5 to P3'' positions. The results were correlated with structural properties like side chain volume, hydrophobicity, and secondary structure propensities of substituting residues. All 3CL^pro prefer Gln at P1 position, Leu at P2 position, basic residues at P3 position, small hydrophobic residues at P4 position, and small residues at P1'' and P2'' positions. Despite 3CL^pro from different groups of CoVs share many similarities in substrate specificities, differences in substrate specificities were observed at P4 positions, with IBV 3CL^pro prefers P4-Pro and SARS-CoV 3CL^pro prefers P4-Val. By combining the most favorable residues at P3 to P5 positions, we identified super-active substrate sequences ‘VARLQ↓SGF’ that can be cleaved efficiently by all 3CL^pro with relative activity of 1.7 to 3.2, and ‘VPRLQ↓SGF’ that can be cleaved specifically by IBV 3CL^pro with relative activity of 4.3.

Conclusions/Significance

The comprehensive substrate specificities of 3CL^pro from each of the group 1, 2a, 2b, and 3 CoVs have been profiled in this study, which may provide insights into a rational design of broad-spectrum peptidomimetic inhibitors targeting the proteases.