key: cord-0841025-t4ngqr15
authors: Ng, Chen Seng; Stobart, Christopher C.; Luo, Honglin
title: Innate immune evasion mediated by picornaviral 3C protease: Possible lessons for coronaviral 3C‐like protease?
date: 2021-01-07
journal: Rev Med Virol
DOI: 10.1002/rmv.2206
sha: e4f092ecb58004107930207f6202b1fe40254375
doc_id: 841025
cord_uid: t4ngqr15

Severe acute respiratory syndrome coronavirus‐2 is the etiological agent of the ongoing pandemic of coronavirus disease‐2019, a multi‐organ disease that has triggered an unprecedented global health and economic crisis. The virally encoded 3C‐like protease (3CL(pro)), which is named after picornaviral 3C protease (3C(pro)) due to their similarities in substrate recognition and enzymatic activity, is essential for viral replication and has been considered as the primary drug target. However, information regarding the cellular substrates of 3CL(pro) and its interaction with the host remains scarce, though recent work has begun to shape our understanding more clearly. Here we summarized and compared the mechanisms by which picornaviruses and coronaviruses have evolved to evade innate immune surveillance, with a focus on the established role of 3C(pro) in this process. Through this comparison, we hope to highlight the potential action and mechanisms that are conserved and shared between 3C(pro) and 3CL(pro). In this review, we also briefly discussed current advances in the development of broad‐spectrum antivirals targeting both 3C(pro) and 3CL(pro).

Coronavirus disease-2019 (Covid-19) is a multiple organ disease that has posed an unprecedented health and economic threat worldwide since its emergence in late 2019. Covid-19 is caused by a novel virus strain, 1,2 namely severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2) 3 , categorized within the family Coronaviridae. It can infect various hosts and target multiple organs through the body. 4 Coronaviruses are a family of enveloped, single-stranded RNA (ssRNA) viruses with positive polarity that can cause respiratory, enteric, cardiovascular, and central nervous system diseases. 5, 6 This family of RNA viruses features the second largest genome size (27−31 kb) found to date right after planarian nidovirus (∼41.1 kb). 7 Together with SARS-CoV and Middle East Respiratory Syndrome-CoV (MERS-CoV) that caused SARS and MERS outbreaks in 2003 and 2012, respectively, SARS-CoV-2 belongs to the genus betacoronavirus. The genome of betacoronavirus encodes more than 20 proteins, including four major structural proteins (i.e., a spike (S) protein that binds to the cell receptor and mediates fusion between virus and cell membrane, a small envelope (E) protein, a highly hydrophobic membrane (M) protein, and a nucleocapsid (N) protein that interacts with viral RNA to form a helical nucleocapsid structure), two cysteine proteases (i.e., a papain-like cysteine protease (PL pro ) and a 3-chymotrypsin-like cysteine protease (3CL pro , also known as the main protease, M pro ) that processes viral polyproteins into individual functional proteins, a helicase required for unwinding doublestranded RNA (dsRNA), a RNA-dependent RNA polymerase (RdRp) that catalyzes the replication of RNA from RNA template, and other enzymes such as endo-and exonucleases essential for viral nucleic acid metabolism. 8 Among these proteins, SARS-CoV-2 proteases play a vital role in viral replication and transcription, thereby being recognized as attractive antiviral targets for Covid-19 treatment. 9, 10 Of the two known CoV proteases that are encoded by open reading frame 1a (ORF1a), 3CL pro [corresponding to nonstructural protein 5 (NSP5)], which is highly conserved among all CoV 3CL pro , has been identified to be structurally analogous to the 3C pro of picornaviruses (3CL pro is named after the picornaviral 3C pro ). 11, 12 Despite subtle structural differences in the active sites, 3CL pro and 3C pro share a similar chymotrypsin-like tertiary structure with a catalytic triad (or dyad) site containing a cysteine nucleophile ( Figure 1 ). Moreover, both of the enzymes have a strong preference for glutamine (Gln) at the P1 position of their targets, the most key determining factor for their substrate recognition. The conserved active sites of 3C pro and 3CL pro have been confirmed by high-resolution three-dimensional structural analysis. Therefore, it is proposed to serve as an attractive target for the design of broad-spectrum antiviral drugs. [13] [14] [15] Picornaviruses are small, non-enveloped viruses containing a positive-sense, ssRNA genome with a length of 7.0-8.5 kb. This family comprises 29 genera, including Apthovirus (e.g., foot-and-mouth disease virus, FMDV),

Cardiovirus (e.g., encephalomyocarditis virus, EMCV), Enterovirus (e.g., poliovirus, PV; coxsackievirus A16/B3, CVA16; CVB3; enterovirus-A71/D68, EV-A71; EV-D68), Rhinovirus (e.g., human rhinovirus, HRV), and Hepatovirus (e.g., hepatitis A virus, HAV) genera. 16 Picornavirus genomic RNA at its 5 0 end is covalently linked to a small viral protein (VPg, also known as 3B) that serves as a primer for the initiation of viral RNA replication. Further, instead of a cap structure, the genome of picornaviruses possesses an element termed internal ribosome entry site (IRES) in their 5 0 -untranslated region (UTR), which is necessary for initiating a cap-independent translation of viral RNA.

The viral genome of picornaviruses contains one open reading frame encoding a single viral polyprotein that undergoes proteolysis by two viral proteases, 2A pro and 3C pro , with the latter being responsible for the majority of the maturation cleavage events of viral polyprotein similar to coronaviral 3CL pro17 . In addition to processing viral polyprotein, picornaviral proteases also target cellular proteins to evade the human immune surveillance and facilitate viral infection. 18 Given the common characteristics of 3C pro and 3CL pro , we postulate that SARS-CoV-2, like picornaviruses, is capable of regulating host innate antiviral processes through the catalytic activity of its 3CL pro . The delay or inhibition of multiple host antiviral machineries would allow effective viral growth and subsequently optimal release and infection. Here we will recapitulate some of the scenarios on how picornaviruses utilize its 3C pro to target major host antiviral mechanisms.

Early during the viral replication cycle, the positive-sense ssRNA (+ssRNA) genomes of picornaviruses and coronaviruses are translated into one or more polyproteins, which include integrated viral protease domains. Maturation cleavage events mediated by virally encoded proteases in both backgrounds are indispensable for virus replication. The picornaviral 3C pro (working in concert with the 2A pro ) mediates the majority of viral cleavage events including autocleavage from the 3D polymerase (3D pol ) domain of the virus. Similarly, coronaviral 3CL pro is responsible for at least 11 maturation cleavages of the viral replicase polyproteins, including its own autoproteolytic cleavage. Beyond these requisite viral polyprotein cleavages, both 3C pro and 3CL pro target and cleave host cellular proteins. Due to the importance in their respective viral backgrounds, these proteases have been extensively studied as primary targets for viral inhibition for over 30 years.

The picornaviral 3C pro is a chymotrypsin-like cysteine protease comprised of two β-barrel domains of six antiparallel strands which surrounds a conserved Cys-His-Asp/Glu catalytic triad ( Figure 1 ). 19, 20 The protease preferentially cleaves after a P1-Gln with greater cleavage site variability in the other cleavage site residue positions. 21 During picornaviral replication, the 3CD pro precursor, which is able to process the P1 structural precursor but lacks polymerase activity, is cleaved to release 3C pro and viral RdRP 3D pol22 . The conversion of 3CD pro to 3C pro plays a critical role in facilitating the transition and regulation from viral translation to replication. 23, 24 Structurally and functionally analogous to the picornaviral 3C pro , the coronaviral 3CL pro is an approximately 300 residue, 3-domain protease. Domains 1 and 2 comprise the substrate-binding and enzymatic active sites of the protease with dimerization driven by interactions between the structurally unique and largely helical domain 3. 25,26 Domains 1 and 2 of 3CL pro form a chymotrypsin-like fold comprised of antiparallel β-barrels housing the His-Cys catalytic dyad residues. 26, 27 The antiparallel β-barrel conformation within domains 1 and 2 surrounding and forming the active site of 3CL pro shares structural similarity to the core structure of the picornaviral 3C pro , albeit with subtle differences in strand positioning ( Figure 1 ). Unlike the picornaviral 3C pro (monomer with only two catalytic domains), an attached helical third-domain in 3CL pro facilitates dimerization of the protease, an essential event for its enzymatic activity and viral replication ( Figure 1C , Domain 3). 28 While targeting the third domain of 3CL pro serves as a valid strategy to disrupt its dimerization and catalytic activity, any inhibitors identified or designed to do so likely have no impact on picornaviral 3C pro activity since dimerization is not necessary for its function. In addition, among coronaviruses, there is considerably more structural conservation for the chymotrypsin-like fold of 3CL pro compared to that of the picornaviral 3C pro . Based on large part to their structural and functional similarities between the 3C pro and 3CL pro , it remains unclear to what extent these proteases share common host cellular targets during viral replication and what role these potentially shared cleavages play in viral replication.

The effectiveness of an antiviral innate immunity depends on the accurate recognition of viral moieties, known as the pathogen-associated molecular patterns (PAMPs), by pattern-recognition receptors (PRRs) composed of at least three classes: retinoic acid-inducible gene-I (RIG-I)-like receptors (RLRs), Toll-like receptors (TLRs), and nucleotide-binding oligomerization domain (NOD)-like receptors (NLRs). 29 Upon RNA viral infection, dsRNAs often accumulate in cells in the form of the viral genome or its replication intermediates. The dsRNAs can be recognized by cytosolic viral RNA sensor, RLRs (e.g., RIG-I (encoded by Ddx58 gene) and melanoma differentiation-associated proteins (MDA5, encoded by Ifih1 gene)), and/or endosomal viral RNA sensor (e.g., TLR3) to initiate type I interferon (IFN-I) immune response. While RIG-I preferentially binds to shorter dsRNA (<1-2 kb) bearing 5 0 -triphosphate group, MDA5 primarily recognizes long dsRNA. 30 Similar to picornavirus, SARS-CoV-2 has a long RNA genome. It is therefore expected that SARS-CoV-2 RNA favorably binds to MDA5 rather than its paralog RIG-I. Indeed, MDA5 has been previously shown to be the specific PRR that recognizes murine coronavirus RNA. 31 Interaction between viral RNA and MDA5 forms MDA5 filaments along dsRNAs, which brings together neighboring caspase activation and recruitment domain (CARD) in close proximity to induce oligomerization and activation of the adapter mitochondrial-antiviral signaling protein (MAVS). 32 Activated MAVS then transmits the signals to its downstream transcription factors, interferon regulatory factor-3/7 (IRF3/7) and nuclear-factor-κB (NF-κB), through TANK-binding kinase-1 (TBK1) and IκB kinase-ε (IKKε). The homo-or hetero-dimerized IRF3/7 subsequently translocate to the nucleus and induce the expression of IFN-I-associated genes (Ifna and Ifnb1), which could further activate the Janus kinase/signal transducers and activators of transcription (JAK/STAT) signaling cascade to trigger the expression of antiviral genes, called interferonstimulated genes (ISGs). 30 Please see Figure 2 for the details.

To antagonize human antiviral innate immune response, picornaviruses target critical components of the RLR signaling pathway for degradation. It was reported that infections with Seneca Valley virus (SVV), PV, EV-A71, EV-D68, CVB3, CVA16, CVA6, HRV-1A, EMCV, and HAV cleave MDA5, MAVS, RIG-I, IRF7, and/or IRF9 through the actions of 3C pro , leading to a disruption of RLR-mediated IFN-I immune responses. [33] [34] [35] [36] [37] [38] [39] In addition to 3C pro , studies have found that MDA5 and MAVS can also be targeted by 2A pro upon PV, EV-A71, CVB3, and HRV-1Ainfections. 34, 40, 41 Moreover, both TAK1 binding protein-1 (TAB1) and NF-κB essential modulator (NEMO, an adapter protein bridging the canonical IKKα/β kinases and the noncanonical kinases TBK1/IKKε via the TANK adapter 42 ) are cleaved by 3C pro following EV-A71, FMDV and HAV infections, resulting in reduced production of IFN-I. [43] [44] [45] In addition, FMDV utilizes its 3C pro to inhibit STAT2 function, a component of the IFN-stimulated gene factor 3 complex, which is also mirrored by the 3CL pro of PDCoV (Table 1 , yellow highlighted row). 57, 58 Another interesting finding associated with this topic is 3C pro -induced cleavage of Toll/IL-1 receptor (TIR)-domain-containing adapter-inducing interferon-β (TRIF) during CVB3, EV-A71, and HAV infections. 36, 47, 48 TRIF is an adapter protein mediating type I IFN antiviral response downstream of the endosomal TLR3 (a viral RNA sensor) 114 and cleavage causes the loss of its function in host defense.

In addition to directly targeting components of the IFN-I pathway, 3C pro can also modulate the function of proteins that participate in the regulation of this pathway. For instance, the 3C pro of EV-A71 downregulates miRNA-526a, consequently leading to increased expression of cylindromatosis (CYLD, a target of miRNA-526a). 115 CYLD is a deubiquitinating enzyme negatively regulating the function of RIG-I by removing K63-linked polyubiquitin chains from RIG-I. 116 Ubiquitination is a post-translational modification required for RIG-I activation, 117 and deubiquitination results in its inactivation. Together, available evidence reveals that 3C pro plays a key role in the efforts of picornaviruses in counteracting the host antiviral immune response by cleaving or inactivating essential adapter proteins in the RIG-I/MDA5-MAVS and/or the TLR3-TRIF signaling pathways ( Figure 2 ).

Like picornaviruses, both SARS-CoV and MERS-CoV trigger a limited IFN-I response. [118] [119] [120] A mouse model infected with SARS-CoV demonstrated that a significant delay in IFN production contributes to disease progression and severity. 121 Using different model systems including cells and ferrets infected with SARS-CoV-2 and post-mortem lung tissues from COVID patients, a recent study showed that SARS-CoV-2 infections elicit low levels of IFN-I and no activation of TBK1 and ISGs. 122 Of note, it was found that SARS-CoV-2 is highly sensitive to IFN-I, suggesting an important role for IFN-I in antiviral defense. 122, 123 Although experimental data on SARS-CoV-2 is still limited, current evidence from SARS-CoV and MERS-CoV research revealed that CoVs develop different strategies to overcome the host innate immunity. For example, the PL pro of SARS-CoV, which has deubiquitinating activities, acts as an IFN-I antagonist by removing ubiquitin chains from IRF3 and through preventing the phosphorylation of IRF3 124, 125 . It was also discovered that the ORF3b, ORF6, and N proteins of SARS-CoV inhibit production and action of IFN-I. 126 Moreover, M protein of SARS-CoV was shown to physically associate with RIG-I, TBK1, IKKε, and TRAF3 and inhibit gene transcription of IFN-I. 127 Most interestingly, several coronaviruses, including SARS-CoV-2, porcine deltacoronavirus (PDCoV), and porcine epidemic diarrhea virus (PEDV) have been reported to cause the cleavage of TAB1 or NEMO through their individual 3CL pro (Table 1 , yellow highlighted row), 49, 59, 60 suggesting an important role for 3CL pro in antagonizing the host antiviral innate immune response.

The importance of virus-induced cytoplasmic aggregates, termed antiviral stress granules (avSGs), 128 in RLRs-mediated innate immunity has been increasingly recognized. Upon viral infection, dsRNAs are generated and accumulate in the cytoplasm, which activate the viral RNA sensor protein kinase R (PKR) and cause phosphorylation of eukaryotic initiation factor 2α and consequent formation of avSGs.

Together with key molecules (i.e., Ras-GTPase-activating protein SH3 domain-binding protein 1 (G3BP1) and T cell intracellular antigen 1), avSGs are formed by recruiting multiple antiviral effectors, such as RLRs, Pumilio, DEAH-box helicase 36, Mex-3 RNA binding family member C, tripartite motif containing 25, OAS and RNaseL. [128] [129] [130] [131] [132] The close-proximity of 5 0 -triphosphate containing ssRNA and dsRNA with RLRs within the compact avSG compartment facilitates a more robust RLR-mediated IFN-I responses to suppress viral replication. [128] [129] [130] [131] [132] To bypass this, picornaviruses, including PV, EMCV, CVB3, FMDV, and EV-D68, utilize 3C pro to cleave G3BP1 and block avSG formation to prolong viral survival. [108] [109] [110] [111] 133 CoVs were also found to be able to modulate the formation of avSGs. It was reported that MERS-CoV accessory protein 4a prevents PKR activation by directly binding viral dsRNA, thereby inhibiting avSG formation allowing for effective viral replication. 134, 135 However, the role of protein 4a in avSG formation F I G U R E 2 Picornaviruses evade type I interferon immune response via the function of 3C protease (3C pro ). Binding of picornaviruses to their respective receptors facilitates their entry into the cells and release of the 5 0 -viral protein genome-linked-containing genomic RNA into cytoplasm. Long double-stranded RNA generated during the replication process binds to MDA5, exposing its CARD and allowing homotypic CARD-CARD interactions with its downstream adapter, MAVS. Subsequently, MAVS triggers the expression of IFN-I genes (Ifnb1 and Ifna in dendritic cells) and ISGs for antiviral purposes through the activation of transcription factor IRF3/7 and NF-κB. To facilitate a robust signaling, more efficient detection of dsRNA occurs in antiviral stress granules. Targets of viral encoded 3Cpro are indicated. CARD, caspase activation and recruitment domain; CTD, C-terminal binding domain; G3BP1, Ras GTPase-activating protein-binding protein 1; IFN-I, type-I interferon; IKKε, inhibitor of nuclear factor-κB (IκB)-kinase-ε; IRF3/7, interferon regulatory factors-3/7; ISGs, interferon-stimulating genes; MAVS, mitochondrial antiviral signaling protein; MDA5, melanoma-differentiation associated protein-5; NF-κB, nuclear factor-κB; NEMO, NF-κB essential modulator; P, phosphate-group; RIG-I, retinoic acid-inducible gene-I; TBK1, TANK binding kinase-1; TLR3, Toll-like receptor 3; TRAF3, TNF-receptor associated factor-3; TRIF, Toll/IL-1 receptor domain-containing adapter-inducing interferon-β appears to be cell type-specific. MERS-CoV mutant with deletion of 4a gene still impedes avSG formation in certain cell types, 134, 135 suggesting that additional proteins (possibly viral proteases) encoded by MERS-CoV are required to antagonize avSGs. In a recent report, Grogan and colleagues 8 utilized an affinity purification mass spectrometry proteomics approach to screen for cellular proteins that interact with individual SARS-CoV-2 proteins, including 3CL pro . Using both wild-type and catalytically inactive (C145A) 3CL pro , they identified two high-confidence interactions with the histone deacetylase 2 (HDAC2) and tRNA methyltransferase 1 (TRMT1), respectively. Of interest, HDAC2 has been previously shown to be required for IFN-I signaling through histone modification. 136, 137 Remarkably, in addition to 3CL pro , other SARS-CoV-2-encoded proteins were also found to interact with cellular proteins involved in regulating host innate immunity, including the core avSG protein G3BP1 8 , a known antiviral protein that induces the innate immune response. 129, 138 Clinical evidence revealed that SARS-CoV-2 infections predis- As NLRP3 plays a vital role in antiviral response, numerous viruses, including picornaviruses, have adopted strategies to counteract its functions. 145 It was reported that NLRP3-, ASC-, and caspase-1-deficient mice infected with EV-A71 display more severe disease phenotype and higher virus titers as compared to wild-type control mice, suggesting a defensive role for the NLRP3 inflammasome against EV-A71 infection. 52 inflammasome. [153] [154] [155] Recent evidence has also revealed that NLRP3

inflammasome is activated in response to SARS-CoV-2 infection. 156 It is speculated that overactivation of the inflammasome may be responsible, at least in part, for the observed cytokine storm in Covid-19 patients.

The NLRP inflammasome pathway and viral manipulation are summarized in Figure 4 .

In eukaryotic cells, posttranscriptional processes (e.g., mRNA sur- 103 The interplay between the cellular RNA degradation pathway and the viruses is illustrated in Figure 5 .

While both the genomic and subgenomic SARS-CoV-2 mRNAs contain 5 0 -cap structure, previous studies have shown that inhibiting several eukaryotic initiation factors family proteins (eIF4E, eIF4F, and eIF4G) could impair coronavirus replication, 160, 161 highlighting the importance of cap-dependent translation in SARS-CoV-2 mRNA synthesis. Thus, it is not surprising that SARS-CoV-2 would intervene the function of host decapping enzyme DCP1/2 and XRN1, possibly through its 3CL pro . However, to date, antiviral roles XRN1 in coronaviral mRNA translation have not been reported.

Macroautophagy (or autophagy in short) is a conserved intracellular degradation pathway that is essential in maintaining cellular homeostasis by removing unwanted or dysfunctional cellular components. 162 The process of autophagy is highly regulated by more than 30 "autophagy-related" proteins and includes three major steps. First, the substrates are sequestered by a crescent-shaped doublemembrane vesicle called a phagophore. Then, the two ends of the phagophore fuse to form an autophagosome. Finally, autophagosome fuses with a lysosome while the enwrapped cargo is degraded by hydrolysis.

Subversion of host autophagy through picornaviral 3C pro . Schematic diagram depicted the molecular mechanism for the initiation of host autophagy pathway upon the presence of RNA virus for the clearance of viral-associated molecules. Picornaviruses utilize its own encoded 2A pro (not shown here) and 3C pro to cleave key components such as p62, NBR1, SNAP29 and PLEKHM1 to facilitate a more robust replication. ATGs, autophagy-related genes; DFCP1, double FYVE-containing protein-1, FIP200, focal adhesion kinase family interacting protein of 200kD; NBR1, neighbor of BRCA1; SNAP29, synaptosomal-associated protein-29; PLEKHM1, Pleckstrin homology and RUN domain containing M1; p62, also known as sequestosome 1 (SQSTM1); STX17, Syntaxin 17; ULK, Unc-51-like kinase-1; UVRAG, UV radiation resistance-associated gene protein; VAMP8, vesicle-associated membrane protein-8; WIPI2, WD-repeat domain phosphoinositideinteracting protein-2; 2A pro , 2A-protease; 3C pro , 3C protease Autophagy plays a significant role in antiviral host defense by directly targeting invading viruses through a process, call virophagy, for clearance. 163, 164 Virophagy is mediated through the function of autophagy cargo receptors, including sequestosome 1 (SQSTM1)/p62, neighbor of BRCA1 (NBR1), calcium binding and coiled-coil domain-containing protein 2 (CALCOCO2)/nuclear dot 10 protein 52 (NDP52), which recruit viral components/particles to autophagosome for degradation. 165 To evade the antiviral efforts of virophagy, many viruses, including picornaviruses, have evolved to disrupt the function of autophagy receptors. 166 For instance, SQSTM1/p62 and CALCOCO2/NDP52 are targeted for degradation by CVB3 2A pro and 3C pro , respectively. 66, 67 Cleavage of SQSTM1/p62 was later confirmed upon PV, HRV-1A, and EV-D68

infection. 68 Furthermore, NBR1, a homolog of SQSTM1/p62, can also be cleaved by 3C pro69 . Remarkably, it was found that cleavage of SQSTM1/p62 and NBR1 not only causes a loss-of-function, but also generates dominant-negative mutants against the function of native proteins. 69 Loss of both SQSTM1/p62 and NBR1 would also impair mitophagy and results in mass production of reactive oxygen species, and IL-1β through constitutive NLRP3-independent inflammasome activation (potentially other NLRP family members). 167 The resulting cleavage fragments of SQSTM1/p62 and NBR1 will accumulate to serve as DAMPs signals and further amplify the inflammatory cascade. Acute respiratory pneumonia due to cytokine storm is a hall mark of SARS-CoV-2 infection, whether its 3CL pro would manipulate host autophagic system in particular to mass produced IL-1β in Covid-19 pathogenesis is certainly worth further investigation.

F I G U R E 7 Schematic workflow of TAILS N-terminomics screening of 3C pro and 3CL pro substrates. Schematic diagram depicts the TAILS workflow and scheme for identification of 3C pro or 3CL pro substrates. In brief, protein samples from whole cell lysates were subjected to in vitro cleavage by either recombinant purified WT 3C pro /3CL pro or mutant (C147A) 3C pro /(C145A) 3CL pro , followed by N-terminal enrichment using TAILS (left panel). Samples were then combined and subjected to pre-TAILS shotgun-like mass-spectrometry analysis after complete digestion with trypsin. The exposed amine groups of N-termini generated by the trypsin digestion were then removed by covalently coupling to a high-molecular weight polyaldehyde polyglycerol polymer. This process allowed for selection via negative enrichment of blocked N termini (middle panel). Peptides were subsequently identified and quantified using high-resolution mass spectrometry (indicated in the right panel). The resultant high-confidence candidate substrates were determined through the analysis of the quantified heavy/light (H/L) ratio of dimethylation-labeled semitryptic neo-N terminus peptides. They will be subjected to further validation through similar in vitro cleavage assay by 3C pro /3CL pro , followed by immunoblotting using specific antibodies; TAILS, terminal amine isotopic labeling of substrates; 3C pro , 3C protease; 3CL pro , 3C-like protease CoV 3CL pro has a role in regulation of autophagic flux and cargo recognition in general as some picornaviral 3C pro does has not been explored and requires further investigations. The list of substrates of 3C pro and 3CL pro is summarized in Table 1 .

A proteomics-based quantitative method, termed N-terminomic terminal amine isotopic labeling of substrates (TAILS) (Figure 7) , 172 has been exploited to globally search for novel cellular substrates of picornaviral proteases. 63, 85 TAILS, developed by the Overall laboratory at the University of British Columbia, uses an unbiased negative selection approach to identify neo-N and -C termini (named N-terminomic and C-terminomic TAILS, respectively). 172, [173] [174] [175] This state-of-the-art approach has several advantages, including quantitative, highly sensitive, and concurrent identification of both the substrates and the cleavage sites, 176 and has been used to analyze substrates of many types of proteases. 177, 178 Jan and colleagues conducted N-terminomic TAILS on HeLa cell or mouse cardiomyocyte extracts subjected to incubation with purified recombinant PV or CVB3 3C pro , respectively, to identify cleaved neo-N-terminal peptides (see Figure 7 for the detailed procedure). 63 Given the effectiveness of the N-terminomic TAILS in identifying the cellular targets of 3C pro , this unbiased proteomics approach is currently being utilized to analyze the cellular targets of MHV and SARS-CoV-2 proteases, including 3CL pro . It is anticipated that identification of the full repertoire of host substrates of CoV proteases will provide a more comprehensive understanding of viral tropism, interaction with host cells, and pathogenesis, as well as assist in the development of novel antiviral drugs.

As discussed earlier, both picornaviral 3C pro and coronaviral 3CL pro are chymotrypsin-like cysteine proteases with conserved substrate specificity (P1-P1 0 and P4 cleavage sites of the substrates are highly conserved between two enzymes) and active sites. 179 Owing to these similarities, efforts have been made to explore the potential of developing broad-spectrum antiviral compounds. [13] [14] [15] The fact that no known human homologs further increases the feasibility of this strategy.

Rupintrivir (AG-7088, a protease inhibitor originally developed for HRV to treat common cold) and/or its derivatives/analogs compounds (11a and 11b) targeting 3CL pro and solved the X-ray crystal structure of these inhibitors bound to 3CL. 9 Although these compounds serve as promising drug candidates, their effectiveness in nature infection remain to be investigated. The list of inhibitors targeting 3C pro and 3CL pro is summarized in Table 2 .

To date, we know very little about the 3CL pro of SARS-CoV-2, especially the molecular mechanism of the pathways by which SARS-CoV-2 3CL pro blocks. Certainly, we cannot rule out that there are some differences but also a lot of similarities among both families. While we are in the process of understanding the structure and functions of SARS-CoV-2 3CL pro , a comparison with the 3C pro from picornaviruses can provide more insights into the pathogenesis and regulatory mechanisms of Covid-19. These will serve as a critical foundation for the design of broad-spectrum anti-coronaviruses inhibitors, or perhaps anti-3C/3CL pro expressing viruses.

We thank all the members in Luo lab for insights and discussion. We apologize for not including all related references due to space con- 

The authors declare that they have no conflict of interest.

Chen Seng Ng and Honglin Luo designed and outlined the review.

Chen Seng Ng prepared the initial manuscript and figures. Christopher C. Stobart prepared the structural figure and relevant information. All authors contributed to revising and writing the final version.

Data sharing not applicable to this article as no datasets were generated or analyzed during the current study.

Honglin Luo https://orcid.org/0000-0002-7708-7840

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Porcine reproductive and respiratory syndrome virus 3C protease cleaves the mitochondrial antiviral signalling complex to antagonize IFN-β expression

Cleavage of the adaptor protein TRIF by enterovirus 71 3C inhibits antiviral responses mediated by Toll-like receptor 3

Disruption of TLR3 signaling due to cleavage of TRIF by the hepatitis A virus protease-polymerase processing intermediate

SARS-CoV-2 proteases cleave IRF3 and critical modulators of inflammatory pathways (NLRP12 and TAB1): implications for disease presentation across species and the search for reservoir hosts

3C protease of enterovirus D68 inhibits cellular defense mediated by interferon regulatory factor 7

Synergistic inhibition of enterovirus 71 replication by interferon and rupintrivir

Reciprocal regulation between enterovirus 71 and the NLRP3 inflammasome

NLRP3 deficiency exacerbates enterovirus infection in mice

Encephalomyocarditis virus 3C protease relieves TRAF family member-associated NF-κB activator (TANK) inhibitory effect on TRAF6-mediated NF-κB signaling through cleavage of TANK

Enterovirus 71 disrupts interferon signaling by reducing the level of interferon receptor 1

Intracellular sensing of complement C3 activates cell autonomous immunity

Porcine deltacoronavirus nsp5 antagonizes type I interferon signaling by cleaving STAT2

3Cpro of foot-and-mouth disease virus antagonizes the interferon signaling pathway by blocking STAT1/STAT2 nuclear translocation

Porcine epidemic diarrhea virus 3C-like protease regulates its interferon antagonism by cleaving NEMO

Porcine deltacoronavirus nsp5 inhibits interferon-β production through the cleavage of NEMO

Porcine reproductive and respiratory syndrome virus nonstructural protein 4 antagonizes beta interferon expression by targeting the NF-κB essential modulator

RIP3 regulates autophagy and promotes coxsackievirus B3 infection of intestinal epithelial cells

N-terminomics TAILS identifies host cell substrates of poliovirus and coxsackievirus B3 3C proteinases that modulate virus infection

The 3C protease of enterovirus A71 counteracts the activity of host zinc-finger antiviral protein (ZAP)

Enterovirus 71 inhibits pyroptosis through cleavage of gasdermin D

CALCOCO2/ NDP52 and SQSTM1/p62 differentially regulate coxsackievirus B3 propagation

Cleavage of sequestosome 1/p62 by an enteroviral protease results in disrupted selective autophagy and impaired NFKB signaling

Enteroviruses remodel autophagic trafficking through regulation of host SNARE proteins to promote virus replication and cell exit

Dominant-negative function of the C-terminal fragments of NBR1 and SQSTM1 generated during enteroviral infection

Enteroviral infection inhibits autophagic flux via disruption of the SNARE complex to enhance viral replication

Dysferlin deficiency confers increased susceptibility to coxsackievirus-induced cardiomyopathy

Enteroviral protease 2A cleaves dystrophin: evidence of cytoskeletal disruption in an acquired cardiomyopathy

2A proteinase of human rhinovirus cleaves cytokeratin 8 in infected HeLa cells

Poliovirus protease 3C mediates cleavage of microtubule-associated protein 4

Cleavage of transcriptional activator Oct-1 by poliovirus encoded protease 3Cpro

Intracellular redistribution of truncated La protein produced by poliovirus 3Cpro-mediated cleavage

Direct cleavage of human TATA-binding protein by poliovirus protease 3C in vivo and in vitro

Shutoff of RNA polymerase II transcription by poliovirus involves 3C proteasemediated cleavage of the TATA-binding protein at an alternative site: incomplete shutoff of transcription interferes with efficient viral replication

Identification of the cleavage site and determinants required for poliovirus 3CPro-catalyzed cleavage of human TATA-binding transcription factor TBP

Poliovirus proteinase 3C converts an active form of transcription factor IIIC to an inactive form: a mechanism for inhibition of host cell polymerase III transcription by poliovirus

Inhibition of host cell transcription by poliovirus: cleavage of transcription factor CREB by poliovirus-encoded protease 3Cpro

Inhibition of basal transcription by poliovirus: a virus-encoded protease (3Cpro) inhibits formation of TBP-TATA box complex in vitro

Cleavage of serum response factor mediated by enteroviral protease 2A contributes to impaired cardiac function

Translation of polioviral mRNA is inhibited by cleavage of polypyrimidine tract-binding proteins executed by polioviral 3C(pro)

Heterogeneous nuclear ribonucleoprotein M facilitates enterovirus infection

Inhibition of U snRNP assembly by a virusencoded proteinase

Cytoplasmic translocation, aggregation, and cleavage of TDP-43 by enteroviral proteases modulate viral pathogenesis

Proteolytic cleavage of the p65-RelA subunit of NF-kappaB during poliovirus infection

Cytoplasmic redistribution and cleavage of AUF1 during coxsackievirus infection enhance the stability of its viral genome

Cleavage of poly(A)-binding protein by enterovirus proteases concurrent with inhibition of translation in vitro

Cleavage of poly(A)-binding protein by duck hepatitis A virus 3C protease

A) binding protein, C-terminally truncated by the hepatitis A virus proteinase 3C, inhibits viral translation

Site-specific cleavage of the host poly(A) binding protein by the encephalomyocarditis virus 3C proteinase stimulates viral replication

Foot-and-mouth disease virus 3C protease induces cleavage of translation initiation factors eIF4A and eIF4G within infected cells

Leader protein of foot-and-mouth disease virus is required for cleavage of the p220 component of the cap-binding protein complex

The two species of the foot-and-mouth disease virus leader protein, expressed individually, exhibit the same activities

Coxsackievirus B3 proteases 2A and 3C induce apoptotic cell death through mitochondrial injury and cleavage of eIF4GI but not DAP5/p97/NAT1

A single amino acid change in protein synthesis initiation factor 4G renders cap-dependent translation resistant to picornaviral 2A proteases

Mapping of functional domains in eukaryotic protein synthesis initiation factor 4G (eIF4G) with picornaviral proteases. Implications for capdependent and cap-independent translational initiation

Cleavage of eukaryotic initiation factor eIF5B by enterovirus 3C proteases

Cleavage of DAP5 by coxsackievirus B3 2A protease facilitates viral replication and enhances apoptosis by altering translation of IRES-containing genes

Cellular protein modification by poliovirus: the two faces of poly(rC)-binding protein

Poliovirus-mediated disruption of cytoplasmic processing bodies

Viral protease cleavage of inhibitor of kappaBalpha triggers host cell apoptosis

Enhanced enteroviral infectivity via viral protease-mediated cleavage of Grb2-associated binder 1

Specific cleavage of the nuclear pore complex protein Nup62 by a viral protease

Differential processing of nuclear pore complex proteins by rhinovirus 2A proteases from different species and serotypes

Production of a dominant-negative fragment due to G3BP1 cleavage contributes to the disruption of mitochondria-associated protective stress granules during CVB3 infection

Encephalomyocarditis virus disrupts stress granules, the critical platform for triggering antiviral innate immune responses

Inhibition of cytoplasmic mRNA stress granule formation by a viral proteinase

Foot-and-Mouth disease virus counteracts on internal ribosome entry site suppression by G3BP1 and inhibits G3BP1-mediated stress granule assembly via post-translational mechanisms

Essential role of enterovirus 2A protease in counteracting stress granule formation and the induction of type I interferon

Foot-and-Mouth disease virus leader protease cleaves G3BP1 and G3BP2 and inhibits stress granule formation

TRIF-dependent TLR signaling, its functions in host defense and inflammation, and its potential as a therapeutic target

Downregulation of microRNA miR-526a by enterovirus inhibits RIG-I-dependent innate immune response

Regulation of IkappaB kinase-related kinases and antiviral responses by tumor suppressor CYLD

Reconstitution of the RIG-I pathway reveals a signaling role of unanchored polyubiquitin chains in innate immunity

Tropism of and innate immune responses to the novel human betacoronavirus lineage C virus in human ex vivo respiratory organ cultures

Pathogenic influenza viruses and coronaviruses utilize similar and contrasting approaches to control interferon-stimulated gene responses

Active replication of Middle East respiratory syndrome coronavirus and aberrant induction of inflammatory cytokines and chemokines in human macrophages: implications for pathogenesis

Dysregulated type I interferon and inflammatory monocyte-macrophage responses cause lethal pneumonia in SARS-CoV-infected mice

Imbalanced Host Response to SARS-CoV-2 Drives Development of COVID-19

Type I interferon susceptibility distinguishes SARS-CoV-2 from SARS-CoV

Deubiquitinating and interferon antagonism activities of coronavirus papain-like proteases

The SARS coronavirus papain like protease can inhibit IRF3 at a post activation step that requires deubiquitination activity

Severe acute respiratory syndrome coronavirus open reading frame (ORF) 3b, ORF 6, and nucleocapsid proteins function as interferon antagonists

Severe acute respiratory syndrome coronavirus M protein inhibits type I interferon production by impeding the formation of TRAF3.TANK.TBK1/IKKepsilon complex

Critical role of an antiviral stress granule containing RIG-I and PKR in viral detection and innate immunity

The stress granule protein G3BP1 binds viral dsRNA and RIG-I to enhance interferon-β response

Pivotal role of RNA-binding E3 ubiquitin ligase MEX3C in RIG-I-mediated antiviral innate immunity

A novel function of human Pumilio proteins in cytoplasmic sensing of viral infection

DHX36 enhances RIG-I signaling by facilitating PKRmediated antiviral stress granule formation

Typical stress granule proteins interact with the 3' untranslated region of enterovirus D68 to inhibit viral replication

Middle East respiratory coronavirus accessory protein 4a inhibits PKR-mediated antiviral stress responses

Inhibition of stress granule formation by middle east respiratory syndrome coronavirus 4a accessory protein facilitates viral translation, leading to efficient virus replication

Opposed regulation of type I IFN-induced STAT3 and ISGF3 transcriptional activities by histone deacetylases (HDACS) 1 and 2

Requirement of histone deacetylase activity for signaling by STAT1

The stress granule protein G3BP1 recruits protein kinase R to promote multiple innate immune antiviral responses

Epidemiological and clinical characteristics of 99 cases of 2019 novel coronavirus pneumonia in Wuhan, China: a descriptive study

Clinical course and risk factors for mortality of adult inpatients with COVID-19 in Wuhan, China: a retrospective cohort study

A unique host defense pathway: TRIF mediates both antiviral and antibacterial immune responses

PKR-dependent cytosolic cGAS foci are necessary for intracellular DNA sensing

G3BP1 promotes DNA binding and activation of cGAS

Pivotal roles of cGAS-cGAMP signaling in antiviral defense and immune adjuvant effects

NLRP3 inflammasome-A key player in antiviral responses

Cleavage of GSDMD by inflammatory caspases determines pyroptotic cell death

Inflammasome-activated gasdermin D causes pyroptosis by forming membrane pores

RNA viruses promote activation of the NLRP3 inflammasome through a RIP1-RIP3-DRP1 signaling pathway

IL-1β production through the NLRP3 inflammasome by hepatic macrophages links hepatitis C virus infection with liver inflammation and disease

NOD2 (nucleotide-binding oligomerization domain 2) is a major pathogenic mediator of coxsackievirus B3-induced myocarditis

Involvement of NLRP3 inflammasome in CVB3-induced viral myocarditis

Clinical and immunological features of severe and moderate coronavirus disease 2019

Severe acute respiratory syndrome coronavirus ORF3a protein activates the NLRP3 inflammasome by promoting TRAF3-dependent ubiquitination of ASC

SARS-Coronavirus Open Reading Frame-8b triggers intracellular stress pathways and activates NLRP3 inflammasomes

Severe acute respiratory syndrome coronavirus E protein transports calcium ions and activates the NLRP3 inflammasome

Inflammasome activation in COVID-19 patients

Cytoplasmic RNA granules and viral infection

Emerging roles for RNA degradation in viral replication and antiviral defense

Spatio-temporal characterization of the antiviral activity of the XRN1-DCP1/2 aggregation against cytoplasmic RNA viruses to prevent cell death

Blocking eIF4E-eIF4G interaction as a strategy to impair coronavirus replication

Reversing chemoresistance by small molecule inhibition of the translation initiation complex eIF4F

Molecular definitions of autophagy and related processes

Autophagy in infection, inflammation and immunity

Autophagy balances inflammation in innate immunity

Structure biology of selective autophagy receptors

The intertwined life cycles of enterovirus and autophagy

Mitochondria and mitophagy: the yin and yang of cell death control

New insights into autophagosomelysosome fusion

Analysis of SARS-CoV-2-controlled autophagy reveals spermidine, MK-2206, and niclosamide as putative antiviral therapeutics

SKP2 attenuates autophagy through Beclin1-ubiquitination and its inhibition reduces MERS-Coronavirus infection

Coronavirus membrane-associated papain-like proteases induce autophagy through interacting with Beclin1 to negatively regulate antiviral innate immunity

Identifying and quantifying proteolytic events and the natural N terminome by terminal amine isotopic labeling of substrates

Proteome-derived, database-searchable peptide libraries for identifying protease cleavage sites

Auf dem Keller U, Overall C M. Characterization of the prime and non-prime active site specificities of proteases by proteome-derived peptide libraries and tandem mass spectrometry

Isotopic labeling of terminal amines in complex samples identifies protein N-termini and protease cleavage products

N-and C-terminal degradomics: new approaches to reveal biological roles for plant proteases from substrate identification

The paracaspase MALT1 cleaves HOIL1 reducing linear ubiquitination by LUBAC to dampen lymphocyte NF-κB signalling

Discovery of a proteolytic flagellin family in diverse bacterial phyla that assembles enzymatically active flagella

An overview of severe acute respiratory syndrome-coronavirus (SARS-CoV) 3CL protease inhibitors: peptidomimetics and small molecule chemotherapy

Enterovirus 71 and coxsackievirus A16 3C proteases: binding to rupintrivir and their substrates and antihand, foot, and mouth disease virus drug design

Conservation of amino acids in human rhinovirus 3C protease correlates with broad-spectrum antiviral activity of rupintrivir, a novel human rhinovirus 3C protease inhibitor

3C protease of enterovirus 68: structure-based design of Michael acceptor inhibitors and their broad-spectrum antiviral effects against picornaviruses

Application of a cellbased protease assay for testing inhibitors of picornavirus 3C proteases

Design of wide-spectrum inhibitors targeting coronavirus main proteases

Phase II, randomized, double-blind, placebo-controlled studies of ruprintrivir nasal spray 2-percent suspension for prevention and treatment of experimentally induced rhinovirus colds in healthy volunteers

Of chloroquine and COVID-19

Compassionate Use of Remdesivir for Patients with Severe Covid-19

Remdesivir and chloroquine effectively inhibit the recently emerged novel coronavirus (2019-nCoV) in vitro

Chloroquine does not inhibit infection of human lung cells with SARS-CoV-2

Tricuspid regurgitation and long-term clinical outcomes

Synthesis, crystal structure, structure-activity relationships, and antiviral activity of a potent SARS coronavirus 3CL protease inhibitor

Inhibition of SARS-CoV 3CL protease by flavonoids

Synthesis and evaluation of pyrazolone compounds as SARS-coronavirus 3C-like protease inhibitors

Aryl methylene ketones and fluorinated methylene ketones as reversible inhibitors for severe acute respiratory syndrome (SARS) 3C-like proteinase

High-throughput screening identifies inhibitors of the SARS coronavirus main proteinase

Stable benzotriazole esters as mechanism-based inactivators of the severe acute respiratory syndrome 3CL protease

A structural view of the inactivation of the SARS coronavirus main proteinase by benzotriazole esters

Identification of novel inhibitors of the SARS coronavirus main protease 3CLpro

Crystal structures reveal an induced-fit binding of a substrate-like Aza-peptide epoxide to SARS coronavirus main peptidase

Crystal structures of the main peptidase from the SARS coronavirus inhibited by a substrate-like aza-peptide epoxide

Aza-peptide epoxides: a new class of inhibitors selective for clan CD cysteine proteases

A new lead for nonpeptidic active-site-directed inhibitors of the severe acute respiratory syndrome coronavirus main protease discovered by a combination of screening and docking methods

New non-peptidic inhibitors of papain derived from etacrynic acid

Structure-based design and synthesis of highly potent SARS-CoV 3CL protease inhibitors

Discovery of a novel family of SARS-CoV protease inhibitors by virtual screening and 3D-QSAR studies

Structure-based drug design and structural biology study of novel nonpeptide inhibitors of severe acute respiratory syndrome coronavirus main protease

Design and synthesis of peptidomimetic severe acute respiratory syndrome chymotrypsin-like protease inhibitors

Inhibition of the severe acute respiratory syndrome 3CL protease by peptidomimetic alpha,betaunsaturated esters

Biflavonoids from Torreya nucifera displaying SARS-CoV 3CL(pro) inhibition

Specific plant terpenoids and lignoids possess potent antiviral activities against severe acute respiratory syndrome coronavirus

SARS-CoV 3CLpro inhibitory effects of quinone-methide triterpenes from Tripterygium regelii

Structural basis of mercury-and zinc-conjugated complexes as SARS-CoV 3C-like protease inhibitors

Evaluation of metal-conjugated compounds as inhibitors of 3CL protease of SARS-CoV

α-Ketoamides as broad-spectrum inhibitors of coronavirus and enterovirus replication: structurebased design, synthesis, and activity assessment

Discovery, synthesis, and structure-based optimization of a series of N-(tert-butyl)-2-(Narylamido)-2-(pyridin-3-yl) acetamides (ML188) as potent noncovalent small molecule inhibitors of the severe acute respiratory syndrome coronavirus (SARS-CoV) 3CL protease

Discovery of N-(benzo[1,2,3]triazol-1-yl)-N-(benzyl)acetamido)phenyl) carboxamides as severe acute respiratory syndrome coronavirus (SARS-CoV) 3CLpro inhibitors: identification of ML300 and noncovalent nanomolar inhibitors with an induced-fit binding

The papain-like protease of severe acute respiratory syndrome coronavirus has deubiquitinating activity

Structure-based design, synthesis, and biological evaluation of irreversible human rhinovirus 3C protease inhibitors. 4. Incorporation of P1 lactam moieties as L-glutamine replacements

Structure of the HRV-C 3C-rupintrivir complex provides new insights for inhibitor design

GC-376, and calpain inhibitors II, XII inhibit SARS-CoV-2 viral replication by targeting the viral main protease

Design, synthesis, and evaluation of 3C protease inhibitors as anti-enterovirus 71 agents

Learning from the past: possible urgent prevention and treatment options for severe acute respiratory infections caused by 2019-nCoV

Synthesis, modification and docking studies of 5-sulfonyl isatin derivatives as SARS-CoV 3C-like protease inhibitors

Synthesis and evaluation of isatin derivatives as effective SARS coronavirus 3CL protease inhibitors

Discovery of potent anilide inhibitors against the severe acute respiratory syndrome 3CL protease

Insight derived from molecular docking and molecular dynamics simulations into the binding interactions between HIV-1 protease inhibitors and SARS-CoV-2 3CLpro

Fused-ring structure of decahydroisoquinolin as a novel scaffold for SARS 3CL protease inhibitors

Design and synthesis of dipeptidyl glutaminyl fluoromethyl ketones as potent severe acute respiratory syndrome coronovirus (SARS-CoV) inhibitors

Innate immune evasion mediated by picornaviral 3C protease: Possible lessons for coronaviral 3C-like protease