Profiling of coronaviral Mpro and enteroviral 3Cpro specificity provides a framework for the development of broad-spectrum antiviral compounds.

The main protease from coronaviruses and the 3C protease from enteroviruses play a crucial role in processing viral polyproteins, making them attractive targets for the development of antiviral agents. In this study, we employed a combinatorial chemistry approach-HyCoSuL-to compare the substrate specificity profiles of the main and 3C proteases from alphacoronaviruses, betacoronaviruses, and enteroviruses. The obtained data demonstrate that coronavirus Mpros exhibit overlapping substrate specificity in all binding pockets, whereas the 3Cpro from enterovirus displays slightly different preferences toward natural and unnatural amino acids at the P4-P2 positions. However, chemical tools such as substrates, inhibitors, and activity-based probes developed for SARS-CoV-2 Mpro can be successfully applied to investigate the activity of the Mpro from other coronaviruses as well as the 3Cpro from enteroviruses. Our study provides a structural framework for the development of broad-spectrum antiviral compounds.

Structural and Biochemical Characterization of Porcine Epidemic Diarrhea Virus Papain-Like Protease 2.

Coronaviral papain-like proteases (PLpros) are essential enzymes that mediate not only the proteolytic processes of viral polyproteins during virus replication but also the deubiquitination and deISGylation of cellular proteins that attenuate host innate immune responses. Therefore, PLpros are attractive targets for antiviral drug development. Here, we report the crystal structure of papain-like protease 2 (PLP2) of porcine epidemic diarrhea virus (PEDV) in complex with ubiquitin (Ub). The X-ray structural analyses reveal that PEDV PLP2 interacts with the Ub substrate mainly through the Ub core region and C-terminal tail. Mutations of Ub-interacting residues resulted in a moderately or completely abolished deubiquitinylating function of PEDV PLP2. In addition, our analyses also indicate that 2-residue-extended blocking loop 2 at the S4 subsite contributes to the substrate selectivity and binding affinity of PEDV PLP2. Furthermore, the PEDV PLP2 Glu99 residue, conserved in alphacoronavirus PLpros, was found to govern the preference of a positively charged P4 residue of peptidyl substrates. Collectively, our data provided structure-based information for the substrate binding and selectivity of PEDV PLP2. These findings may help us gain insights into the deubiquitinating (DUB) and proteolytic functions of PEDV PLP2 from a structural perspective. IMPORTANCE Current challenges in coronaviruses (CoVs) include a comprehensive understanding of the mechanistic effects of associated enzymes, including the 3C-like and papain-like proteases. We have previously reported that the PEDV PLP2 exhibits a broader substrate preference, superior DUB function, and inferior peptidase activity. However, the structural basis for these functions remains largely unclear. Here, we show the high-resolution X-ray crystal structure of PEDV PLP2 in complex with Ub. Integrated structural and biochemical analyses revealed that (i) three Ub core-interacting residues are essential for DUB function, (ii) 2-residue-elongated blocking loop 2 regulates substrate selectivity, and (iii) a conserved glutamate residue governs the substrate specificity of PEDV PLP2. Collectively, our findings provide not only structural insights into the catalytic mechanism of PEDV PLP2 but also a model for developing antiviral strategies.

Enteroviruses and coronaviruses: similarities and therapeutic targets.

Introduction: Enteroviruses are common viruses causing a huge number of acute and chronic infections and producing towering economic costs. Similarly, coronaviruses cause seasonal mild infections, epidemics, and even pandemics and can lead to severe respiratory symptoms. It is important to develop broadly acting antiviral molecules to efficiently tackle the infections caused by thes.Areas covered: This review illuminates the differences and similarities between enteroviruses and coronaviruses and examines the most appealing therapeutic targets to combat both virus groups. Publications of both virus groups and deposited structures discovered through PubMed to March 2021 for viral proteases have been evaluated.Expert opinion: The main protease of coronaviruses and enteroviruses share similarities in their structure and function. These proteases process their viral polyproteins and thus drugs that bind to the active site have potential to target both virus groups. It is important to develop drugs that target more evolutionarily conserved processes and proteins. Moreover, it is a wise strategy to concentrate on processes that are similar between several virus families.

ADP-ribose and analogues bound to the deMARylating macrodomain from the bat coronavirus HKU4.

Macrodomains are proteins that recognize and hydrolyze ADP ribose (ADPR) modifications of intracellular proteins. Macrodomains are implicated in viral genome replication and interference with host cell immune responses. They are important to the infectious cycle of Coronaviridae and Togaviridae viruses. We describe crystal structures of the conserved macrodomain from the bat coronavirus (CoV) HKU4 in complex with ligands. The structures reveal a binding cavity that accommodates ADPR and analogs via local structural changes within the pocket. Using a radioactive assay, we present evidence of mono-ADPR (MAR) hydrolase activity. In silico analysis presents further evidence on recognition of the ADPR modification for hydrolysis. Mutational analysis of residues within the binding pocket resulted in diminished enzymatic activity and binding affinity. We conclude that the common structural features observed in the macrodomain in a bat CoV contribute to a conserved function that can be extended to other known macrodomains.

Targeting novel structural and functional features of coronavirus protease nsp5 (3CLpro, Mpro) in the age of COVID-19.

Coronavirus protease nsp5 (Mpro, 3CLpro) remains a primary target for coronavirus therapeutics due to its indispensable and conserved role in the proteolytic processing of the viral replicase polyproteins. In this review, we discuss the diversity of known coronaviruses, the role of nsp5 in coronavirus biology, and the structure and function of this protease across the diversity of known coronaviruses, and evaluate past and present efforts to develop inhibitors to the nsp5 protease with a particular emphasis on new and mostly unexplored potential targets of inhibition. With the recent emergence of pandemic SARS-CoV-2, this review provides novel and potentially innovative strategies and directions to develop effective therapeutics against the coronavirus protease nsp5.

An insight into the interaction between α-ketoamide- based inhibitor and coronavirus main protease: A detailed in silico study.

The search for therapeutic drugs that can neutralize the effects of COVID-2019 (SARS-CoV-2) infection is the main focus of current research. The coronavirus main protease (Mpro) is an attractive target for anti-coronavirus drug design. Further, α-ketoamide is proved to be very effective as a reversible covalent-inhibitor against cysteine proteases. Herein, we report on the non-covalent to the covalent adduct formation mechanism of α-ketoamide-based inhibitor with the enzyme active site amino acids by QM/SQM model (QM = quantum mechanical, SQM = semi-empirical QM). To uncover the mechanism, we focused on two approaches: a concerted and a stepwise fashion. The concerted pathway proceeds via deprotonation of the thiol of cysteine (here, Cys145 SγH) and simultaneous reversible nucleophilic attack of sulfur onto the α-ketoamide warhead. In this work, we propose three plausible concerted pathways. On the contrary, in a traditional two-stage pathway, the first step is proton transfer from Cys145 SγH to His41 Nδ forming an ion pair, and consecutively, in the second step, the thiolate ion attacks the α-keto group to form a thiohemiketal. In this reaction, we find that the stability of the tetrahedral intermediate oxyanion/hydroxyl group plays an important role. Moreover, as the α-keto group has two faces Si or Re for the nucleophilic attack, we considered both possibilities of attack leading to S- and R-thiohemiketal. We computed the structural, electronic, and energetic parameters of all stationary points including transition states via ONIOM and pure DFT method. Additionally, to characterize covalent, weak noncovalent interaction (NCI) and hydrogen-bonds, we applied NCI-reduced density gradient (NCI-RDG) methods along with Bader's Quantum Theory of Atoms-in-Molecules (QTAIM) and natural bonding orbital (NBO) analysis.

The SARS-CoV-2 Conserved Macrodomain Is a Mono-ADP-Ribosylhydrolase.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and other SARS-related CoVs encode 3 tandem macrodomains within nonstructural protein 3 (nsp3). The first macrodomain, Mac1, is conserved throughout CoVs and binds to and hydrolyzes mono-ADP-ribose (MAR) from target proteins. Mac1 likely counters host-mediated antiviral ADP-ribosylation, a posttranslational modification that is part of the host response to viral infections. Mac1 is essential for pathogenesis in multiple animal models of CoV infection, implicating it as a virulence factor and potential therapeutic target. Here, we report the crystal structure of SARS-CoV-2 Mac1 in complex with ADP-ribose. SARS-CoV-2, SARS-CoV, and Middle East respiratory syndrome coronavirus (MERS-CoV) Mac1 domains exhibit similar structural folds, and all 3 proteins bound to ADP-ribose with affinities in the low micromolar range. Importantly, using ADP-ribose-detecting binding reagents in both a gel-based assay and novel enzyme-linked immunosorbent assays (ELISAs), we demonstrated de-MARylating activity for all 3 CoV Mac1 proteins, with the SARS-CoV-2 Mac1 protein leading to a more rapid loss of substrate than the others. In addition, none of these enzymes could hydrolyze poly-ADP-ribose. We conclude that the SARS-CoV-2 and other CoV Mac1 proteins are MAR-hydrolases with similar functions, indicating that compounds targeting CoV Mac1 proteins may have broad anti-CoV activity.IMPORTANCE SARS-CoV-2 has recently emerged into the human population and has led to a worldwide pandemic of COVID-19 that has caused more than 1.2 million deaths worldwide. With no currently approved treatments, novel therapeutic strategies are desperately needed. All coronaviruses encode a highly conserved macrodomain (Mac1) that binds to and removes ADP-ribose adducts from proteins in a dynamic posttranslational process that is increasingly being recognized as an important factor that regulates viral infection. The macrodomain is essential for CoV pathogenesis and may be a novel therapeutic target. Thus, understanding its biochemistry and enzyme activity are critical first steps for these efforts. Here, we report the crystal structure of SARS-CoV-2 Mac1 in complex with ADP-ribose and describe its ADP-ribose binding and hydrolysis activities in direct comparison to those of SARS-CoV and MERS-CoV Mac1 proteins. These results are an important first step for the design and testing of potential therapies targeting this unique protein domain.

Viral Ubiquitin and Ubiquitin-Like Deconjugases-Swiss Army Knives for Infection.

Posttranslational modifications of cellular proteins by covalent conjugation of ubiquitin and ubiquitin-like polypeptides regulate numerous cellular processes that are captured by viruses to promote infection, replication, and spreading. The importance of these protein modifications for the viral life cycle is underscored by the discovery that many viruses encode deconjugases that reverse their functions. The structural and functional characterization of these viral enzymes and the identification of their viral and cellular substrates is providing valuable insights into the biology of viral infections and the host's antiviral defense. Given the growing body of evidence demonstrating their key contribution to pathogenesis, the viral deconjugases are now recognized as attractive targets for the design of novel antiviral therapeutics.

Deubiquitinating Enzymes in Coronaviruses and Possible Therapeutic Opportunities for COVID-19.

Following the outbreak of novel severe acute respiratory syndrome (SARS)-coronavirus (CoV)2, the majority of nations are struggling with countermeasures to fight infection, prevent spread and improve patient survival. Considering that the pandemic is a recent event, no large clinical trials have been possible and since coronavirus specific drug are not yet available, there is no strong consensus on how to treat the coronavirus disease 2019 (COVID-19) associated viral pneumonia. Coronaviruses code for an important multifunctional enzyme named papain-like protease (PLP), that has many roles in pathogenesis. First, PLP is one of the two viral cysteine proteases, along with 3-chymotripsin-like protease, that is responsible for the production of the replicase proteins required for viral replication. Second, its intrinsic deubiquitinating and deISGylating activities serve to antagonize the host's immune response that would otherwise hinder infection. Both deubiquitinating and deISGylating functions involve the removal of the small regulatory polypeptides, ubiquitin and ISG15, respectively, from target proteins. Ubiquitin modifications can regulate the innate immune response by affecting regulatory proteins, either by altering their stability via the ubiquitin proteasome pathway or by directly regulating their activity. ISG15 is a ubiquitin-like modifier with pleiotropic effects, typically expressed during the host cell immune response. PLP inhibitors have been evaluated during past coronavirus epidemics, and have showed promising results as an antiviral therapy in vitro. In this review, we recapitulate the roles of PLPs in coronavirus infections, report a list of PLP inhibitors and suggest possible therapeutic strategies for COVID-19 treatment, using both clinical and preclinical drugs.

Halting coronavirus polymerase.

The nucleotide analogue remdesivir is an investigational drug for the treatment of human coronavirus infection. Remdesivir is a phosphoramidate prodrug and is known to target viral RNA-dependent RNA polymerases. In this issue, Gordon et al. identify that remdesivir acts as a delayed RNA chain terminator for MERS-CoV polymerase complexes.

Structural and biochemical characterization of SADS-CoV papain-like protease 2.

Swine acute diarrhea syndrome coronavirus (SADS-CoV) is a novel coronavirus that is involved in severe diarrhea disease in piglets, causing considerable agricultural and economic loss in China. The emergence of this new coronavirus increases the importance of understanding SADS-CoV as well as antivirals. Coronaviral proteases, including main proteases and papain-like proteases (PLP), are attractive antiviral targets because of their essential roles in polyprotein processing and thus viral maturation. Here, we describe the biochemical and structural identification of recombinant SADS papain-like protease 2 (PLP2) domain of nsp3. The SADS-CoV PLP2 was shown to cleave nsp1 proteins and also peptides mimicking the nsp2|nsp3 cleavage site and also had deubiquitinating and deISGynating activity by in vitro assays. The crystal structure adopts an architecture resembling that of PLPs from other coronaviruses. We characterize both conserved and unique structural features likely directing the interaction of PLP2 with the substrates, including the tentative mapping of active site and other essential residues. These results provide a foundation for understanding the molecular basis of coronaviral PLPs' catalytic mechanism and for the screening and design of therapeutics to combat infection by SADS coronavirus.

The antiviral compound remdesivir potently inhibits RNA-dependent RNA polymerase from Middle East respiratory syndrome coronavirus.

Antiviral drugs for managing infections with human coronaviruses are not yet approved, posing a serious challenge to current global efforts aimed at containing the outbreak of severe acute respiratory syndrome-coronavirus 2 (CoV-2). Remdesivir (RDV) is an investigational compound with a broad spectrum of antiviral activities against RNA viruses, including severe acute respiratory syndrome-CoV and Middle East respiratory syndrome (MERS-CoV). RDV is a nucleotide analog inhibitor of RNA-dependent RNA polymerases (RdRps). Here, we co-expressed the MERS-CoV nonstructural proteins nsp5, nsp7, nsp8, and nsp12 (RdRp) in insect cells as a part a polyprotein to study the mechanism of inhibition of MERS-CoV RdRp by RDV. We initially demonstrated that nsp8 and nsp12 form an active complex. The triphosphate form of the inhibitor (RDV-TP) competes with its natural counterpart ATP. Of note, the selectivity value for RDV-TP obtained here with a steady-state approach suggests that it is more efficiently incorporated than ATP and two other nucleotide analogs. Once incorporated at position i, the inhibitor caused RNA synthesis arrest at position i + 3. Hence, the likely mechanism of action is delayed RNA chain termination. The additional three nucleotides may protect the inhibitor from excision by the viral 3'-5' exonuclease activity. Together, these results help to explain the high potency of RDV against RNA viruses in cell-based assays.

α-Ketoamides as Broad-Spectrum Inhibitors of Coronavirus and Enterovirus Replication: Structure-Based Design, Synthesis, and Activity Assessment.

The main protease of coronaviruses and the 3C protease of enteroviruses share a similar active-site architecture and a unique requirement for glutamine in the P1 position of the substrate. Because of their unique specificity and essential role in viral polyprotein processing, these proteases are suitable targets for the development of antiviral drugs. In order to obtain near-equipotent, broad-spectrum antivirals against alphacoronaviruses, betacoronaviruses, and enteroviruses, we pursued a structure-based design of peptidomimetic α-ketoamides as inhibitors of main and 3C proteases. Six crystal structures of protease-inhibitor complexes were determined as part of this study. Compounds synthesized were tested against the recombinant proteases as well as in viral replicons and virus-infected cell cultures; most of them were not cell-toxic. Optimization of the P2 substituent of the α-ketoamides proved crucial for achieving near-equipotency against the three virus genera. The best near-equipotent inhibitors, 11u (P2 = cyclopentylmethyl) and 11r (P2 = cyclohexylmethyl), display low-micromolar EC50 values against enteroviruses, alphacoronaviruses, and betacoronaviruses in cell cultures. In Huh7 cells, 11r exhibits three-digit picomolar activity against the Middle East Respiratory Syndrome coronavirus.

Identification of novel proteolytically inactive mutations in coronavirus 3C-like protease using a combined approach.

Coronaviruses (CoVs) infect humans and multiple other animal species, causing highly prevalent and severe diseases. 3C-like proteases (3CLpros) from CoVs (also called main proteases) are essential for viral replication and are also involved in polyprotein cleavage and immune regulation, making them attractive and effective targets for the development of antiviral drugs. Herein, the 3CLpro from the porcine epidemic diarrhea virus, an enteropathogenic CoV, was used as a model to identify novel crucial residues for enzyme activity. First, we established a rapid, sensitive, and efficient luciferase-based biosensor to monitor the activity of PDEV 3CLproin vivo. Using this luciferase biosensor, along with confirming the well-known catalytic residues (His41 and Cys144), we identified 4 novel proteolytically inactivated mutants of PDEV 3CLpro, which was also confirmed in mammalian cells by biochemical experiments. Our molecular dynamics (MD) simulations showed that the hydrogen bonding interactions occurring within and outside of the protease's active site and the dynamic fluctuations of the substrate, especially the van der Waals contacts, were drastically altered, a situation related to the loss of 3CLpro activity. These data suggest that changing the intermolecular dynamics in protein-substrate complexes eliminates the mechanism underlying the protease activity. The discovery of novel crucial residues for enzyme activity in the binding pocket could potentially provide more druggable sites for the design of protease inhibitors. In addition, our in-depth study of the dynamic substrate's envelope model using MD simulations is an approach that could augment the discovery of new inhibitors against 3CLpro in CoVs and other viral 3C proteases.-Zhou, J., Fang, L., Yang, Z., Xu, S., Lv, M., Sun, Z., Chen, J., Wang, D., Gao, J., Xiao, S. Identification of novel proteolytically inactive mutations in coronavirus 3C-like protease using a combined approach.

Protease inhibitors broadly effective against feline, ferret and mink coronaviruses.

Ferret and mink coronaviruses typically cause catarrhal diarrhea in ferrets and minks, respectively. In recent years, however, systemic fatal coronavirus infection has emerged in ferrets, which resembles feline infectious peritonitis (FIP) in cats. FIP is a highly fatal systemic disease caused by a virulent feline coronavirus infection in cats. Despite the importance of coronavirus infections in these animals, there are no effective commercial vaccines or antiviral drugs available for these infections. We have previously reported the efficacy of a protease inhibitor in cats with FIP, demonstrating that a virally encoded 3C-like protease (3CLpro) is a valid target for antiviral drug development for coronavirus infections. In this study, we extended our previous work on coronavirus inhibitors and investigated the structure-activity relationships of a focused library of protease inhibitors for ferret and mink 3CLpro. Using the fluorescence resonance energy transfer assay, we identified potent inhibitors broadly effective against feline, ferret and mink coronavirus 3CLpro. Multiple amino acid sequence analysis and modelling of 3CLpro of ferret and mink coronaviruses were conducted to probe the structural basis for these findings. The results of this study provide support for further research to develop broad-spectrum antiviral agents for multiple coronavirus infections. To the best of our knowledge, this is the first report on small molecule inhibitors of ferret and mink coronaviruses.

Coronavirus Susceptibility to the Antiviral Remdesivir (GS-5734) Is Mediated by the Viral Polymerase and the Proofreading Exoribonuclease.

Emerging coronaviruses (CoVs) cause severe disease in humans, but no approved therapeutics are available. The CoV nsp14 exoribonuclease (ExoN) has complicated development of antiviral nucleosides due to its proofreading activity. We recently reported that the nucleoside analogue GS-5734 (remdesivir) potently inhibits human and zoonotic CoVs in vitro and in a severe acute respiratory syndrome coronavirus (SARS-CoV) mouse model. However, studies with GS-5734 have not reported resistance associated with GS-5734, nor do we understand the action of GS-5734 in wild-type (WT) proofreading CoVs. Here, we show that GS-5734 inhibits murine hepatitis virus (MHV) with similar 50% effective concentration values (EC50) as SARS-CoV and Middle East respiratory syndrome coronavirus (MERS-CoV). Passage of WT MHV in the presence of the GS-5734 parent nucleoside selected two mutations in the nsp12 polymerase at residues conserved across all CoVs that conferred up to 5.6-fold resistance to GS-5734, as determined by EC50 The resistant viruses were unable to compete with WT in direct coinfection passage in the absence of GS-5734. Introduction of the MHV resistance mutations into SARS-CoV resulted in the same in vitro resistance phenotype and attenuated SARS-CoV pathogenesis in a mouse model. Finally, we demonstrate that an MHV mutant lacking ExoN proofreading was significantly more sensitive to GS-5734. Combined, the results indicate that GS-5734 interferes with the nsp12 polymerase even in the setting of intact ExoN proofreading activity and that resistance can be overcome with increased, nontoxic concentrations of GS-5734, further supporting the development of GS-5734 as a broad-spectrum therapeutic to protect against contemporary and emerging CoVs.IMPORTANCE Coronaviruses (CoVs) cause severe human infections, but there are no approved antivirals to treat these infections. Development of nucleoside-based therapeutics for CoV infections has been hampered by the presence of a proofreading exoribonuclease. Here, we expand the known efficacy of the nucleotide prodrug remdesivir (GS-5734) to include a group ß-2a CoV. Further, GS-5734 potently inhibits CoVs with intact proofreading. Following selection with the GS-5734 parent nucleoside, 2 amino acid substitutions in the nsp12 polymerase at residues that are identical across CoVs provide low-level resistance to GS-5734. The resistance mutations decrease viral fitness of MHV in vitro and attenuate pathogenesis in a SARS-CoV animal model of infection. Together, these studies define the target of GS-5734 activity and demonstrate that resistance is difficult to select, only partial, and impairs fitness and virulence of MHV and SARS-CoV, supporting further development of GS-5734 as a potential effective pan-CoV antiviral.

Evaluation of polyphenols from Broussonetia papyrifera as coronavirus protease inhibitors.

The current study was designed to assess the inhibitory activity of Broussonetia papyrifera-derived polyphenols against 3-chymotrypsin-like and papain-like coronavirus cysteine proteases. The isolated compounds were broussochalcone B (1), broussochalcone A (2), 4-hydroxyisolonchocarpin (3), papyriflavonol A (4), 3'-(3-methylbut-2-enyl)-3',4,7-trihydroxyflavane (5), kazinol A (6), kazinol B (7), broussoflavan A (8), kazinol F (9), and kazinol J (10). All polyphenols were more potent against papain-like protease (PLpro) than against 3-chymotripsin-like protease (3CLpro); therefore, we investigated their structural features that were responsible for this selectivity. Compound 4 was the most potent inhibitor of PLpro with an IC50 value of 3.7 µM. The active compounds displayed kinetic behaviors, and the binding constants of their interaction with PLpro were determined from surface plasmon resonance analysis. Our results suggest B. papyrifera constituents as promising candidates for development into potential anti-coronaviral agents.

Mutagenesis of S-Adenosyl-l-Methionine-Binding Residues in Coronavirus nsp14 N7-Methyltransferase Demonstrates Differing Requirements for Genome Translation and Resistance to Innate Immunity.

UNLABELLED: Eukaryotic mRNAs possess a methylated 5'-guanosine cap that is required for RNA stability, efficient translation, and protection from cell-intrinsic defenses. Many viruses use 5' caps or other mechanisms to mimic a cap structure to limit detection of viral RNAs by intracellular innate sensors and to direct efficient translation of viral proteins. The coronavirus (CoV) nonstructural protein 14 (nsp14) is a multifunctional protein with N7-methyltransferase (N7-MTase) activity. The highly conserved S-adenosyl-l-methionine (SAM)-binding residues of the DxG motif are required for nsp14 N7-MTase activity in vitro However, the requirement for CoV N7-MTase activity and the importance of the SAM-binding residues during viral replication have not been determined. Here, we engineered mutations in murine hepatitis virus (MHV) nsp14 N7-MTase at residues D330 and G332 and determined the effects of these mutations on viral replication, sensitivity to mutagen, inhibition by type I interferon (IFN), and translation efficiency. Virus encoding a G332A substitution in nsp14 displayed delayed replication kinetics and decreased peak titers relative to wild-type (WT) MHV. In addition, replication of nsp14 G332A virus was diminished following treatment of cells with IFN-ß, and nsp14 G332A genomes were translated less efficiently both in vitro and during viral infection. In contrast, substitution of alanine at MHV nsp14 D330 did not affect viral replication, sensitivity to mutagen, or inhibition by IFN-ß compared to WT MHV. Our results demonstrate that the conserved MHV N7-MTase SAM-binding-site residues are not required for MHV viability and suggest that the determinants of CoV N7-MTase activity differ in vitro and during virus infection. IMPORTANCE: Human coronaviruses, most notably severe acute respiratory syndrome (SARS)-CoV and Middle East respiratory syndrome (MERS)-CoV, cause severe and lethal human disease. Since specific antiviral therapies are not available for the treatment of human coronavirus infections, it is essential to understand the functions of conserved CoV proteins in viral replication. Here, we show that substitution of alanine at G332 in the N7-MTase domain of nsp14 impairs viral replication, enhances sensitivity to the innate immune response, and reduces viral RNA translation efficiency. Our data support the idea that coronavirus RNA capping could be targeted for development of antiviral therapeutics.

Coronavirus receptor switch explained from the stereochemistry of protein-carbohydrate interactions and a single mutation.

Hemagglutinin-esterases (HEs) are bimodular envelope proteins of orthomyxoviruses, toroviruses, and coronaviruses with a carbohydrate-binding "lectin" domain appended to a receptor-destroying sialate-O-acetylesterase ("esterase"). In concert, these domains facilitate dynamic virion attachment to cell-surface sialoglycans. Most HEs (type I) target 9-O-acetylated sialic acids (9-O-Ac-Sias), but one group of coronaviruses switched to using 4-O-Ac-Sias instead (type II). This specificity shift required quasisynchronous adaptations in the Sia-binding sites of both lectin and esterase domains. Previously, a partially disordered crystal structure of a type II HE revealed how the shift in lectin ligand specificity was achieved. How the switch in esterase substrate specificity was realized remained unresolved, however. Here, we present a complete structure of a type II HE with a receptor analog in the catalytic site and identify the mutations underlying the 9-O- to 4-O-Ac-Sia substrate switch. We show that (i) common principles pertaining to the stereochemistry of protein-carbohydrate interactions were at the core of the transition in lectin ligand and esterase substrate specificity; (ii) in consequence, the switch in O-Ac-Sia specificity could be readily accomplished via convergent intramolecular coevolution with only modest architectural changes in lectin and esterase domains; and (iii) a single, inconspicuous Ala-to-Ser substitution in the catalytic site was key to the emergence of the type II HEs. Our findings provide fundamental insights into how proteins "see" sugars and how this affects protein and virus evolution.

Recent progress in the discovery of inhibitors targeting coronavirus proteases.

Coronaviruses (CoVs) can cause highly prevalent diseases in humans and animals. The fatal outbreak of severe acute respiratory syndrome (SARS) and Middle East respiratory syndrome (MERS) highlights the threat posed by this unique virus subfamily. However, no specific drugs have been approved to treat CoV-associated diseases to date. The CoV proteases, which play pivotal roles in viral gene expression and replication through a highly complex cascade involving the proteolytic processing of replicase polyproteins, are attractive targets for drug design. This review summarizes the recent advances in biological and structural studies, together with the development of inhibitors targeting CoV proteases, particularly main proteases (M(pro)s), which could help develop effective treatments to prevent CoV infection.