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Viruses have evolved a range of strategies to utilize or manipulate the host’s cellular translational machinery for efficient infection, although the mechanisms by which infectious bronchitis virus (IBV) manipulates the host translation machinery remain unclear. In this study, we firstly demonstrate that IBV infection causes host shutoff, although viral protein synthesis is not affected. We then screened 23 viral proteins, and identified that more than one viral protein is responsible for IBV-induced host shutoff, the inhibitory effects of proteins Nsp15 were particularly pronounced. Ribosome profiling was used to draw the landscape of viral mRNA and cellular genes expression model, and the results showed that IBV mRNAs gradually dominated the cellular mRNA pool, the translation efficiency of the viral mRNAs was lower than the median efficiency (about 1) of cellular mRNAs. In the analysis of viral transcription and translation, higher densities of RNA sequencing (RNA-seq) and ribosome profiling (Ribo-seq) reads were observed for structural proteins and 5′ untranslated regions, which conformed to the typical transcriptional characteristics of nested viruses. Translational halt events and the number of host genes increased significantly after viral infection. The translationally paused genes were enriched in translation, unfolded-protein-related response, and activation of immune response pathways. Immune- and inflammation related mRNAs were inefficiently translated in infected cells, and IBV infection delayed the production of IFN-β and IFN-λ. Our results describe the translational landscape of IBV-infected cells and demonstrate new strategies by which IBV induces host gene shutoff to promote its replication. IMPORTANCE Infectious bronchitis virus (IBV) is a γ-coronavirus that causes huge economic losses to the poultry industry. Understanding how the virus manipulates cellular biological processes to facilitate its replication is critical for controlling viral infections. Here, we used Ribo-seq to determine how IBV infection remodels the host’s biological processes and identified multiple viral proteins involved in host gene shutoff. Immune- and inflammation-related mRNAs were inefficiently translated, the translation halt of unfolded proteins and immune activation-related genes increased significantly, benefitting IBV replication. These data provide new insights into how IBV modulates its host’s antiviral responses. [ABSTRACT FROM AUTHOR]

NendoU from SARS-CoV-2 is responsible for the virus's ability to evade the innate immune system by cleaving the polyuridine leader sequence of antisense viral RNA. Here we report the room-temperature structure of NendoU, solved by serial femtosecond crystallography at an X-ray free-electron laser to 2.6 Å resolution. The room-temperature structure provides insight into the flexibility, dynamics, and other intrinsic properties of NendoU, with indications that the enzyme functions as an allosteric switch. Functional studies examining cleavage specificity in solution and in crystals support the uridine-purine cleavage preference, and we demonstrate that enzyme activity is fully maintained in crystal form. Optimizing the purification of NendoU and identifying suitable crystallization conditions set the benchmark for future time-resolved serial femtosecond crystallography studies. This could advance the design of antivirals with higher efficacy in treating coronaviral infections, since drugs that block allosteric conformational changes are less prone to drug resistance.

Severe acute respiratory syndrome coronavirus (SARS-CoV)-2 has caused millions of deaths since its emergence in 2019. Innate immune antagonism by lethal CoVs such as SARS-CoV-2 is crucial for optimal replication and pathogenesis. The conserved nonstructural protein 15 (nsp15) endoribonuclease (EndoU) limits activation of double-stranded (ds)RNA-induced pathways, including interferon (IFN) signaling, protein kinase R (PKR), and oligoadenylate synthetase/ribonuclease L (OAS/RNase L) during diverse CoV infections including murine coronavirus and Middle East respiratory syndrome (MERS)-CoV. To determine how nsp15 functions during SARS-CoV-2 infection, we constructed a recombinant SARS-CoV-2 (nsp15mut) expressing catalytically inactivated nsp15, which we show promoted increased dsRNA accumulation. Infection with SARS-CoV-2 nsp15mut led to increased activation of the IFN signaling and PKR pathways in lung-derived epithelial cell lines and primary nasal epithelial air-liquid interface (ALI) cultures as well as significant attenuation of replication in ALI cultures compared to wild-type virus. This replication defect was rescued when IFN signaling was inhibited with the Janus activated kinase (JAK) inhibitor ruxolitinib. Finally, to assess nsp15 function in the context of minimal (MERS-CoV) or moderate (SARS-CoV-2) innate immune induction, we compared infections with SARS-CoV-2 nsp15mut and previously described MERS-CoV nsp15 mutants. Inactivation of nsp15 had a more dramatic impact on MERS-CoV replication than SARS-CoV-2 in both Calu3 cells and nasal ALI cultures suggesting that SARS-CoV-2 can better tolerate innate immune responses. Taken together, SARS-CoV-2 nsp15 is a potent inhibitor of dsRNA-induced innate immune response and its antagonism of IFN signaling is necessary for optimal viral replication in primary nasal ALI cultures. [ABSTRACT FROM AUTHOR]

The genus Laurencia, a category of marine red algae, is well recognized for producing a large variety of natural products (NPs) that are both chemically intriguing and structurally distinct. The aim of this research was to identify NPs with potential anti-SARS-CoV-2 activity. The crystals of the proteins RdRp and nsp15 were obtained from the RCSB protein database. About 300 NPs were discovered using the PubChem, ChemSpider, and CMNPD databases. The program Autodock Vina was used to conduct the molecular docking procedure once the proteins and ligands were prepared. Before running MD simulations using the CABS-flex 2.0 website, binding affinity assessments and interactions between amino acids were carefully reviewed. Only nine NPs were shortlisted to be examined further. Bromophycolide R, S, and bromophycoic acid C show the tendency to inhibit RdRp by β-hairpin motif binding at the N-terminal known as Active site 2 (AS2), whereas the other four NPs, bromophycolide E, H, P, and thyrsenol A, may effectively inhibit RdRp through interactions via C-terminal, also known as the Active site 1 (AS1). For the enzyme nsp15, bromophycoic B, C, and floridoside showed plausible interactions. In conclusion, out of nine, seven candidates shortlisted for RdRp exhibited strong interactions with the key residues in the AS1 and AS2 regions. Bromophycoic acid C may work as a dual inhibitor due to its favorable interactions with the nsp15 protein and RdRp’s N-terminal, with affinities of −8.5 and −8.2 kcal/mol, respectively.

SARS–CoV–2 is accountable for severe social and economic disruption around the world causing COVID–19. Non–structural protein–15 (NSP15) possesses a domain that is vital to the viral life cycle and is known as uridylate–specific endoribonuclease (EndoU). This domain binds to the uridine 5′–monophosphate (U5P) so that the protein may carry out its native activity. It is considered a vital drug target to inhibit the growth of the virus. Thus, in this current study, ML–based QSAR and virtual screening of U5P analogues targeting Nsp15 were performed to identify potential molecules against SARS–CoV–2. Screening of 816 unique U5P analogues using ML–based QSAR identified 397 compounds ranked on their predicted bioactivity (pIC50). Further, molecular docking and hydrogen bond interaction analysis resulted in the selection of the top three compounds (53309102, 57398422, and 76314921). Molecular dynamics simulation of the most promising compounds showed that two molecules 53309102 and 57398422 acted as potential binders of Nsp15. The compound was able to inhibit nsp15 activity as it was successfully bound to the active site of the nsp15 protein. This was achieved by the formation of relevant contacts with enzymatically critical amino acid residues (His235, His250, and Lys290). Principal component analysis and free energy landscape studies showed stable complex formation while MM/GBSA calculation showed lower binding energies for 53309102 (ΔGTOTAL = –29.4 kcal/mol) and 57398422 (ΔGTOTAL = –39.4 kcal/mol) compared to the control U5P (ΔGTOTAL = –18.8 kcal/mol). This study aimed to identify analogues of U5P inhibiting the NSP15 function that potentially could be used for treating COVID–19.

ABSTRACT Endoribonuclease non-structural protein 15 (nsp15) (EndoU) is conserved among coronaviruses (CoVs) and is crucial for viral replication, evasion of the innate immune system, and virulence. EndoU-deficient CoVs can activate the interferon (IFN) response and attenuate their virulence, and nsp15 is considered the target of attenuated vaccine development. Among alpha-CoVs, transmissible gastroenteritis virus (TGEV) and feline infectious peritonitis virus (FIPV) cause lethal diseases in piglets and cats, but the role of EndoU in viral propagation and virulence remains unclear. Here, we verified the TGEV and FIPV EndoU active sites His226 and His241 and found that the antagonization of SeV-induced IFN-β production by nsp15 depends on its EndoU activity. Furthermore, we constructed infectious clones of wild-type (WT) and EndoU-deficient (EnUmt) TGEV and FIPV. Unexpectedly, we found that both the WT and EnUmt viruses propagated efficiently in multiple types of immunocompetent (PK-15, IPI-2I, ST, CRFK, F81, and Fcwf-4) cells. Moreover, the results of infection experiments showed that compared with piglets and cats infected with the WT, the EnUmt virus-infected piglets and cats did not exhibit significantly reduced mortality, tissue injury, or viral shedding. Specially, the death of cats infected with EnUmt-FIPV occurred earlier than that of cats infected with WT. Hence, our results suggest that the function of EndoU is conserved, but nsp15-mediated regulation of the propagation and pathogenesis of CoVs are diverse. Our findings provide a reference for an in-depth understanding of EndoU-mediated immune escape and pathogenicity in CoVs. IMPORTANCE Understanding the role of the endoribonuclease non-structural protein 15 (nsp15) (EndoU) in coronavirus (CoV) infection and pathogenesis is essential for vaccine target discovery. Whether the EndoU activity of CoV nsp15, as a virulence-related protein, has a diverse effect on viral virulence needs to be further explored. Here, we found that the transmissible gastroenteritis virus (TGEV) and feline infectious peritonitis virus (FIPV) nsp15 proteins antagonize SeV-induced interferon-β (IFN-β) production in human embryonic kidney 293 cells. Interestingly, compared with wild-type infection, infection with EnUmt-TGEV or EnUmt-FIPV did not change the IFN-β response or reduce viral propagation in immunocompetent cells. The results of animal experiments showed that EnUmt viruses did not reduce the clinical presentation and mortality caused by TGEV and FIPV. Our findings enrich the understanding of nsp15-mediated regulation of alpha-CoV propagation and virulence and reveal that the conserved functions of nonstructural proteins have diverse effects on the pathogenicity of CoVs.

The emergence of SARS-CoV-1 in 2003 followed by MERS-CoV and now SARS-CoV-2 has proven the latent threat these viruses pose to humanity. While the SARS-CoV-2 pandemic has shifted to a stage of endemicity, the threat of new coronaviruses emerging from animal reservoirs remains. To address this issue, the global community must develop small molecule drugs targeting highly conserved structures in the coronavirus proteome. Here, we characterized existing drugs for their ability to inhibit the endoribonuclease activity of the SARS-CoV-2 non-structural protein 15 (nsp15) via in silico, in vitro, and in vivo techniques. We have identified nsp15 inhibition by the drugs pibrentasvir and atovaquone which effectively inhibit SARS-CoV-2 and HCoV-OC43 at low micromolar concentrations in cell cultures. Furthermore, atovaquone, but not pibrentasvir, is observed to modulate HCoV-OC43 dsRNA and infection in a manner consistent with nsp15 inhibition. Although neither pibrentasvir nor atovaquone translate to clinical efficacy in a murine prophylaxis model of SARS-CoV-2 infection, atovaquone may serve as a basis for the design of future nsp15 inhibitors. [ABSTRACT FROM AUTHOR]

The genus Laurencia, a category of marine red algae, is well recognized for producing a large variety of natural products (NPs) that are both chemically intriguing and structurally distinct. The aim of this research was to identify NPs with potential anti-SARS-CoV-2 activity. The crystals of the proteins RdRp and nsp15 were obtained from the RCSB protein database. About 300 NPs were discovered using the PubChem, ChemSpider, and CMNPD databases. The program Autodock Vina was used to conduct the molecular docking procedure once the proteins and ligands were prepared. Before running MD simulations using the CABS-flex 2.0 website, binding affinity assessments and interactions between amino acids were carefully reviewed. Only nine NPs were shortlisted to be examined further. Bromophycolide R, S, and bromophycoic acid C show the tendency to inhibit RdRp by β-hairpin motif binding at the N-terminal known as Active site 2 (AS2), whereas the other four NPs, bromophycolide E, H, P, and thyrsenol A, may effectively inhibit RdRp through interactions via C-terminal, also known as the Active site 1 (AS1). For the enzyme nsp15, bromophycoic B, C, and floridoside showed plausible interactions. In conclusion, out of nine, seven candidates shortlisted for RdRp exhibited strong interactions with the key residues in the AS1 and AS2 regions. Bromophycoic acid C may work as a dual inhibitor due to its favorable interactions with the nsp15 protein and RdRp's N-terminal, with affinities of −8.5 and −8.2 kcal/mol, respectively. [ABSTRACT FROM AUTHOR]

Severe Acute Respiratory Syndrome coronavirus 2 (SARS-CoV-2) is rapidly spreading around the world. There is no existing vaccine or proven drug to prevent infections and stop virus proliferation. Although this virus is similar to human and animal SARS-CoVs and Middle East Respiratory Syndrome coronavirus (MERS-CoVs), the detailed information about SARS-CoV-2 proteins structures and functions is urgently needed to rapidly develop effective vaccines, antibodies, and antivirals. We applied high-throughput protein production and structure determination pipeline at the Center for Structural Genomics of Infectious Diseases to produce SARS-CoV-2 proteins and structures. Here we report two high-resolution crystal structures of endoribonuclease Nsp15/NendoU. We compare these structures with previously reported homologs from SARS and MERS coronaviruses.

Two active coronaviral proteins (3CLpro and Nsp15) have been studied using both the GCMS and docking methods. These coronaviral proteins have been examined with the methanol extract generated from leaves of the Arum palaestinum. According to the GC-MS findings, 19 major natural compounds are present in the plant's methanolic extract. The lowest Binding Energy (LBE) and the inhibition constant (Ki) have been used to identify and classify the potential of these lead drugs with their pharmacological properties. The affinity of these compounds with coronaviral proteins has been evaluated to reveal the usage of these compounds at the active sites of the receptors, 3CLpro (PDB ID: 6LU7) and Nsp15 (PDB ID: 6VWW). The results of β-Sitosterol, Androstan-3-one, Phenobarbital, Maltose, and α-Tocopherol show more affinity to Nsp15 and 3CLpro than to the supporting control drugs. Furthermore, an evaluation of the interactions of these components with the amino acids of 3CLpro and Nsp15 revealed that β-Sitosterol has the best LBE score and Ki value as compared with those of the approved medication and all other compounds under investigation. Consequently, these potential compounds may be modern inhibitors of coronavirus. Further in vitro and in vivo studies are needed for such computational findings. [ABSTRACT FROM AUTHOR]

Researchers worldwide are looking for molecules that might disrupt the COVID-19 life cycle. Endoribonuclease, which is responsible for processing viral RNA to avoid detection by the host defense system, and helicase, which is responsible for unwinding the RNA helices for replication, are two key non-structural proteins. This study performs a hierarchical structure-based virtual screening approach for NSP15 and helicase to reach compounds with high binding probabilities. In this investigation, we incorporated a variety of filtering strategies for predicting compound interactions. First, we evaluated 756,275 chemicals from four databases using a deep learning method (NCI, Drug Bank, Maybridge, and COCONUT). Following that, two docking techniques (extra precision and induced fit) were utilized to evaluate the compounds' binding affinity, followed by molecular dynamic simulation supported by the MM-GBSA free binding energy calculation. Remarkably, two compounds (90616 and CNP0111740) exhibited high binding affinity values of -66.03 and -12.34 kcal/mol for helicase and NSP15, respectively. The VERO-E6 cell line was employed to test their in vitro therapeutic impact. The CC 50 for CNP0111740 and 90616 were determined to be 102.767 μg/ml and 379.526 μg/ml, while the IC 50 values were 140.176 μg/ml and 5.147 μg/ml, respectively. As a result, the selectivity index for CNP0111740 and 90616 is 0.73 and 73.73, respectively. Finally, these compounds were found to be novel, effective inhibitors for the virus; however, further in vivo validation is needed.Communicated by Ramaswamy H. Sarma.

Coronavirus (CoV) Nsp15 is a viral endoribonuclease (EndoU) with a preference for uridine residues. CoV Nsp15 is an innate immune antagonist which prevents dsRNA sensor recognition and stress granule formation by targeting viral and host RNAs. SARS-CoV-2 restricts and delays the host antiviral innate immune responses through multiple viral proteins, but the role of SARS-CoV-2 Nsp15 in innate immune evasion is not completely understood. Here, we generate an EndoU activity knockout rSARS-CoV-2 Nsp15-H234A to elucidate the biological functions of Nsp15. Relative to wild-type rSARS-CoV-2, replication of rSARS-CoV-2 Nsp15-H234A was significantly decreased in IFN-responsive A549-ACE2 cells but not in its STAT1 knockout counterpart. Transcriptomic analysis revealed upregulation of innate immune response genes in cells infected with rSARS-CoV-2 Nsp15-H234A relative to wild-type virus, including cGAS-STING, cytosolic DNA sensors activated by both DNA and RNA viruses. Treatment with STING inhibitors H-151 and SN-011 rescued the attenuated phenotype of rSARS-CoV-2 Nsp15-H234A . SARS-CoV-2 Nsp15 inhibited cGAS-STING-mediated IFN-β promoter and NF-κB reporter activity, as well as facilitated the replication of EV-D68 and NDV by diminishing cGAS and STING expression and downstream innate immune responses. Notably, the decline in cGAS and STING was also apparent during SARS-CoV-2 infection. The EndoU activity was essential for SARS-CoV-2 Nsp15-mediated cGAS and STING downregulation, but not all HCoV Nsp15 share the consistent substrate selectivity. In the hamster model, rSARS-CoV-2 Nsp15-H234A replicated to lower titers in the nasal turbinates and lungs and induced higher innate immune responses. Collectively, our findings exhibit that SARS-CoV-2 Nsp15 serves as a host innate immune antagonist by targeting host cGAS and STING., Competing Interests: Competing Interest Statement: S.J. is a co-founder of Elucidate Bio Inc, has received speaking honorariums from Cell Signaling Technology, and has received research support from Roche unrelated to this work.

The emergence of SARS-CoV-1 in 2003 followed by MERS-CoV and now SARS-CoV-2 has proven the latent threat these viruses pose to humanity. While the SARS-CoV-2 pandemic has shifted to a stage of endemicity, the threat of new coronaviruses emerging from animal reservoirs remains. To address this issue, the global community must develop small molecule drugs targeting highly conserved structures in the coronavirus proteome. Here, we characterized existing drugs for their ability to inhibit the endoribonuclease activity of the SARS-CoV-2 non-structural protein 15 (nsp15) via in silico, in vitro, and in vivo techniques. We have identified nsp15 inhibition by the drugs pibrentasvir and atovaquone which effectively inhibit SARS-CoV-2 and HCoV-OC43 at low micromolar concentrations in cell cultures. Furthermore, atovaquone, but not pibrentasvir, is observed to modulate HCoV-OC43 dsRNA and infection in a manner consistent with nsp15 inhibition. Although neither pibrentasvir nor atovaquone translate to clinical efficacy in a murine prophylaxis model of SARS-CoV-2 infection, atovaquone may serve as a basis for the design of future nsp15 inhibitors.

The current research has centered on the use of pharmacological and binding affinity methods to test the 36 compounds as bioactive constituents' inhibitors for COVID-19. Six compounds out of 36 phytoconstituents (rutin, quercetin, catechin gallate, rhamnetin, campesterol and stigmasterol) have demonstrated outstanding molecular docking and drug-like properties as HIV inhibitors Lopinavir and Indinavir. Interestingly, the lowest binding energies (LBE) and the inhibition constant (Ki) have showed that these compounds are able to bind to the P-glycoprotein substrate of 3CLpro and Nsp15. Interestingly, rutin has been found to be an excellent potential inhibitor for COVID-19 proteins because it has the best LBE score and Ki value than those of other compounds, and of its ability to form strong H-bonds with COVID-19 proteins. The compounds that come next to the rutin compound are stigmasterol and campesterol. As a result, these compounds are considered possible novel inhibitors of COVID-19. In order to validate the computational results, more in vitro and in vivo investigations are required to support the findings of this research. [ABSTRACT FROM AUTHOR]

The gut microbiome profile of COVID-19 patients was found to correlate with a viral load of SARS-CoV-2, COVID-19 severity, and dysfunctional immune responses, suggesting that gut microbiota may be involved in anti-infection. In order to investigate the role of gut microbiota in anti-infection against SARS-CoV-2, we established a high-throughput in vitro screening system for COVID-19 therapeutics by targeting the endoribonuclease (Nsp15). We also evaluated the activity inhibition of the target by substances of intestinal origin, using a mouse model in an attempt to explore the interactions between gut microbiota and SARS-CoV-2. The results unexpectedly revealed that antibiotic treatment induced the appearance of substances with Nsp15 activity inhibition in the intestine of mice. Comprehensive analysis based on functional profiling of the fecal metagenomes and endoribonuclease assay of antibiotic-enriched bacteria and metabolites demonstrated that the Nsp15 inhibitors were the primary bile acids that accumulated in the gut as a result of antibiotic-induced deficiency of bile acid metabolizing microbes. This study provides a new perspective on the development of COVID-19 therapeutics using primary bile acids.

The SARS-CoV-2 pandemic has bolstered unprecedented research efforts to better understand the pathogenesis of coronavirus (CoV) infections and develop effective therapeutics. We here focus on non-structural protein nsp15, a hexameric component of the viral replication-transcription complex (RTC). Nsp15 possesses uridine-specific endoribonuclease (EndoU) activity for which some specific cleavage sites were recently identified in viral RNA. By preventing accumulation of viral dsRNA, EndoU helps the virus to evade RNA sensors of the innate immune response. The immune-evading property of nsp15 was firmly established in several CoV animal models and makes it a pertinent target for antiviral therapy. The search for nsp15 inhibitors typically proceeds via compound screenings and is aided by the rapidly evolving insight in the protein structure of nsp15. In this overview, we broadly cover this fascinating protein, starting with its structure, biochemical properties and functions in CoV immune evasion. Next, we summarize the reported studies in which compound screening or a more rational method was used to identify suitable leads for nsp15 inhibitor development. In this way, we hope to raise awareness on the relevance and druggability of this unique CoV protein. • Coronavirus nsp15 is a conserved hexameric protein that is part of the RTC. • Its EndoU function contributes to viral evasion from innate immunity and represents a pertinent drug target. • Insight in the structure of nsp15 is rapidly evolving and creates an opportunity for rational design of nsp15 inhibitors. • Besides the EndoU catalytic core, such inhibitors may target a protein-protein or protein-RNA interface in hexameric nsp15. • Nsp15 inhibitors can be valuable tools to establish the functions of nsp15 in coronavirus replication and pathogenesis. [ABSTRACT FROM AUTHOR]

SARS‐CoV‐2 wreaks havoc around the world, triggering the COVID‐19 pandemic. It has been confirmed that the endoribonuclease NSP15 is crucial to the viral replication, and thus identified as a potential drug target against COVID‐19. The NSP15 protein was used as the target to conduct high‐throughput virtual screening on 30,926 natural products from the NPASS database to identify potential NSP15 inhibitors. And 100 ns molecular dynamics simulations were performed on the NSP15 and NSP15‐NPC198199 system. In all, 10 natural products with high docking scores with NSP15 protein were obtained, among which compound NPC198199 scored the highest. The analysis of the binding mode between NPC198199 and NSP15 found that NPC198199 would form H‐bond interactions with multiple key residues at the catalytic site. Subsequently, a series of post‐dynamics simulation analyses (including RMSD, RMSF, PCA, DCCM, RIN, binding free energy, and H‐bond occupancy) were performed to further explore inhibitory mechanism of compound NPC198199 on NSP15 protein at the molecular level. The research strongly indicates that the 10 natural compounds screened can be used as potential inhibitors of NSP15, and provides valuable information for the subsequent drug discovery of anti‐SARS‐CoV‐2. Practical applications: Natural products play an important role in the treatment of many difficult diseases. In this study, high‐throughput virtual screening technology was used to screen the natural product database to obtain potential inhibitors against endoribonuclease NSP15. The binding mechanism between natural products and NSP15 was investigated at the molecular level by molecular dynamics technology so that it is expected to become candidate drugs for the treatment of SARS‐CoV‐2. We hope that our research can provide new clue to combat COVID‐19 and overcome the epidemic situation as soon as possible. [ABSTRACT FROM AUTHOR]

The SARS-CoV-2's endoribonuclease (NendoU) nsp15, is an Mn2+ dependent endoribonuclease specific to uridylate that SARS-CoV-2 uses to avoid the innate immune response by managing the stray RNA generated during replication. As of the writing of this review 20 structures of SARS-CoV-2 nsp15 have been deposited into the PDB, largely solved using X-ray crystallography and some through Cryo-EM. These structures show that an nsp15 monomer consist of three conserved domains, the N-terminal oligomerization domain, the middle domain, and the catalytic NendoU domain. Enzymatically active nsp15 forms a hexamer through a dimer of trimers (point group 32), whose assembly is facilitated by the oligomerization domain. This review summarises the structural and functional information gained from SARs-CoV-2, SARs-CoV and MERS-CoV nsp15 structures, compiles the current structure-based drug design efforts, and complementary knowledge with a view to provide a clear starting point for downstream structure users interested in studying nsp15 as a novel drug target to treat COVID-19. [ABSTRACT FROM AUTHOR]

Viruses have evolved a range of strategies to utilize or manipulate the host's cellular translational machinery for efficient infection, although the mechanisms by which infectious bronchitis virus (IBV) manipulates the host translation machinery remain unclear. In this study, we firstly demonstrate that IBV infection causes host shutoff, although viral protein synthesis is not affected. We then screened 23 viral proteins, and identified that more than one viral protein is responsible for IBV-induced host shutoff, the inhibitory effects of proteins Nsp15 were particularly pronounced. Ribosome profiling was used to draw the landscape of viral mRNA and cellular genes expression model, and the results showed that IBV mRNAs gradually dominated the cellular mRNA pool, the translation efficiency of the viral mRNAs was lower than the median efficiency (about 1) of cellular mRNAs. In the analysis of viral transcription and translation, higher densities of RNA sequencing (RNA-seq) and ribosome profiling (Ribo-seq) reads were observed for structural proteins and 5' untranslated regions, which conformed to the typical transcriptional characteristics of nested viruses. Translational halt events and the number of host genes increased significantly after viral infection. The translationally paused genes were enriched in translation, unfolded-protein-related response, and activation of immune response pathways. Immune- and inflammation-related mRNAs were inefficiently translated in infected cells, and IBV infection delayed the production of IFN-β and IFN-λ. Our results describe the translational landscape of IBV-infected cells and demonstrate new strategies by which IBV induces host gene shutoff to promote its replication., Importance: Infectious bronchitis virus (IBV) is a γ-coronavirus that causes huge economic losses to the poultry industry. Understanding how the virus manipulates cellular biological processes to facilitate its replication is critical for controlling viral infections. Here, we used Ribo-seq to determine how IBV infection remodels the host's biological processes and identified multiple viral proteins involved in host gene shutoff. Immune- and inflammation-related mRNAs were inefficiently translated, the translation halt of unfolded proteins and immune activation-related genes increased significantly, benefitting IBV replication. These data provide new insights into how IBV modulates its host's antiviral responses., Competing Interests: The authors declare no conflict of interest.

In this study, molecular modelling study of previously synthesized compounds against SARS-CoV-2 target enzyme was performed. A subset of 156 compounds from an in-house database has been subjected to molecular modelling studies against the SARS-CoV-2 ADP-ribose phosphatase (ADRP, NSP3), Papain-like protease (PLpro), and uridine specific endoribonuclease (NSP15) enzymes. We have identified one compound that is expected to inhibit the SARS-CoV-2 ADRP enzyme and one compound that is expected to inhibit the NSP15 enzyme. [ABSTRACT FROM AUTHOR]

Two active coronaviral proteins (3CLpro and Nsp15) have been studied using both the GCMS and docking methods. These coronaviral proteins have been examined with the methanol extract generated from leaves of the Arum palaestinum. According to the GC-MS findings, 19 major natural compounds are present in the plant's methanolic extract. The lowest Binding Energy (LBE) and the inhibition constant (Ki) have been used to identify and classify the potential of these lead drugs with their pharmacological properties. The affinity of these compounds with coronaviral proteins has been evaluated to reveal the usage of these compounds at the active sites of the receptors, 3CLpro (PDB ID: 6LU7) and Nsp15 (PDB ID: 6VWW). The results of β-Sitosterol, Androstan-3-one, Phenobarbital, Maltose, and α-Tocopherol show more affinity to Nsp15 and 3CLpro than to the supporting control drugs. Furthermore, an evaluation of the interactions of these components with the amino acids of 3CLpro and Nsp15 revealed that β-Sitosterol has the best LBE score and Ki value as compared with those of the approved medication and all other compounds under investigation. Consequently, these potential compounds may be modern inhibitors of coronavirus. Further in vitro and in vivo studies are needed for such computational findings. [ABSTRACT FROM AUTHOR]

The newly emerged human coronavirus, SARS-CoV-2, had begun to spread last year and sparked worldwide. In this study, molecular docking is utilized to test some previously approved drugs against the SARS-CoV-2 nonstructural protein 15 (Nsp15). We screened 23 drugs, from which three (saquinavir, valrubicin and aprepitant) show a paramount predicted binding affinity (-9.1, -9.6 and -9.2 kcal/mol, respectively) against SARS-CoV-2 Nsp15. Moreover, saquinavir and aprepitant make nonbonded interactions with Leu201 in the active site cavity of Nsp15, while the drug valrubicin interacts with Arg199 and Leu201. This binding pattern may be effective against the targeted protein, leading to Nsp15 blockage and virus abolition. Additionally, the pharmacological properties of the screened drugs are known since they have been approved against different viruses. [ABSTRACT FROM AUTHOR]

Coronavirus infectious disease 2019 (COVID-19), a viral infection caused by a novel coronavirus (nCoV), continues to emerge as a serious threat to public health. This pandemic caused by SARS-CoV-2 (severe acute respiratory syndrome–coronavirus-2) has infected globally with 1,550,000 plus deaths to date, representing a high risk to public health. No effective drug or vaccine is available to curb down this deadly virus. The expedition for searching for a potential drug or vaccine against COVID-19 is of massive potential and favour to the community. This study is focused on finding an effective natural compound that can be processed further into a potential inhibitor to check the activity of SARS-CoV-2 with minimal side effects targeting NSP15 protein, which belongs to the EndoU enzyme family. The natural screening suggested two efficient compounds (PubChem ID: 95372568 and 1776037) with dihydroxyphenyl region of the compound, found to be important in the interaction with the viral protein showing promising activity which may act as a potent lead inhibitory molecule against the virus. In combination with virtual screening, modelling, drug likeliness, molecular docking, and 500 ns cumulative molecular dynamics simulations (100 ns for each complex) along with the decomposition analysis to calculate and confirm the stability and fold, we propose 95372568 and 1776037 as novel compounds of natural origin capable of getting developed into potent lead molecules against SARS-CoV-2 target protein NSP15. [ABSTRACT FROM AUTHOR]

SARS‐CoV and SARS‐CoV‐2 belong to the subfamily Coronaviridae and infect humans, they are constituted by four structural proteins: Spike glycoprotein (S), membrane (M), envelope (E) and nucleocapsid (N), and nonstructural proteins, such as Nsp15 protein which is exclusively present on nidoviruses and is absent in other RNA viruses, making it an ideal target in the field of drug design. A virtual screening strategy to search for potential drugs was proposed, using molecular docking to explore a library of approved drugs available in the DrugBank database in order to identify possible NSP15 inhibitors to treat Covid19 disease. We found from the docking analysis that the antiviral drugs: Paritaprevir and Elbasvir, currently both approved for hepatitis C treatment which showed some of the lowest free binding energy values were considered as repositioning drugs to combat SARS‐CoV‐2. Furthermore, molecular dynamics simulations of the Apo and Holo‐Nsp15 systems were performed in order to get insights about the stability of these protein‐ligand complexes. [ABSTRACT FROM AUTHOR]

BackgroundCoronavirus disease 2019 (COVID-19) is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Although a preliminary understanding of the replication and transcription of SARS-CoV-2 has recently emerged, their regulation remains unknown.ResultsBy comprehensive analysis of genome sequence and protein structure data, we propose a negative feedback model to explain the regulation of CoV replication and transcription, providing a molecular basis of the “leader-to-body fusion” model. The key step leading to the proposal of our model was that the transcription regulatory sequence (TRS) motifs were identified as the cleavage sites of nsp15, a nidoviral RNA uridylate-specific endoribonuclease (NendoU). According to this model, nsp15 regulates the synthesis of subgenomic RNAs (sgRNAs), and genomic RNAs (gRNAs) by cleaving TRSs. The expression level of nsp15 controls the relative proportions of sgRNAs and gRNAs, which in turn change the expression level of nsp15 to reach equilibrium between the CoV replication and transcription.ConclusionThe replication and transcription of CoVs are regulated by a negative feedback mechanism that influences the persistence of CoVs in hosts. Our findings enrich fundamental knowledge in the field of gene expression and its regulation, and provide new clues for future studies. One important clue is that nsp15 may be an important and ideal target for the development of drugs (e.g., uridine derivatives) against CoVs.

Background: Coronavirus disease 2019 (COVID-19) is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Although a preliminary understanding of the replication and transcription of SARS-CoV-2 has recently emerged, their regulation remains unknown. Results: By comprehensive analysis of genome sequence and protein structure data, we propose a negative feedback model to explain the regulation of CoV replication and transcription, providing a molecular basis of the "leader-to-body fusion" model. The key step leading to the proposal of our model was that the transcription regulatory sequence (TRS) motifs were identified as the cleavage sites of nsp15, a nidoviral RNA uridylate-specific endoribonuclease (NendoU). According to this model, nsp15 regulates the synthesis of subgenomic RNAs (sgRNAs), and genomic RNAs (gRNAs) by cleaving TRSs. The expression level of nsp15 controls the relative proportions of sgRNAs and gRNAs, which in turn change the expression level of nsp15 to reach equilibrium between the CoV replication and transcription. Conclusion: The replication and transcription of CoVs are regulated by a negative feedback mechanism that influences the persistence of CoVs in hosts. Our findings enrich fundamental knowledge in the field of gene expression and its regulation, and provide new clues for future studies. One important clue is that nsp15 may be an important and ideal target for the development of drugs (e.g., uridine derivatives) against CoVs. [ABSTRACT FROM AUTHOR]

The pandemic caused by SARS-CoV-2 is not over yet, despite all the efforts from the scientific community. Vaccination is a crucial weapon to fight this virus; however, we still urge the development of antivirals to reduce the severity and progression of the COVID-19 disease. For that, a deep understanding of the mechanisms involved in viral replication is necessary. nsp15 is an endoribonuclease critical for the degradation of viral polyuridine sequences that activate host immune sensors. This enzyme is known as one of the major interferon antagonists from SARS-CoV-2. In this work, a biochemical characterization of SARS-CoV-2 nsp15 was performed. We saw that nsp15 is active as a hexamer, and zinc can block its activity. The role of conserved residues from SARS-CoV-2 nsp15 was investigated, and N164 was found to be important for protein hexamerization and to contribute to the specificity to degrade uridines. Several chemical groups that impact the activity of this ribonuclease were also identified. Additionally, FDA-approved drugs with the capacity to inhibit the in vitro activity of nsp15 are reported in this work. This study is of utmost importance by adding highly valuable information that can be used for the development and rational design of therapeutic strategies.

Coronaviruses (CoVs) have repeatedly emerged from wildlife hosts and infected humans and livestock animals to cause epidemics with significant morbidity and mortality. CoVs infect various organs, including respiratory and enteric systems, as exemplified by newly emerged severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The constellation of viral factors that contribute to developing enteric disease remains elusive. Here, we investigated CoV interferon antagonists for their contribution to enteric pathogenesis. Using an infectious clone of an enteric CoV, porcine epidemic diarrhea virus (icPEDV), we generated viruses with inactive versions of interferon antagonist nonstructural protein 1 (nsp1), nsp15, and nsp16 individually or combined into one virus designated icPEDV-mut4. Interferon-responsive PK1 cells were infected with these viruses and produced higher levels of interferon responses than were seen with wild-type icPEDV infection. icPEDV-mut4 elicited robust interferon responses and was severely impaired for replication in PK1 cells. To evaluate viral pathogenesis, piglets were infected with either icPEDV or icPEDVmut4. While the icPEDV-infected piglets exhibited clinical disease, the icPEDV-mut4- infected piglets showed no clinical symptoms and exhibited normal intestinal pathology at day 2 postinfection. icPEDV-mut4 replicated in the intestinal tract, as revealed by detection of viral RNA in fecal swabs, with sequence analysis documenting genetic stability of the input strain. Importantly, icPEDV-mut4 infection elicited IgG and neutralizing antibody responses to PEDV. These results identify nsp1, nsp15, and nsp16 as virulence factors that contribute to the development of PEDV-induced diarrhea in swine. Inactivation of these CoV interferon antagonists is a rational approach for generating candidate vaccines to prevent disease and spread of enteric CoVs, including SARS-CoV-2. [ABSTRACT FROM AUTHOR]

Coronaviruses (CoVs) encode multiple interferon (IFN) antagonists that modulate the host response to virus replication. Here, we evaluated the host transcriptional response to infection with murine coronaviruses encoding independent mutations in one of two different viral antagonists, the deubiquitinase (DUB) within nonstructural protein 3 or the endoribonuclease (EndoU) within nonstructural protein 15. We used transcriptomics approaches to compare the scope and kinetics of the host response to the wild-type (WT), DUBmut, and EndoUmut viruses in infected macrophages. We found that the EndoUmut virus activates a focused response that predominantly involves type I interferons and interferon-related genes, whereas the WT and DUBmut viruses more broadly stimulate upregulation of over 2,800 genes, including networks associated with activating the unfolded protein response (UPR) and the proinflammatory response associated with viral pathogenesis. This study highlights the role of viral interferon antagonists in shaping the kinetics and magnitude of the host response during virus infection and demonstrates that inactivating a dominant viral antagonist, the coronavirus endoribonuclease, dramatically alters the host response in macrophages. [ABSTRACT FROM AUTHOR]

Coronaviruses (CoVs) are positive-sense RNA viruses that can emerge from endemic reservoirs and infect zoonotically, causing significant morbidity and mortality. CoVs encode an endoribonuclease designated EndoU that facilitates evasion of host pattern recognition receptor MDA5, but the target of EndoU activity was not known. Here, we report that EndoU cleaves the 5′-polyuridines fromnegative-sense viral RNA, termed PUN RNA, which is the product of polyA-templated RNA synthesis. Using a virus containing an EndoU catalytic-inactive mutation, we detected a higher abundance of PUN RNA in the cytoplasm compared to wildtype− infected cells. Furthermore, we found that transfecting PUN RNA into cells stimulates a robust, MDA5-dependent interferon response, and that removal of the polyuridine extension on the RNA dampens the response. Overall, the results of this study reveal the PUN RNA to be a CoV MDA5-dependent pathogen-associated molecular pattern (PAMP). We also establish a mechanism for EndoU activity to cleave and limit the accumulation of this PAMP. Since EndoU activity is highly conserved in all CoVs, inhibiting this activity may serve as an approach for therapeutic interventions against existing and emerging CoV infections. [ABSTRACT FROM AUTHOR]

SARS–CoV–2 is accountable for severe social and economic disruption around the world causing COVID–19. Non–structural protein–15 (NSP15) possesses a domain that is vital to the viral life cycle and is known as uridylate–specific endoribonuclease (EndoU). This domain binds to the uridine 5′–monophosphate (U5P) so that the protein may carry out its native activity. It is considered a vital drug target to inhibit the growth of the virus. Thus, in this current study, ML–based QSAR and virtual screening of U5P analogues targeting Nsp15 were performed to identify potential molecules against SARS–CoV–2. Screening of 816 unique U5P analogues using ML–based QSAR identified 397 compounds ranked on their predicted bioactivity (pIC50). Further, molecular docking and hydrogen bond interaction analysis resulted in the selection of the top three compounds (53309102 , 57398422 , and 76314921). Molecular dynamics simulation of the most promising compounds showed that two molecules 53309102 and 57398422 acted as potential binders of Nsp15. The compound was able to inhibit nsp15 activity as it was successfully bound to the active site of the nsp15 protein. This was achieved by the formation of relevant contacts with enzymatically critical amino acid residues (His235, His250, and Lys290). Principal component analysis and free energy landscape studies showed stable complex formation while MM/GBSA calculation showed lower binding energies for 53309102 (ΔG TOTAL = –29.4 kcal/mol) and 57398422 (ΔG TOTAL = –39.4 kcal/mol) compared to the control U5P (ΔG TOTAL = –18.8 kcal/mol). This study aimed to identify analogues of U5P inhibiting the NSP15 function that potentially could be used for treating COVID–19. [ABSTRACT FROM AUTHOR]

The coronavirus disease-19 (COVID-19) pandemic has raised major interest in innovative drug concepts to suppress human coronavirus (HCoV) infections. We previously reported on a class of 1,2,3-triazolo fused betulonic acid derivatives causing strong inhibition of HCoV-229E replication via the viral nsp15 protein, which is proposedly related to compound binding at an intermonomer interface in hexameric nsp15. In the present study, we further explored the structure-activity relationship (SAR), by varying the substituent at the 1,2,3-triazolo ring as well as the triterpenoid skeleton. The 1,2,3-triazolo fused triterpenoids were synthesized by a multicomponent triazolization reaction, which has been developed in-house. Several analogs possessing a betulin, oleanolic acid, or ursolic acid core displayed favorable activity and selectivity (EC 50 values for HCoV-229E: 1.6-3.5 μM), but neither of them proved as effective as the lead compound containing betulonic acid. The 18β-glycyrrhetinic acid-containing analogs had low selectivity. The antiviral findings were rationalized by in silico docking in the available structure of the HCoV-229E nsp15 protein. The new SAR insights will aid the further development of these 1,2,3-triazolo fused triterpenoid compounds as a unique type of coronavirus inhibitors., (© 2023 Deutsche Pharmazeutische Gesellschaft.)

Importance: Understanding the role of the endoribonuclease non-structural protein 15 (nsp15) (EndoU) in coronavirus (CoV) infection and pathogenesis is essential for vaccine target discovery. Whether the EndoU activity of CoV nsp15, as a virulence-related protein, has a diverse effect on viral virulence needs to be further explored. Here, we found that the transmissible gastroenteritis virus (TGEV) and feline infectious peritonitis virus (FIPV) nsp15 proteins antagonize SeV-induced interferon-β (IFN-β) production in human embryonic kidney 293 cells. Interestingly, compared with wild-type infection, infection with EnUmt-TGEV or EnUmt-FIPV did not change the IFN-β response or reduce viral propagation in immunocompetent cells. The results of animal experiments showed that EnUmt viruses did not reduce the clinical presentation and mortality caused by TGEV and FIPV. Our findings enrich the understanding of nsp15-mediated regulation of alpha-CoV propagation and virulence and reveal that the conserved functions of nonstructural proteins have diverse effects on the pathogenicity of CoVs., Competing Interests: The authors declare no conflict of interest.

SARS-CoV-2 is a positive-stranded RNA virus that bundles its genomic material as messenger-sense RNA in infectious virions and replicates these genomes through RNA intermediates. Several virus-encoded nonstructural proteins play a key role during the viral life cycle. Endoribonuclease NSP15 is vital for the replication and life cycle of the virus, and is thus considered a compelling druggable target. Here, we performed a combination of multiscoring virtual screening and molecular docking of a library of 1624 natural compounds (Nuclei of Bioassays, Ecophysiology and Biosynthesis of Natural Products (NuBBE) database) on the active sites of NSP15 (PDB:6VWW). After sequential high-throughput screening by LibDock and GOLD, docking optimization by CDOCKER, and final scoring by calculating binding energies, top-ranked compounds NuBBE-1970 and NuBBE-242 were further investigated via an indepth molecular-docking and molecular-dynamics simulation of 60 ns, which revealed that the binding of these two compounds with active site residues of NSP15 was sufficiently strong and stable. The findings strongly suggest that further optimization and clinical investigations of these potent compounds may lead to effective SARS-CoV-2 treatment.

The emergence of severe acute respiratory syndrome coronavirus 2, the causative agent of coronavirus disease 2019, has resulted in the largest pandemic in recent history. Current therapeutic strategies to mitigate this disease have focused on the development of vaccines and on drugs that inhibit the viral 3CL protease or RNA-dependent RNA polymerase enzymes. A less-explored and potentially complementary drug target is Nsp15, a uracil-specific RNA endonuclease that shields coronaviruses and other nidoviruses from mammalian innate immune defenses. Here, we perform a high-throughput screen of over 100,000 small molecules to identify Nsp15 inhibitors. We characterize the potency, mechanism, selectivity, and predicted binding mode of five lead compounds. We show that one of these, IPA-3, is an irreversible inhibitor that might act via covalent modification of Cys residues within Nsp15. Moreover, we demonstrate that three of these inhibitors (hexachlorophene, IPA-3, and CID5675221) block severe acute respiratory syndrome coronavirus 2 replication in cells at subtoxic doses. This study provides a pipeline for the identification of Nsp15 inhibitors and pinpoints lead compounds for further development against coronavirus disease 2019 and related coronavirus infections., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Here we review the evolving story of the coronavirus endoribonuclease (EndoU). Coronavirus EndoU is encoded within the sequence of nonstructural protein (nsp) 15, which was initially identified as a component of the viral replication complex. Biochemical and structural studies revealed the enzymatic nature of nsp15/EndoU, which was postulated to be essential for the unique replication cycle of viruses in the order Nidovirales . However, the role of nsp15 in coronavirus replication was enigmatic as EndoU-deficient coronaviruses were viable and replicated to near wild-type virus levels in fibroblast cells. A breakthrough in our understanding of the role of EndoU was revealed in recent studies, which showed that EndoU mediates the evasion of viral double-stranded RNA recognition by host sensors in macrophages. This new discovery of nsp15/EndoU function leads to new opportunities for investigating how a viral EndoU contributes to pathogenesis and exploiting this enzyme for therapeutics and vaccine design against pathogenic coronaviruses. [ABSTRACT FROM AUTHOR]

Coronaviruses are positive-sense RNA viruses that generate double-stranded RNA (dsRNA) intermediates during replication, yet evade detection by host innate immune sensors. Here we report that coronavirus nonstructural protein 15 (nsp15), an endoribonuclease, is required for evasion of dsRNA sensors. We evaluated two independent nsp15 mutant mouse coronaviruses, designated N15m1 and N15m3, and found that these viruses replicated poorly and induced rapid cell death in mouse bone marrow-derived macrophages. Infection of macrophages with N15m1, which expresses an unstable nsp15, or N15m3, which expresses a catalysis-deficient nsp15, activated MDA5, PKR, and the OAS/RNase L system, resulting in an early, robust induction of type I IFN, PKR-mediated apoptosis, and RNA degradation. Immunofluorescence imaging of nsp15 mutant virus-infected macrophages revealed significant dispersal of dsRNA early during infection, whereas in WT virus-infected cells, the majority of the dsRNA was associated with replication complexes. The loss of nsp15 activity also resulted in greatly attenuated disease in mice and stimulated a protective immune response. Taken together, our findings demonstrate that coronavirus nsp15 is critical for evasion of host dsRNA sensors in macrophages and reveal that modulating nsp15 stability and activity is a strategy for generating liveattenuated vaccines. [ABSTRACT FROM AUTHOR]

COVID-19 (Corona Virus Disease of 2019) caused by the novel 'Severe Acute Respiratory Syndrome Coronavirus-2' (SARS-CoV-2) has wreaked havoc on human health and the global economy. As a result, for new medication development, it's critical to investigate possible therapeutic targets against the novel virus. 'Non-structural protein 15' (Nsp15) endonuclease is one of the crucial targets which helps in the replication of virus and virulence in the host immune system. Here, in the current study, we developed the structure-based pharmacophore model based on Nsp15-UMP interactions and virtually screened several databases against the selected model. To validate the screening process, we docked the top hits obtained after secondary filtering (Lipinski's rule of five, ADMET & Topkat) followed by 100 ns molecular dynamics (MD) simulations. Next, to revalidate the MD simulation studies, we have calculated the binding free energy of each complex using the MM-PBSA procedure. The discovered repurposed drugs can aid the rational design of novel inhibitors for Nsp15 of the SARS-CoV-2 enzyme and may be considered for immediate drug development.