ANALYSIS OF THREE NON-STRUCTURAL PROTEINS, NSP1, NSP2, AND NSP10 OF SARS-COV-2 AS PIVOTAL TARGET PROTEINS FOR COMPUTATIONAL DRUG SCREENING

Coronaviruses cause mild to severe respiratory infections. The highly contagious SARS-CoV-2 new coronavirus caused a global outbreak of atypical viral pneumonia in late 2019. Acute respiratory distress syndrome, multiple organ failure, respiratory failure, and death can result from the infection. This study aimed to evaluate the possible inhibition activity from numerous medicinal plants' bioactive compounds against three non-structural proteins, namely Nsp1, Nsp2, and Nsp10 of SARS-CoV-2, through the computational study. Molecular docking was performed on the ligands and the target protein. This study investigated multiple criteria, including binding affinity value, location, and chemical interaction. In this present study, we found the top three highest binding affinity values of bioactive compounds against Nisp1, namely cafestol, crocin, and ledene;the top three highest binding affinity values of bioactive compounds against Nisp2, including cafestol, kahweol, and theaflavin 3,3′-digallate;and top three highest binding affinity value of bioactive compounds against Nisp10 namely cafestol, kahweol, and theaflavin-3,3′-digallate. Interestingly, we also found that cafestol, crocin, and theaflavin-3,3'-digallate binds to all target proteins © 2023, Journal of Microbiology, Biotechnology and Food Sciences.All Rights Reserved.

In-Silico Structure-Based Drug Discovery of Candidate Drugs Against Novel Protein Receptor Complex Nsp10-Nsp16 of SARS-CoV-2 Using Drug Repurposing Approach

Background: Several therapeutic possibilities have been explored against Severe Acute Respiratory Syndrome-2 (SARS-CoV-2), such as convalescent plasma (CP), intravenous immunoglobulin (IVIG) and monoclonal antibodies. Compounds such as hydroxychloroquine have also been found to have fatal drawbacks. Repurposing of existing antiviral drugs can be an effective strategy, which could fasten up the process of drug discovery. Objective(s): The present study is designed to predict the computational efficacy of pre-existing antiviral drugs as inhibitors for the Nsp10-Nsp16 complex protein of SARS-CoV-2. Method(s): Twenty-six known antiviral drugs along with their similar structures based on Tanimoto simi-larity, were screened towards the Nsp10-Nsp16 complex's active site. Result(s): Our study reports competitive binding of 1-[3-[2-(2-Ethoxyphenoxy) ethylamino]-2-hydroxypropyl]-9H-carbazol-4-ol against AdoMet binding site in Nsp10-Nsp16 complex. Formation of the stable ligand-receptor complex with 1-[3-[2-(2-Ethoxyphenoxy) ethylamino]-2-hydroxypropyl]-9H-carbazol-4-ol could functionally inhibit the Nsp10-Nsp16 complex, thereby making the SARS-CoV-2 vulnerable to host immuno-surveillance mechanisms. Conclusion(s): We conclude that these computational hits can display positive results in in-vitro trials against SARS-CoV-2.Copyright © 2021 Bentham Science Publishers.

Molecular interactions of Zyesami with the SARS-CoV-2 nsp10/nsp16 protein complex.

Background SARS-CoV-2 emerged in late 2019, causes COVID-19. Patients treated with Zyesami were found to be 3-fold decrease in respiratory failure and improvement in clinical outcome. It was reported that Zyesami inhibits RNA replication of SARS-CoV-2, including several non-structural proteins that essential in viral RNA replication. SARS-CoV-2 is a distinctive virus that required nsp10 and nsp16 for its methyltransferases activity which is crucial for RNA stability and protein synthesis. Objective We aimed the in silico determination of inhibitory consequences of Zyesami on the SARS-CoV-2 nsp10/nsp16 complex. Targeting SARS-CoV-2 nsp10/ nsp16 protein complex may be used for the development of drug against the COVID-19. Methods I-TASSER was used for secondary structure prediction of Zyesami. CABS-dock was used for modelling of Zyesami with SARS-CoV-2 nsp16 interaction. The docked complex was visualized using PyMol. The quality of the docking model was checked by using ProQdock. Results The 3D structure of SARS-CoV 2, nsp10/nsp16 showed that essential interactions exist between nsp10 and nsp16. Significant contact areas of Zyesami exist across amino acid residues of nsp10; Asn40-Thr47, Val57-Pro59, Gly69-Ser72, Cys77-Pro84, Lys93-Tyr96. In addition, polar contacts between nsp16 and Zyesami are Asn299-Ser440, Val297-Asn443, Gly149-Tyr437, Gln159-Lys430, Asn178-Arg429, Ser146-Arg429, Ser146-Arg429, Lys147-Arg429, Asr221-Thr422, Lys183-Asp423, Lys183-Asp423, and Gln219-Asp423 the residues are shown of nsp16 and Zyesami respectively. Conclusion The structural bioinformatics analyses have indicated the potential binding specificity of Zyesami and nsp16. Data predict how the initial binding of Zyesami with nsp10 and nsp16 may occur. Moreover, this binding could significantly inhibit the 2 -O-MTase activity of the SARS-CoV nsp10/16 complex.

SS148 and WZ16 inhibit the activities of nsp10-nsp16 complexes from all seven human pathogenic coronaviruses.

Seven coronaviruses have infected humans (HCoVs) to-date. SARS-CoV-2 caused the current COVID-19 pandemic with the well-known high mortality and severe socioeconomic consequences. MERS-CoV and SARS-CoV caused epidemic of MERS and SARS, respectively, with severe respiratory symptoms and significant fatality. However, HCoV-229E, HCoV-NL63, HCoV-HKU1, and HCoV-OC43 cause respiratory illnesses with less severe symptoms in most cases. All coronaviruses use RNA capping to evade the immune systems of humans. Two viral methyltransferases, nsp14 and nsp16, play key roles in RNA capping and are considered valuable targets for development of anti-coronavirus therapeutics. But little is known about the kinetics of nsp10-nsp16 methyltransferase activities of most HCoVs, and reliable assays for screening are not available. Here, we report the expression, purification, and kinetic characterization of nsp10-nsp16 complexes from six HCoVs in parallel with previously characterized SARS-CoV-2. Probing the active sites of all seven by SS148 and WZ16, the two recently reported dual nsp14 / nsp10-nsp16 inhibitors, revealed pan-inhibition. Overall, our study show feasibility of developing broad-spectrum dual nsp14 / nsp10-nsp16-inhibitor therapeutics.

A Non-Structural Protein of Severe Acute Respiratory Syndrome Coronavirus 2 with a Potential Ability to Reduce Blood Glucose for Use in Controlling Type 2 Diabetes Mellitus.

It is estimated that more than 400 million people worldwide are suffering from diabetes. There are two types of diabetes. Type 1 diabetes is the result of insufficient insulin secretion into the bloodstream, most often due to an autoimmune attack on the pancreas glands. Type 2 diabetes is caused by the inability of the surface ligands to adsorb the insulin from the bloodstream. The conventional medicines for diabetes mellitus include sulfonylureas, biguanide, thiazolidinediones, alpha-glucosidase inhibitors, and meglitinide. By February 2022, Severe acute respiratory syndrome coronavirus 2 (SARSCoV2) had infected more than 391 million people worldwide, claiming 5.7 million lives and imposing heavy costs on the healthcare system. The present study aimed to assess the potential use of this non-structural SARS-CoV-2 protein in the treatment of type 2 diabetes mellitus. The nsp10 was structurally aligned with GoDrugBank therapeutic agents, and lixisenatide was found to have the most similar chemical structure. This drug is a glucagon-like peptide-1 (GLP1) receptor agonist used for the treatment of type 2 diabetes mellitus. The best molecular docking energy score for these two proteins was -301.47, and the ligand root mean square deviation was calculated to be 107.93 Å. The molecular dynamics for the stability of the nsp10 and GLP1R binding in triplicate for 150 ns demonstrated that the nsp10-GLP1R remained bound for more than 80 ns. This study indicated that the nsp10 protein can be further studied to be used as an antidiabetic medication.

Computational Analysis of SAM Analogs as Methyltransferase Inhibitors of nsp16/nsp10 Complex from SARS-CoV-2.

Methyltransferases (MTases) enzymes, responsible for RNA capping into severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), are emerging important targets for the design of new anti-SARS-CoV-2 agents. Here, analogs of S-adenosylmethionine (SAM), obtained from the bioisosteric substitution of the sulfonium and amino acid groups, were evaluated by rigorous computational modeling techniques such as molecular dynamics (MD) simulations followed by relative binding free analysis against nsp16/nsp10 complex from SARS-CoV-2. The most potent inhibitor (2a) shows the lowest binding free energy (-58.75 Kcal/mol) and more potency than Sinefungin (SFG) (-39.8 Kcal/mol), a pan-MTase inhibitor, which agrees with experimental observations. Besides, our results suggest that the total binding free energy of each evaluated SAM analog is driven by van der Waals interactions which can explain their poor cell permeability, as observed in experimental essays. Overall, we provide a structural and energetic analysis for the inhibition of the nsp16/nsp10 complex involving the evaluated SAM analogs as potential inhibitors.

Identifying inhibitors of NSP16-NSP10 of SARS-CoV-2 from large databases.

The COVID-19 pandemic, which has already claimed millions of lives, continues to pose a serious threat to human health, requiring the development of new effective drugs. Non-structural proteins of SARS-CoV-2 play an important role in viral replication and infection. Among them, NSP16 (non-structured protein 16) and its cofactor NSP10 (non-structured protein 10) perform C2'-O methylation at the 5' end of the viral RNA, which promotes efficient virus replication. Therefore, the NSP16-NSP10 complex becomes an attractive target for drug development. Using a multi-step virtual screening protocol which includes Lipinski's rule, docking, steered molecular dynamics and umbrella sampling, we searched for potential inhibitors from the PubChem and anti-HIV databases. It has been shown that CID 135566620 compound from PubChem is the best candidate with an inhibition constant in the sub-µM range. The Van der Waals interaction was found to be more important than the electrostatic interaction in the binding affinity of this compound to NSP16-NSP10. Further in vitro and in vivo studies are needed to test the activity of the identified compound against COVID-19.Communicated by Ramaswamy H. Sarma.

Crystal structure of SARS-CoV-2 nsp10-nsp16 in complex with small molecule inhibitors, SS148 and WZ16.

SARS-CoV-2 nsp10-nsp16 complex is a 2'-O-methyltransferase (MTase) involved in viral RNA capping, enabling the virus to evade the immune system in humans. It has been considered a valuable target in the discovery of antiviral therapeutics, as the RNA cap formation is crucial for viral propagation. Through cross-screening of the inhibitors that we previously reported for SARS-CoV-2 nsp14 MTase activity against nsp10-nsp16 complex, we identified two compounds (SS148 and WZ16) that also inhibited nsp16 MTase activity. To further enable the chemical optimization of these two compounds towards more potent and selective dual nsp14/nsp16 MTase inhibitors, we determined the crystal structure of nsp10-nsp16 in complex with each of SS148 and WZ16. As expected, the structures revealed the binding of both compounds to S-adenosyl-L-methionine (SAM) binding pocket of nsp16. However, our structural data along with the biochemical mechanism of action determination revealed an RNA-dependent SAM-competitive pattern of inhibition for WZ16, clearly suggesting that binding of the RNA first may help the binding of some SAM competitive inhibitors. Both compounds also showed some degree of selectivity against human protein MTases, an indication of great potential for chemical optimization towards more potent and selective inhibitors of coronavirus MTases.

Virtual screening and molecular dynamics simulation for identification of natural antiviral agents targeting SARS-CoV-2 NSP10.

New variations of SARS-CoV-2 continue to emerge in the global pandemic, which may be resistant to at least some vaccines in COVID-19, indicating that drug and vaccine development must be continuously strengthened. NSP10 plays an essential role in SARS-CoV-2 viral life cycle. It stimulates the enzymatic activities of NSP14-ExoN and NSP16-O-MTase by the formation of NSP10/NSP14 and NSP10/NSP16 complexes. Inhibiting NSP10 can block the binding of NSP10 to NSP14 and NSP16. This study has identified potential natural NSP10 inhibitors from ZINC database. The protein druggable pocket was identified for screening candidates. Molecular docking of the selected compounds was performed and MM-GBSA binding energy was calculated. After ADMET assessment, 4 hits were obtained for favorable druggability. The analysis of site interactions suggested that the hits all had excellent binding. Molecular dynamics studies revealed that selected natural compounds stably bind to NSP10. These compounds were identified as potential leads against NSP10 for the development of strategies to combat SARS-CoV-2 replication and could serve as the basis for further studies.

Refolding of lid subdomain of SARS-CoV-2 nsp14 upon nsp10 interaction releases exonuclease activity.

During RNA replication, coronaviruses require proofreading to maintain the integrity of their large genomes. Nsp14 associates with viral polymerase complex to excise the mismatched nucleotides. Aside from the exonuclease activity, nsp14 methyltransferase domain mediates cap methylation, facilitating translation initiation and protecting viral RNA from recognition by the innate immune sensors. The nsp14 exonuclease activity is modulated by a protein co-factor nsp10. While the nsp10/nsp14 complex structure is available, the mechanistic basis for nsp10-mediated modulation remains unclear in the absence of the nsp14 structure. Here, we provide a crystal structure of nsp14 in an apo-form. Comparative analysis of the apo- and nsp10-bound structures explain the modulatory role of the co-factor protein and reveal the allosteric nsp14 control mechanism essential for drug discovery. Further, the flexibility of the N-terminal lid of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) nsp14 structure presented in this study rationalizes the recently proposed idea of nsp14/nsp10/nsp16 ternary complex.

Virtual screening of molecular databases for potential inhibitors of the NSP16/NSP10 methyltransferase from SARS-CoV-2.

COVID-19 is a disease caused by the SARS-CoV-2 virus and represents one of the greatest health problems that humanity faces at the moment. Therefore, efforts have been made with the objective of seeking therapies that could be effective in combating this problematic. In the search for ligands, computational chemistry plays an essential role, since it allows the screening of thousands of molecules on a given target, in order to save time and money for the in vitro or in vivo pharmacological stage. In this paper, we perform a virtual screening by docking looking for potential inhibitors of the NSP16-NSP10 protein dimer (methyltransferase) from SARS-CoV-2, by evaluating a homemade databank of molecules found in plants of the Caatinga Brazilian biome, compounds from ZINC online molecular database, as well as structural analogues of the enzymatic cofactor s-adenosylmethionine (SAM) and a known inhibitor in the literature, sinefungin (SFG), provided at PubChem database. All the evaluated sets presented molecules that deserve attention, highlighting four compounds from ZINC as the most promising ligands. These results contribute to the discovery of new molecular hits, in the search of potential agents against SARS-CoV-2 virus, still unveiling a pathway that can be used in combined therapies.

In silico study of potential antiviral activity of copper(II) complexes with non-steroidal anti-inflammatory drugs on various SARS-CoV-2 target proteins.

In silico molecular docking studies, in vitro toxicity and in silico predictions on the biological activity profile, pharmacokinetic properties, drug-likeness, ADMET (absorption, distribution, metabolism, excretion, and toxicity) physicochemical pharmacokinetic data, and target proteins and toxicity predictions were performed on six copper(II) complexes with the non-steroidal anti-inflammatory drugs ibuprofen, loxoprofen, fenoprofen and clonixin as ligands, in order to investigate the ability of these complexes to interact with the key therapeutic target proteins of SARS-CoV-2 (Severe Acute Respiratory Syndrome Coronavirus 2) 3C-like cysteine main protease (3CLpro/Mpro), viral papain-like protease (PLpro), RNA-dependent RNA polymerase (RdRp), and non-structural proteins (Nsps) Nsp16-Nsp10 2'-O-methyltransferase complex, and their capacity to act as antiviral agents, contributing thus to understanding the role they can play in the context of coronavirus 2019 (COVID-19) pandemic. Cytotoxic activity against five human cancer and normal cell lines were also evaluated.

Molecular Docking Studies to Identify Promising Natural Inhibitors Targeting SARS-CoV-2 Nsp10-Nsp16 Protein Complex

Objectives: Unavailability of potential drugs/vaccines in the outbreak of the pandemic severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2) have devastated the human population globally. Several druggable targets have been analyzed against different viral proteins such as the spike protein. The study aims to explore the potential of natural compounds as an effective drug against a novel nsp10-nsp16 complex of SARS-CoV-2 using in silico approaches. Materials and Methods: In silico screening (Docking analysis) was performed for 10 shortlisted natural compounds viz. allicin, ajoene, carvacrol, coumarin, curcumin, menthol, eugenol, theaflavin, ursolic acid, and catechin against a novel target of SARS-CoV-2, that has been anticipated to provide valuable lead molecules and potentially druggable compounds for the treatment of SARS-CoV-2. Results: Theaflavin and catechin, the natural components of black tea and green tea, out of 10 shortlisted compounds have shown excellent performance in our docking studies with the minimum binding energy of -11.8 kcal/mol and -9.2 kcal/mol respectively, against a novel nsp10-nsp16 complex of SARS-CoV-2 that indicates their potential for inhibitory molecular interactions against the virus to assist rapid drug designing from natural products. Conclusion: Either consumption of black tea and green tea or repurposing them as drug candidates may help individuals to fight against SARS-CoV-2, subject to their in vivo and in vitro further experimental validations.

Exploring the Catalytic Mechanism of the RNA Cap Modification by nsp16-nsp10 Complex of SARS-CoV-2 through a QM/MM Approach.

The inhibition of key enzymes that may contain the viral replication of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) have assumed central importance in drug discovery projects. Nonstructural proteins (nsps) are essential for RNA capping and coronavirus replication since it protects the virus from host innate immune restriction. In particular, nonstructural protein 16 (nsp16) in complex with nsp10 is a Cap-0 binding enzyme. The heterodimer formed by nsp16-nsp10 methylates the 5'-end of virally encoded mRNAs to mimic cellular mRNAs and thus it is one of the enzymes that is a potential target for antiviral therapy. In this study, we have evaluated the mechanism of the 2'-O methylation of the viral mRNA cap using hybrid quantum mechanics/molecular mechanics (QM/MM) approach. It was found that the calculated free energy barriers obtained at M062X/6-31+G(d,p) is in agreement with experimental observations. Overall, we provide a detailed molecular analysis of the catalytic mechanism involving the 2'-O methylation of the viral mRNA cap and, as expected, the results demonstrate that the TS stabilization is critical for the catalysis.

Activation of the SARS-CoV-2 NSP14 3'-5' exoribonuclease by NSP10 and response to antiviral inhibitors.

Understanding the core replication complex of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is essential to the development of novel coronavirus-specific antiviral therapeutics. Among the proteins required for faithful replication of the SARS-CoV-2 genome are nonstructural protein 14 (NSP14), a bifunctional enzyme with an N-terminal 3'-to-5' exoribonuclease (ExoN) and a C-terminal N7-methyltransferase, and its accessory protein, NSP10. The difficulty in producing pure and high quantities of the NSP10/14 complex has hampered the biochemical and structural study of these important proteins. We developed a straightforward protocol for the expression and purification of both NSP10 and NSP14 from Escherichia coli and for the in vitro assembly and purification of a stoichiometric NSP10/14 complex with high yields. Using these methods, we observe that NSP10 provides a 260-fold increase in kcat/Km in the exoribonucleolytic activity of NSP14 and enhances protein stability. We also probed the effect of two small molecules on NSP10/14 activity, remdesivir monophosphate and the methyltransferase inhibitor S-adenosylhomocysteine. Our analysis highlights two important factors for drug development: first, unlike other exonucleases, the monophosphate nucleoside analog intermediate of remdesivir does not inhibit NSP14 activity; and second, S-adenosylhomocysteine modestly activates NSP14 exonuclease activity. In total, our analysis provides insights for future structure-function studies of SARS-CoV-2 replication fidelity for the treatment of coronavirus disease 2019.

Cellular miR-150-5p may have a crucial role to play in the biology of SARS-CoV-2 infection by regulating nsp10 gene.

The role for circulating miRNAs as biomarkers of the COVID-19 disease remains uncertain. We analysed the circulating miRNA profile in twelve COVID-19 patients with moderate-severe disease. This analysis was conducted by performing next generation sequencing (NGS) followed by real-time polymerase chain reaction (RT-qPCR). Compared with healthy controls, we detected significant changes in the circulating miRNA profile of COVID-19 patients. The miRNAs that were significantly altered in all the COVID-19 patients were miR-150-5p, miR-375, miR-122-5p, miR-494-3p, miR-3197, miR-4690-5p, miR-1915-3p, and miR-3652. Infection assays performed using miRNA mimics in HEK-293 T cells determined miR-150-5p to have a crucial role in SARS-CoV-2 infection and this was based on the following data: (i) miR-150-5p mimic lowered in vitro SARS-CoV-2 infection; (ii) miR-150-5p inhibitor reversed the effects of miR-150-5p mimic on SARS-CoV-2 infection of cells; and (iii) a novel miRNA recognition element (MRE) was identified in the coding strand of SARS-CoV-2 nsp10, the expression of which could be inhibited by miR-150-5p mimic. Our findings identified crucial miRNA footprints in COVID-19 patients with moderate-severe disease. A combination of co-transfection and Western blotting experiments also determined the ability of miR-150-5p to inhibit SARS-CoV-2 infection via directly interacting with MRE in the coding strand of nsp10. Our investigation showed that a sharp decline in the miR-150-5p plasma levels in COVID-19 patients may support enhanced SARS-CoV-2 infection. Furthermore, this study provides insight into one possible mechanism by which COVID-19-induced changes to miR-150-5p levels may promote SARS-CoV-2 infection via modulating nsp10 expression.

In Silico Exploration of Potential Natural Inhibitors against SARS-Cov-2 nsp10.

In continuation of our previous effort, different in silico selection methods were applied to 310 naturally isolated metabolites that exhibited antiviral potentialities before. The applied selection methods aimed to pick the most relevant inhibitor of SARS-CoV-2 nsp10. At first, a structural similarity study against the co-crystallized ligand, S-Adenosyl Methionine (SAM), of SARS-CoV-2 nonstructural protein (nsp10) (PDB ID: 6W4H) was carried out. The similarity analysis culled 30 candidates. Secondly, a fingerprint study against SAM preferred compounds 44, 48, 85, 102, 105, 182, 220, 221, 282, 284, 285, 301, and 302. The docking studies picked 48, 182, 220, 221, and 284. While the ADMET analysis expected the likeness of the five candidates to be drugs, the toxicity study preferred compounds 48 and 182. Finally, a density-functional theory (DFT) study suggested vidarabine (182) to be the most relevant SARS-Cov-2 nsp10 inhibitor.

Search for RNA aptamers against non-structural protein of SARS-CoV-2: Design using molecular dynamics approach.

BACKGROUND: Recent outbreak of deadly Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) urges the scientist to identify the potential vaccine or drug to control the disease. SARS-CoV-2 with its single stranded RNA genome (length ~ 30 kb) is enveloped with active spike proteins. The genome is non-segmental with 5'-cap and 3'-poly tail and acts as a mRNA for the synthesis of replicase polyproteins. The replicase gene lying downstream to 5'-end encodes for non-structural protein, which in turn pose multiple functions ranging from envelope to nucleocapsid development. This study aims to identify the highly stable, effective and less toxic single strand RNA-based aptamers against non-structural protein 10 (NSP10). NSP10 is the significant activator of methyltransferase enzymes (NSP14 and NSP16) in SARS-CoV-2. Inhibiting the activation of methyltransferase leads to partial viral RNA capping or lack of capping, which makes the virus particles susceptible to host defence system. RESULTS: In this study, we focused on designing RNA aptamers through computational approach, docking of protein-aptamer followed by molecular dynamics simulation to perceive the binding stability of complex. Docking study reveals the high binding affinity of three aptamers namely RNA-053, 001, 010 to NSP10 with the HADDOCK score of - 88.5 ± 7.0, - 87.7 ± 11.5, - 86.1 ± 12 respectively. Molecular Dynamics suggests high conformational stability between the aptamer and the protein. Among the screened aptamers two aptamers maintained at least 3-4 intermolecular H-bonds throughout the simulation period. CONCLUSIONS: The study identifies the potential aptamer candidate against less investigated but significant antiviral target i.e., NSP10/NSP16 interface complex. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1186/s43088-021-00152-5.

New targets for drug design: importance of nsp14/nsp10 complex formation for the 3'-5' exoribonucleolytic activity on SARS-CoV-2.

SARS-CoV-2 virus has triggered a global pandemic with devastating consequences. The understanding of fundamental aspects of this virus is of extreme importance. In this work, we studied the viral ribonuclease nsp14, one of the most interferon antagonists from SARS-CoV-2. Nsp14 is a multifunctional protein with two distinct activities, an N-terminal 3'-to-5' exoribonuclease (ExoN) and a C-terminal N7-methyltransferase (N7-MTase), both critical for coronaviruses life cycle, indicating nsp14 as a prominent target for the development of antiviral drugs. In coronaviruses, nsp14 ExoN activity is stimulated through the interaction with the nsp10 protein. We have performed a biochemical characterization of nsp14-nsp10 complex from SARS-CoV-2. We confirm the 3'-5' exoribonuclease and MTase activities of nsp14 and the critical role of nsp10 in upregulating the nsp14 ExoN activity. Furthermore, we demonstrate that SARS-CoV-2 nsp14 N7-MTase activity is functionally independent of the ExoN activity and nsp10. A model from SARS-CoV-2 nsp14-nsp10 complex allowed mapping key nsp10 residues involved in this interaction. Our results show that a stable interaction between nsp10 and nsp14 is required for the nsp14-mediated ExoN activity of SARS-CoV-2. We studied the role of conserved DEDD catalytic residues of SARS-CoV-2 nsp14 ExoN. Our results show that motif I of ExoN domain is essential for the nsp14 function, contrasting to the functionality of these residues in other coronaviruses, which can have important implications regarding the specific pathogenesis of SARS-CoV-2. This work unraveled a basis for discovering inhibitors targeting specific amino acids in order to disrupt the assembly of this complex and interfere with coronaviruses replication.

Localization of SARS-CoV-2 Capping Enzymes Revealed by an Antibody against the nsp10 Subunit.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of the coronavirus disease-19 pandemic. One of the key components of the coronavirus replication complex are the RNA methyltransferases (MTases), RNA-modifying enzymes crucial for RNA cap formation. Recently, the structure of the 2'-O MTase has become available; however, its biological characterization within the infected cells remains largely elusive. Here, we report a novel monoclonal antibody directed against the SARS-CoV-2 non-structural protein nsp10, a subunit of both the 2'-O RNA and N7 MTase protein complexes. Using this antibody, we investigated the subcellular localization of the SARS-CoV-2 MTases in cells infected with the SARS-CoV-2.