Unwinding mechanism of SARS-CoV helicase (nsp13) in the presence of Ca2+, elucidated by biochemical and single-molecular studies.

The recent outbreak of COVID-19 has created a serious health crisis with fatFal infectious viral diseases, such as Severe Acute Respiratory Syndrome (SARS). The nsp13, a helicase of coronaviruses is an essential element for viral replication that unwinds secondary structures of DNA and RNA, and is thus considered a major therapeutic target for treatment. The replication of coronaviruses and other retroviruses occurs in the cytoplasm of infected cells, in association with viral replication organelles, called virus-induced cytosolic double-membrane vesicles (DMVs). In addition, an increase in cytosolic Ca2+ concentration accelerates viral replication. However, the molecular mechanism of nsp13 in the presence of Ca2+ is not well understood. In this study, we applied biochemical methods and single-molecule techniques to demonstrate how nsp13 achieves its unwinding activity while performing ATP hydrolysis in the presence of Ca2+. Our study found that nsp13 could efficiently unwind double stranded (ds) DNA under physiological concentration of Ca2+ of cytosolic DMVs. These findings provide new insights into the properties of nsp13 in the range of calcium in cytosolic DMVs.

Phase-separated nucleocapsid protein of SARS-CoV-2 suppresses cGAS-DNA recognition by disrupting cGAS-G3BP1 complex.

Currently, the incidence and fatality rate of SARS-CoV-2 remain continually high worldwide. COVID-19 patients infected with SARS-CoV-2 exhibited decreased type I interferon (IFN-I) signal, along with limited activation of antiviral immune responses as well as enhanced viral infectivity. Dramatic progresses have been made in revealing the multiple strategies employed by SARS-CoV-2 in impairing canonical RNA sensing pathways. However, it remains to be determined about the SARS-CoV-2 antagonism of cGAS-mediated activation of IFN responses during infection. In the current study, we figure out that SARS-CoV-2 infection leads to the accumulation of released mitochondria DNA (mtDNA), which in turn triggers cGAS to activate IFN-I signaling. As countermeasures, SARS-CoV-2 nucleocapsid (N) protein restricts the DNA recognition capacity of cGAS to impair cGAS-induced IFN-I signaling. Mechanically, N protein disrupts the assembly of cGAS with its co-factor G3BP1 by undergoing DNA-induced liquid-liquid phase separation (LLPS), subsequently impairs the double-strand DNA (dsDNA) detection ability of cGAS. Taken together, our findings unravel a novel antagonistic strategy by which SARS-CoV-2 reduces DNA-triggered IFN-I pathway through interfering with cGAS-DNA phase separation.

Development of FRET and Stress Granule Dual-Based System to Screen for Viral 3C Protease Inhibitors.

3C proteases (3Cpros) of picornaviruses and 3C-like proteases (3CLpros) of coronaviruses and caliciviruses represent a group of structurally and functionally related viral proteases that play pleiotropic roles in supporting the viral life cycle and subverting host antiviral responses. The design and screening for 3C/3CLpro inhibitors may contribute to the development broad-spectrum antiviral therapeutics against viral diseases related to these three families. However, current screening strategies cannot simultaneously assess a compound's cytotoxicity and its impact on enzymatic activity and protease-mediated physiological processes. The viral induction of stress granules (SGs) in host cells acts as an important antiviral stress response by blocking viral translation and stimulating the host immune response. Most of these viruses have evolved 3C/3CLpro-mediated cleavage of SG core protein G3BP1 to counteract SG formation and disrupt the host defense. Yet, there are no SG-based strategies screening for 3C/3CLpro inhibitors. Here, we developed a fluorescence resonance energy transfer (FRET) and SG dual-based system to screen for 3C/3CLpro inhibitors in living cells. We took advantage of FRET to evaluate the protease activity of poliovirus (PV) 3Cpro and live-monitor cellular SG dynamics to cross-verify its effect on the host antiviral response. Our drug screen uncovered a novel role of Telaprevir and Trifluridine as inhibitors of PV 3Cpro. Moreover, Telaprevir and Trifluridine also modulated 3Cpro-mediated physiological processes, including the cleavage of host proteins, inhibition of the innate immune response, and consequent facilitation of viral replication. Taken together, the FRET and SG dual-based system exhibits a promising potential in the screening for inhibitors of viral proteases that cleave G3BP1.

Pharmacological disruption of mSWI/SNF complex activity restricts SARS-CoV-2 infection.

Identification of host determinants of coronavirus infection informs mechanisms of viral pathogenesis and can provide new drug targets. Here we demonstrate that mammalian SWItch/Sucrose Non-Fermentable (mSWI/SNF) chromatin remodeling complexes, specifically canonical BRG1/BRM-associated factor (cBAF) complexes, promote severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection and represent host-directed therapeutic targets. The catalytic activity of SMARCA4 is required for mSWI/SNF-driven chromatin accessibility at the ACE2 locus, ACE2 expression and virus susceptibility. The transcription factors HNF1A/B interact with and recruit mSWI/SNF complexes to ACE2 enhancers, which contain high HNF1A motif density. Notably, small-molecule mSWI/SNF ATPase inhibitors or degraders abrogate angiotensin-converting enzyme 2 (ACE2) expression and confer resistance to SARS-CoV-2 variants and a remdesivir-resistant virus in three cell lines and three primary human cell types, including airway epithelial cells, by up to 5 logs. These data highlight the role of mSWI/SNF complex activities in conferring SARS-CoV-2 susceptibility and identify a potential class of broad-acting antivirals to combat emerging coronaviruses and drug-resistant variants.

SARS-CoV-2 pandemics: An update of CRISPR in diagnosis and host-virus interaction studies.

Since December 2019, the Coronavirus disease 2019 (COVID-19) outbreak caused by severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) has spread rapidly around the world, overburdening healthcare systems and creating significant global health concerns. Rapid detection of infected individuals via early diagnostic tests and administration of effective therapy remains vital in pandemic control, and recent advances in the clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated proteins (Cas) system may support the development of novel diagnostic and therapeutic approaches. Cas-based SARS-CoV-2 detection methods (FnCAS9 Editor Linked Uniform Detection Assay (FELUDA), DNA endonuclease-targeted CRISPR trans reporter (DETECTR), and Specific High-sensitivity Enzymatic Reporter Unlocking (SHERLOCK)) have been developed for easier handling compared to quantitative polymerase chain reaction (qPCR) assays, with good rapidity, high specificity, and reduced need for complex instrumentation. Cas-CRISPR-derived RNA (Cas-crRNA) complexes have been shown to reduce viral loads in the lungs of infected hamsters, by degrading virus genomes and limiting viral replication in host cells. Viral-host interaction screening platforms have been developed using the CRISPR-based system to identify essential cellular factors involved in pathogenesis, and CRISPR knockout (CRISPRKO) and activation screening results have revealed vital pathways in the life cycle of coronaviruses, including host cell entry receptors (ACE2, DPP4, and ANPEP), proteases involved in spike activation and membrane fusion (cathepsin L (CTSL) and transmembrane protease serine 2 (TMPRSS2)), intracellular traffic control routes for virus uncoating and budding, and membrane recruitment for viral replication. Several novel genes (SWI/SNF Related, Matrix Associated, Actin Dependent Regulator of Chromatin, subfamily A, member 4 (SMARCA4), ARIDIA, and KDM6A) have also been identified via systematic data mining analysis as pathogenic factors for severe CoV infection. This review highlights how CRISPR-based systems can be applied to investigate the viral life cycle, detect viral genomes, and develop therapies against SARS-CoV-2 infection.

In Silico Binding of 2-Aminocyclobutanones to SARS-CoV-2 Nsp13 Helicase and Demonstration of Antiviral Activity.

The landscape of viral strains and lineages of SARS-CoV-2 keeps changing and is currently dominated by Delta and Omicron variants. Members of the latest Omicron variants, including BA.1, are showing a high level of immune evasion, and Omicron has become a prominent variant circulating globally. In our search for versatile medicinal chemistry scaffolds, we prepared a library of substituted ɑ-aminocyclobutanones from an ɑ-aminocyclobutanone synthon (11). We performed an in silico screen of this actual chemical library as well as other virtual 2-aminocyclobutanone analogs against seven SARS-CoV-2 nonstructural proteins to identify potential drug leads against SARS-CoV-2, and more broadly against coronavirus antiviral targets. Several of these analogs were initially identified as in silico hits against SARS-CoV-2 nonstructural protein 13 (Nsp13) helicase through molecular docking and dynamics simulations. Antiviral activity of the original hits as well as ɑ-aminocyclobutanone analogs that were predicted to bind more tightly to SARS-CoV-2 Nsp13 helicase are reported. We now report cyclobutanone derivatives that exhibit anti-SARS-CoV-2 activity. Furthermore, the Nsp13 helicase enzyme has been the target of relatively few target-based drug discovery efforts, in part due to a very late release of a high-resolution structure accompanied by a limited understanding of its protein biochemistry. In general, antiviral agents initially efficacious against wild-type SARS-CoV-2 strains have lower activities against variants due to heavy viral loads and greater turnover rates, but the inhibitors we are reporting have higher activities against the later variants than the wild-type (10-20X). We speculate this could be due to Nsp13 helicase being a critical bottleneck in faster replication rates of the new variants, so targeting this enzyme affects these variants to an even greater extent. This work calls attention to cyclobutanones as a useful medicinal chemistry scaffold, and the need for additional focus on the discovery of Nsp13 helicase inhibitors to combat the aggressive and immune-evading variants of concern (VOCs).

Pro-Viral and Anti-Viral Roles of the RNA-Binding Protein G3BP1.

Viruses depend on host cellular resources to replicate. Interaction between viral and host proteins is essential for the pathogens to ward off immune responses as well as for virus propagation within the infected cells. While different viruses employ unique strategies to interact with diverse sets of host proteins, the multifunctional RNA-binding protein G3BP1 is one of the common targets for many viruses. G3BP1 controls several key cellular processes, including mRNA stability, translation, and immune responses. G3BP1 also serves as the central hub for the protein-protein and protein-RNA interactions within a class of biomolecular condensates called stress granules (SGs) during stress conditions, including viral infection. Increasing evidence suggests that viruses utilize distinct strategies to modulate G3BP1 function-either by degradation, sequestration, or redistribution-and control the viral life cycle positively and negatively. In this review, we summarize the pro-viral and anti-viral roles of G3BP1 during infection among different viral families.

Nucleolin mediates SARS-CoV-2 replication and viral-induced apoptosis of host cells.

Host-oriented antiviral therapeutics are promising treatment options to combat COVID-19 and its emerging variants. However, relatively little is known about the cellular proteins hijacked by SARS-CoV-2 for its replication. Here we show that SARS-CoV-2 induces expression and cytoplasmic translocation of the nucleolar protein, nucleolin (NCL). NCL interacts with SARS-CoV-2 viral proteins and co-localizes with N-protein in the nucleolus and in stress granules. Knockdown of NCL decreases the stress granule component G3BP1, viral replication and improved survival of infected host cells. NCL mediates viral-induced apoptosis and stress response via p53. SARS-CoV-2 increases NCL expression and nucleolar size and number in lungs of infected hamsters. Inhibition of NCL with the aptamer AS-1411 decreases viral replication and apoptosis of infected cells. These results suggest nucleolin as a suitable target for anti-COVID therapies.

Nsp1 proteins of human coronaviruses HCoV-OC43 and SARS-CoV2 inhibit stress granule formation.

Stress granules (SGs) are cytoplasmic condensates that often form as part of the cellular antiviral response. Despite the growing interest in understanding the interplay between SGs and other biological condensates and viral replication, the role of SG formation during coronavirus infection remains poorly understood. Several proteins from different coronaviruses have been shown to suppress SG formation upon overexpression, but there are only a handful of studies analyzing SG formation in coronavirus-infected cells. To better understand SG inhibition by coronaviruses, we analyzed SG formation during infection with the human common cold coronavirus OC43 (HCoV-OC43) and the pandemic SARS-CoV2. We did not observe SG induction in infected cells and both viruses inhibited eukaryotic translation initiation factor 2α (eIF2α) phosphorylation and SG formation induced by exogenous stress. Furthermore, in SARS-CoV2 infected cells we observed a sharp decrease in the levels of SG-nucleating protein G3BP1. Ectopic overexpression of nucleocapsid (N) and non-structural protein 1 (Nsp1) from both HCoV-OC43 and SARS-CoV2 inhibited SG formation. The Nsp1 proteins of both viruses inhibited arsenite-induced eIF2α phosphorylation, and the Nsp1 of SARS-CoV2 alone was sufficient to cause a decrease in G3BP1 levels. This phenotype was dependent on the depletion of cytoplasmic mRNA mediated by Nsp1 and associated with nuclear accumulation of the SG-nucleating protein TIAR. To test the role of G3BP1 in coronavirus replication, we infected cells overexpressing EGFP-tagged G3BP1 with HCoV-OC43 and observed a significant decrease in virus replication compared to control cells expressing EGFP. The antiviral role of G3BP1 and the existence of multiple SG suppression mechanisms that are conserved between HCoV-OC43 and SARS-CoV2 suggest that SG formation may represent an important antiviral host defense that coronaviruses target to ensure efficient replication.

The Discovery of Novel Ferulic Acid Derivatives Incorporating Substituted Isopropanolamine Moieties as Potential Tobacco Mosaic Virus Helicase Inhibitors.

Target-based drug design, a high-efficiency strategy used to guide the development of novel pesticide candidates, has attracted widespread attention. Herein, various natural-derived ferulic acid derivatives incorporating substituted isopropanolamine moieties were designed to target the tobacco mosaic virus (TMV) helicase. Bioassays demonstrating the optimized A19, A20, A29, and A31 displayed excellent in vivo antiviral curative abilities, affording corresponding EC50 values of 251.1, 336.2, 347.1, and 385.5 µg/mL, which visibly surpassed those of commercial ribavirin (655.0 µg/mL). Moreover, configurational analysis shows that the R-forms of target compounds were more beneficial to aggrandize antiviral profiles. Mechanism studies indicate that R-A19 had a strong affinity (Kd = 5.4 µM) to the TMV helicase and inhibited its ability to hydrolyze ATP (50.61% at 200 µM). Meanwhile, A19 could down-regulate the expression of the TMV helicase gene in the host to attenuate viral replication. These results illustrate the excellent inhibitory activity of A19 towards the TMV helicase. Additionally, docking simulations uncovered that R-A19 formed more hydrogen bonds with the TMV helicase in the binding pocket. Recent studies have unambiguously manifested that these designed derivatives could be considered as promising potential helicase-based inhibitors for plant disease control.

Targeting SARS-CoV-2 nsp13 Helicase and Assessment of Druggability Pockets: Identification of Two Potent Inhibitors by a Multi-Site In Silico Drug Repurposing Approach.

The SARS-CoV-2 non-structural protein 13 (nsp13) helicase is an essential enzyme for viral replication and has been identified as an attractive target for the development of new antiviral drugs. In detail, the helicase catalyzes the unwinding of double-stranded DNA or RNA in a 5' to 3' direction and acts in concert with the replication-transcription complex (nsp7/nsp8/nsp12). In this work, bioinformatics and computational tools allowed us to perform a detailed conservation analysis of the SARS-CoV-2 helicase genome and to further predict the druggable enzyme's binding pockets. Thus, a structure-based virtual screening was used to identify valuable compounds that are capable of recognizing multiple nsp13 pockets. Starting from a database of around 4000 drugs already approved by the Food and Drug Administration (FDA), we chose 14 shared compounds capable of recognizing three out of four sites. Finally, by means of visual inspection analysis and based on their commercial availability, five promising compounds were submitted to in vitro assays. Among them, PF-03715455 was able to block both the unwinding and NTPase activities of nsp13 in a micromolar range.

Stress granules and mTOR are regulated by membrane atg8ylation during lysosomal damage.

We report that lysosomal damage is a hitherto unknown inducer of stress granule (SG) formation and that the process termed membrane atg8ylation coordinates SG formation with mTOR inactivation during lysosomal stress. SGs were induced by lysosome-damaging agents including SARS-CoV-2ORF3a, Mycobacterium tuberculosis, and proteopathic tau. During damage, mammalian ATG8s directly interacted with the core SG proteins NUFIP2 and G3BP1. Atg8ylation was needed for their recruitment to damaged lysosomes independently of SG condensates whereupon NUFIP2 contributed to mTOR inactivation via the Ragulator-RagA/B complex. Thus, cells employ membrane atg8ylation to control and coordinate SG and mTOR responses to lysosomal damage.

Z-RNA and the Flipside of the SARS Nsp13 Helicase: Is There a Role for Flipons in Coronavirus-Induced Pathology?

We present evidence suggesting that the severe acute respiratory syndrome (SARS) coronavirus non-structural protein 13 (Nsp13) modulates the Z-RNA dependent regulated cell death pathways . We show that Z-prone sequences [called flipons] exist in coronavirus and provide a signature (Z-sig) that enables identification of the animal viruses from which the human pathogens arose. We also identify a potential RIP Homology Interaction Motif (RHIM) in the helicase Nsp13 that resembles those present in proteins that initiate Z-RNA-dependent cell death through interactions with the Z-RNA sensor protein ZBP1. These two observations allow us to suggest a model in which Nsp13 down regulates Z-RNA activated innate immunity by two distinct mechanisms. The first involves a novel ATP-independent Z-flipon helicase (flipase) activity in Nsp13 that differs from that of canonical A-RNA helicases. This flipase prevents formation of Z-RNAs that would otherwise activate cell death pathways. The second mechanism likely inhibits the interactions between ZBP1 and the Receptor Interacting Proteins Kinases RIPK1 and RIPK3 by targeting their RHIM domains. Together the described Nsp13 RHIM and flipase activities have the potential to alter the host response to coronaviruses and impact the design of drugs targeting the Nsp13 protein. The Z-sig and RHIM domains may provide a way of identifying previously uncharacterized viruses that are potentially pathogenic for humans.

Punicalagin as an allosteric NSP13 helicase inhibitor potently suppresses SARS-CoV-2 replication in vitro.

The severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) helicase NSP13 plays a conserved role in the replication of coronaviruses and has been identified as an ideal target for the development of antiviral drugs against SARS-CoV-2. Here, we identify a novel NSP13 helicase inhibitor punicalagin (PUG) through high-throughput screening. Surface plasmon resonance (SPR)-based analysis and molecular docking calculation reveal that PUG directly binds NSP13 on the interface of domains 1A and 2A, with a KD value of 21.6 nM. Further biochemical and structural analyses suggest that PUG inhibits NSP13 on ATP hydrolysis and prevents it binding to DNA substrates. Finally, the antiviral studies show that PUG effectively suppresses the SARS-CoV-2 replication in A549-ACE2 and Vero cells, with EC50 values of 347 nM and 196 nM, respectively. Our work demonstrates the potential application of PUG in the treatment of coronavirus disease 2019 (COVID-19) and identifies an allosteric inhibition mechanism for future drug design targeting the viral helicases.

Phillygenin activates PKR/eIF2α pathway and induces stress granule to exert anti-avian infectious bronchitis virus.

The prevalence of avian infectious bronchitis virus (IBV) is still one of causes inducing severe losses of production in the poultry industry worldwide. Vaccination does not completely prevent IBV infection and spread due to immune failure and viral mutations. ForsythiaeFructus and its compounds have been widely used in a lot of prescriptions of the traditional Chinese medicine for a long history, and it is well-known as safety and efficiency in heat-clearing and detoxifying. This study aims to investigate the anti-IBV activity and mechanism of phillygenin. The results showed that phillygenin inhibited IBV replication by disturbing multiple stages of the virus life cycle, including viral adsorption, invasion, internalization, and release in Vero cells. After being treated with 100, 125 and 150 µg/mL phillygenin, the expression of G3BP1 was significantly increased and the phosphorylation of PKR/eIF2α was activated, which increased stress granule, thereby triggering the antiviral response in Vero cells. The anti-virus activity of PHI was decreased when G3BP1 was interfered by si-RNA, and G3BP1 was down-regulated when PKR/eIF2α was interfered by si-RNA. In conclusion, our findings indicate that phillygenin activates PKR/eIF2α pathway and induces stress granule formation to exert anti-IBV, which holds promise to develop into a novel anti-IBV drug. Further study in vivo is needed to explore phillygenin as a potential and effective drug to prevent IB in poultry.

DEAD/H-Box Helicases in Immunity, Inflammation, Cell Differentiation, and Cell Death and Disease.

DEAD/H-box proteins are the largest family of RNA helicases in mammalian genomes, and they are present in all kingdoms of life. Since their discovery in the late 1980s, DEAD/H-box family proteins have been a major focus of study. They have been found to play central roles in RNA metabolism, gene expression, signal transduction, programmed cell death, and the immune response to bacterial and viral infections. Aberrant functions of DEAD/H-box proteins have been implicated in a wide range of human diseases that include cancer, neurodegeneration, and inherited genetic disorders. In this review, we provide a historical context and discuss the molecular functions of DEAD/H-box proteins, highlighting the recent discoveries linking their dysregulation to human diseases. We will also discuss the state of knowledge regarding two specific DEAD/H-box proteins that have critical roles in immune responses and programmed cell death, DDX3X and DDX58, also known as RIG-I. Given their importance in homeostasis and disease, an improved understanding of DEAD/H-box protein biology and protein-protein interactions will be critical for informing strategies to counteract the pathogenesis associated with several human diseases.

SARS-CoV-2 N Protein Antagonizes Stress Granule Assembly and IFN Production by Interacting with G3BPs to Facilitate Viral Replication.

SARS-CoV-2 is the causative agent of the ongoing pandemic of coronavirus disease 2019 (COVID-19) and poses a significant threat to global health. N protein (NP), which is a major pathogenic protein among betacoronaviruses, binds to the viral RNA genome to allow viral genome packaging and viral particle release. Recent studies showed that NP antagonizes interferon (IFN) induction and mediates phase separation. Using live SARS-CoV-2 viruses, this study provides solid evidence showing that SARS-CoV-2 NP associates with G3BP1 and G3BP2 in vitro and in vivo. NPSARS-CoV-2 could efficiently suppress G3BP-mediated SG formation and potentiate viral infection by overcoming G3BP1-mediated antiviral innate immunity. G3BP1 conditional knockout mice (g3bp1fl/fL, Sftpc-Cre) exhibit significantly higher lung viral loads after SARS-CoV-2 infection than wild-type mice. Our findings contribute to the growing body of knowledge regarding the pathogenicity of NPSARS-CoV-2 and provide insight into new therapeutics targeting NPSARS-CoV-2. IMPORTANCE In this study, by in vitro assay and live SARS-CoV-2 virus infection, we provide solid evidence that the SARS-CoV-2 NP associates with G3BP1 and G3BP2 in vitro and in vivo. NPSARS-CoV-2 could efficiently suppress G3BP-mediated SG formation and potentiate viral infection by overcoming antiviral innate immunity mediated by G3BP1 in A549 cell lines and G3BP1 conditional knockout mice (g3bp1-cKO) mice, which provide in-depth evidence showing the mechanism underlying NP-related SARS-CoV-2 pathogenesis through G3BPs.

The SARS-CoV-2 helicase as a target for antiviral therapy: Identification of potential small molecule inhibitors by in silico modelling.

Although vaccines that provide protection against severe illness from coronavirus disease (COVID-19) have been made available, emerging variant strains of severe acute respiratory syndrome 2 coronavirus 2 (SARS-CoV-2) are of concern. A different research direction involves investigation of antiviral therapeutics. In addition to structural proteins, the SARS-CoV-2 non-structural proteins are of interest and this includes the helicase (nsp13). In this study, an initial screen of 300 ligands was performed to identify potential inhibitors of the SARS-CoV-2 nsp13 examining the nucleoside triphosphatase site (NTPase activity) as the target region. The antiviral activity of polyphenols has been previously reported in the literature and as a result, the phenolic compounds and fatty acids from the OliveNet™ library were utilised. Synthetic compounds with antimicrobial and anti-inflammatory properties were also selected. The structures of the SARS-CoV and MERS-CoV helicases, as well as the human RECQ-like DNA helicase, DHX9 helicase, PcrA helicase, hepatitis C NS3 helicase, and mouse Dna2 nuclease-helicase were used for comparison. As expected, sequence and structural homology between the various species was evident. A number of broad-spectrum and well-known inhibitors interacted with the NTPase active site highlighting the need to potentially identify more specific inhibitors for SARS-CoV-2. Acetylcysteine, clavulanic acid and homovanillic acid were identified as potential lead compounds for the SARS-CoV-2 helicase. Molecular dynamics simulations were performed with the leads bound to the SARS-CoV-2 helicase for 200 ns in triplicate, with favourable binding free energies to the NTPase site. Given their availability, further exploration of their potential inhibitory activity could be considered.

SARS-CoV-2 Nucleocapsid Protein Targets a Conserved Surface Groove of the NTF2-like Domain of G3BP1.

Stress granule (SG) formation mediated by Ras GTPase-activating protein-binding protein 1 (G3BP1) constitutes a key obstacle for viral replication, which makes G3BP1 a frequent target for viruses. For instance, the SARS-CoV-2 nucleocapsid (N) protein interacts with G3BP1 directly to suppress SG assembly and promote viral production. However, the molecular basis for the SARS-CoV-2 N - G3BP1 interaction remains elusive. Here we report biochemical and structural analyses of the SARS-CoV-2 N - G3BP1 interaction, revealing differential contributions of various regions of SARS-CoV-2 N to G3BP1 binding. The crystal structure of the NTF2-like domain of G3BP1 (G3BP1NTF2) in complex with a peptide derived from SARS-CoV-2 N (residues 1-25, N1-25) reveals that SARS-CoV-2 N1-25 occupies a conserved surface groove of G3BP1NTF2 via surface complementarity. We show that a φ-x-F (φ, hydrophobic residue) motif constitutes the primary determinant for G3BP1NTF2-targeting proteins, while the flanking sequence underpins diverse secondary interactions. We demonstrate that mutation of key interaction residues of the SARS-CoV-2 N1-25 - G3BP1NTF2 complex leads to disruption of the SARS-CoV-2 N - G3BP1 interaction in vitro. Together, these results provide a molecular basis of the strain-specific interaction between SARS-CoV-2 N and G3BP1, which has important implications for the development of novel therapeutic strategies against SARS-CoV-2 infection.

Discovery of Zafirlukast as a novel SARS-CoV-2 helicase inhibitor using in silico modelling and a FRET-based assay.

The coronavirus helicase is an essential enzyme required for viral replication/transcription pathways. Structural studies revealed a sulphate moiety that interacts with key residues within the nucleotide-binding site of the helicase. Compounds with a sulphoxide or a sulphone moiety could interfere with these interactions and consequently inhibit the enzyme. The molecular operating environment (MOE) was used to dock 189 sulphoxide and sulphone-containing FDA-approved compounds to the nucleotide-binding site. Zafirlukast, a leukotriene receptor antagonist used to treat chronic asthma, achieved the lowest docking score at -8.75 kcals/mol. The inhibitory effect of the compounds on the SARS-CoV-2 helicase dsDNA unwinding activity was tested by a FRET-based assay. Zafirlukast was the only compound to inhibit the enzyme (IC50 = 16.3 µM). The treatment of Vero E6 cells with 25 µM zafirlukast prior to SARS-CoV-2 infection decreased the cytopathic effects of SARS-CoV-2 significantly. These results suggest that zafirlukast alleviates SARS-CoV-2 pathogenicity by inhibiting the viral helicase and impairing the viral replication/transcription pathway. Zafirlukast could be clinically developed as a new antiviral treatment for SARS-CoV-2 and other coronavirus diseases. This discovery is based on molecular modelling, in vitro inhibition of the SARS-CoV helicase activity and cell-based SARS-CoV-2 viral replication.