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Inhibition of SARS-CoV-2 viral entry upon blocking N- and O-glycan elaboration.

ABSTRACT: The Spike protein of SARS-CoV-2, its receptor-binding domain (RBD), and its primary receptor ACE2 are extensively glycosylated. The impact of this post-translational modification on viral entry is yet unestablished. We expressed different glycoforms of the Spike-protein and ACE2 in CRISPR-Cas9 glycoengineered cells, and developed corresponding SARS-CoV-2 pseudovirus. We observed that N- and O-glycans had only minor contribution to Spike-ACE2 binding. However, these carbohydrates played a major role in regulating viral entry. Blocking N-glycan biosynthesis at the oligomannose stage using both genetic approaches and the small molecule kifunensine dramatically reduced viral entry into ACE2 expressing HEK293T cells. Blocking O-glycan elaboration also partially blocked viral entry. Mechanistic studies suggest multiple roles for glycans during viral entry. Among them, inhibition of N-glycan biosynthesis enhanced Spike-protein proteolysis. This could reduce RBD presentation on virus, lowering binding to host ACE2 and decreasing viral entry. Overall, chemical inhibitors of glycosylation may be evaluated for COVID-19.

SUBMITTER: Yang Q

PROVIDER: S-EPMC7685702 | biostudies-literature | 2020 Oct

REPOSITORIES: biostudies-literature

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Inhibition of SARS-CoV-2 viral entry upon blocking N- and O-glycan elaboration.

Yang Qi Q Hughes Thomas A TA Kelkar Anju A Yu Xinheng X Cheng Kai K Park Sheldon S Huang Wei-Chiao WC Lovell Jonathan F JF Neelamegham Sriram S

eLife 20201026

The Spike protein of SARS-CoV-2, its receptor-binding domain (RBD), and its primary receptor ACE2 are extensively glycosylated. The impact of this post-translational modification on viral entry is yet unestablished. We expressed different glycoforms of the Spike-protein and ACE2 in CRISPR-Cas9 glycoengineered cells, and developed corresponding SARS-CoV-2 pseudovirus. We observed that N- and O-glycans had only minor contribution to Spike-ACE2 binding. However, these carbohydrates played a major r ...[more]

PMID: 33103998

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Project description:BackgroundMyricetin (3,5,7-trihydroxy-2-(3,4,5-tri hydroxyphenyl)-4-benzopyrone) is a common flavonol extracted from many natural plants and Chinese herb medicines and has been demonstrated to have multiple pharmacological activities, such as anti-microbial, anti-thrombotic, neuroprotective, and anti-inflammatory effects. Previously, myricetin was reported to target Mpro and 3CL-Pro-enzymatic activity to SARS-CoV-2. However, the protective value of myricetin on SARS-Cov-2 infection through viral-entry facilitators has not yet been comprehensively understood.PurposeThe aim of the current study was to evaluate the pharmacological efficacy and the mechanisms of action of myricetin against SARS-CoV-2 infection both in vitro and in vivo.MethodsThe inhibitory effects of myricetin on SARS-CoV-2 infection and replication were assessed on Vero E6 cells. Molecular docking analysis and bilayer interferometry (BLI) assays, immunocytochemistry (ICC), and pseudoviruses assays were performed to evaluate the roles of myricetin in the intermolecular interaction between the receptor binding domain (RBD) of the SARS-CoV-2 spike (S) protein and angiotensin-converting enzyme 2 (ACE2). The anti-inflammatory potency and mechanisms of myricetin were examined in THP1 macrophages in vitro, as well as in carrageenan-induced paw edema, delayed-type hypersensitivity (DTH) induced auricle edema, and LPS-induced acute lung injury (ALI) animal models.ResultsThe results showed that myricetin was able to inhibit binding between the RBD of the SARS-CoV-2 S protein and ACE2 through molecular docking analysis and BLI assay, demonstrating its potential as a viral-entry facilitator blocker. Myricetin could also significantly inhibit SASR-CoV-2 infection and replication in Vero E6 cells (EC50 55.18 μM), which was further validated with pseudoviruses containing the RBD (wild-type, N501Y, N439K, Y453F) and an S1 glycoprotein mutant (S-D614G). Moreover, myricetin exhibited a marked suppressive action on the receptor-interacting serine/threonine protein kinase 1 (RIPK1)-driven inflammation and NF-kappa B signaling in THP1 macrophages. In animal model studies, myricetin notably ameliorated carrageenan-induced paw edema in rats, DTH induced auricle edema in mice, and LPS-induced ALI in mice.ConclusionOur findings showed that myricetin inhibited HCoV-229E and SARS-CoV-2 replication in vitro, blocked SARS-CoV-2 virus entry facilitators and relieved inflammation through the RIPK1/NF-κB pathway, suggesting that this flavonol has the potential to be developed as a therapeutic agent against COVID-19.

Dataset Information

Inhibition of SARS-CoV-2 viral entry upon blocking N- and O-glycan elaboration.

Publications

Inhibition of SARS-CoV-2 viral entry upon blocking N- and O-glycan elaboration.

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