Nanoluciferase complementation-based bioreporter reveals the importance of N-linked glycosylation of SARS-CoV-2 S for viral entry.
Angiotensin-Converting Enzyme 2
/ antagonists & inhibitors
Antibodies, Neutralizing
/ pharmacology
Asparagine
/ chemistry
Binding Sites
Biological Assay
COVID-19
/ diagnosis
Genes, Reporter
Glycosylation
/ drug effects
HEK293 Cells
Host-Pathogen Interactions
/ drug effects
Humans
Lectins
/ pharmacology
Luciferases
/ genetics
Luminescent Measurements
Protein Binding
Protein Interaction Domains and Motifs
Protein Structure, Secondary
Receptors, Virus
/ antagonists & inhibitors
SARS-CoV-2
/ drug effects
Spike Glycoprotein, Coronavirus
/ antagonists & inhibitors
Virus Internalization
/ drug effects
COVID-19 Drug Treatment
2019-nCoV
COVID-19
SARS-CoV-2
bioluminescence
bioreporter
coronavirus
high-throughput screening
viral entry
Journal
Molecular therapy : the journal of the American Society of Gene Therapy
ISSN: 1525-0024
Titre abrégé: Mol Ther
Pays: United States
ID NLM: 100890581
Informations de publication
Date de publication:
02 06 2021
02 06 2021
Historique:
received:
17
11
2020
revised:
19
01
2021
accepted:
04
02
2021
pubmed:
13
2
2021
medline:
22
6
2021
entrez:
12
2
2021
Statut:
ppublish
Résumé
The ongoing COVID-19 pandemic has highlighted the immediate need for the development of antiviral therapeutics targeting different stages of the SARS-CoV-2 life cycle. We developed a bioluminescence-based bioreporter to interrogate the interaction between the SARS-CoV-2 viral spike (S) protein and its host entry receptor, angiotensin-converting enzyme 2 (ACE2). The bioreporter assay is based on a nanoluciferase complementation reporter, composed of two subunits, large BiT and small BiT, fused to the S receptor-binding domain (RBD) of the SARS-CoV-2 S protein and ACE2 ectodomain, respectively. Using this bioreporter, we uncovered critical host and viral determinants of the interaction, including a role for glycosylation of asparagine residues within the RBD in mediating successful viral entry. We also demonstrate the importance of N-linked glycosylation to the RBD's antigenicity and immunogenicity. Our study demonstrates the versatility of our bioreporter in mapping key residues mediating viral entry as well as screening inhibitors of the ACE2-RBD interaction. Our findings point toward targeting RBD glycosylation for therapeutic and vaccine strategies against SARS-CoV-2.
Identifiants
pubmed: 33578036
pii: S1525-0016(21)00074-5
doi: 10.1016/j.ymthe.2021.02.007
pmc: PMC7872859
pii:
doi:
Substances chimiques
Antibodies, Neutralizing
0
Lectins
0
Receptors, Virus
0
Spike Glycoprotein, Coronavirus
0
spike protein, SARS-CoV-2
0
Asparagine
7006-34-0
Luciferases
EC 1.13.12.-
ACE2 protein, human
EC 3.4.17.23
Angiotensin-Converting Enzyme 2
EC 3.4.17.23
Types de publication
Journal Article
Research Support, Non-U.S. Gov't
Langues
eng
Sous-ensembles de citation
IM
Pagination
1984-2000Informations de copyright
Copyright © 2021. Published by Elsevier Inc.
Déclaration de conflit d'intérêts
Declaration of interests The authors declare no competing interests.
Références
Science. 2020 Mar 13;367(6483):1260-1263
pubmed: 32075877
Anal Chem. 2003 Apr 1;75(7):1584-9
pubmed: 12705589
Virology. 2006 Jun 20;350(1):15-25
pubmed: 16510163
Nat Methods. 2006 Dec;3(12):977-9
pubmed: 17099704
Sci Immunol. 2020 Jun 11;5(48):
pubmed: 32527802
EMBO J. 2005 Apr 20;24(8):1634-43
pubmed: 15791205
Science. 2009 Oct 9;326(5950):285-9
pubmed: 19729618
Cancers (Basel). 2019 Oct 19;11(10):
pubmed: 31635084
Cell Mol Immunol. 2020 Jun;17(6):613-620
pubmed: 32203189
Cell. 2020 Sep 3;182(5):1295-1310.e20
pubmed: 32841599
Glycobiology. 2020 Dec 9;30(12):981-988
pubmed: 32363391
Cell Host Microbe. 2020 Oct 7;28(4):586-601.e6
pubmed: 32841605
Cell. 2020 May 14;181(4):905-913.e7
pubmed: 32333836
Mol Syst Biol. 2011 Oct 11;7:539
pubmed: 21988835
Nature. 2020 Jul;583(7815):290-295
pubmed: 32422645
FASEB J. 2019 Nov;33(11):12487-12499
pubmed: 31431076
Nature. 2020 Oct;586(7830):567-571
pubmed: 32756549
Nat Commun. 2020 May 4;11(1):2251
pubmed: 32366817
Cell. 2020 Apr 16;181(2):281-292.e6
pubmed: 32155444
J Biol Chem. 2004 Jan 30;279(5):3197-201
pubmed: 14670965
Membranes (Basel). 2020 Aug 30;10(9):
pubmed: 32872641
Antiviral Res. 2007 Sep;75(3):179-87
pubmed: 17428553
Nat Biotechnol. 2020 Sep;38(9):1073-1078
pubmed: 32704169
ACS Chem Biol. 2016 Feb 19;11(2):400-8
pubmed: 26569370
Adv Exp Med Biol. 2018;1062:277-301
pubmed: 29845540
Cell Host Microbe. 2020 Sep 9;28(3):475-485.e5
pubmed: 32735849
Biosens Bioelectron. 2013 Mar 15;41:642-8
pubmed: 23122229
Nature. 2020 Oct;586(7830):516-527
pubmed: 32967006
Science. 2005 Sep 16;309(5742):1864-8
pubmed: 16166518
Nat Commun. 2018 Mar 13;9(1):1061
pubmed: 29535383
Anal Bioanal Chem. 2014 Sep;406(23):5541-60
pubmed: 25002334
Antimicrob Agents Chemother. 2018 Nov 26;62(12):
pubmed: 30224531
Proc Natl Acad Sci U S A. 2020 Jan 28;117(4):2122-2132
pubmed: 31932446
Cell. 2020 Sep 3;182(5):1284-1294.e9
pubmed: 32730807
Nature. 2020 Dec;588(7839):682-687
pubmed: 33045718
PLoS Pathog. 2013 Oct;9(10):e1003738
pubmed: 24204277
Science. 2020 Jul 17;369(6501):330-333
pubmed: 32366695
ACS Chem Biol. 2012 Nov 16;7(11):1848-57
pubmed: 22894855
Cell. 2020 Nov 12;183(4):1024-1042.e21
pubmed: 32991844
J Virol Methods. 2011 Sep;176(1-2):108-11
pubmed: 21645548
Cell. 2020 Apr 16;181(2):271-280.e8
pubmed: 32142651
Viruses. 2020 May 06;12(5):
pubmed: 32384820
Nature. 2020 May;581(7807):215-220
pubmed: 32225176