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Tytuł pozycji:

An optimized and robust SARS-CoV-2 pseudovirus system for viral entry research.

Tytuł:
An optimized and robust SARS-CoV-2 pseudovirus system for viral entry research.
Autorzy:
Yang P; College of Life Sciences, Henan Normal University, Xinxiang 453007, China; Beijing Institute of Radiation Medicine, Beijing 100850, China.
Yang Y; Beijing Institute of Radiation Medicine, Beijing 100850, China.
Wu Y; Beijing Institute of Radiation Medicine, Beijing 100850, China.
Huang C; Beijing Institute of Radiation Medicine, Beijing 100850, China; Cooperative Innovation Center for Molecular Target New Drug Study, University of South China, Hengyang 421001, China.
Ding Y; Beijing Institute of Radiation Medicine, Beijing 100850, China.
Wang X; Beijing Institute of Radiation Medicine, Beijing 100850, China. Electronic address: .
Wang S; College of Life Sciences, Henan Normal University, Xinxiang 453007, China; Beijing Institute of Radiation Medicine, Beijing 100850, China. Electronic address: .
Źródło:
Journal of virological methods [J Virol Methods] 2021 Sep; Vol. 295, pp. 114221. Date of Electronic Publication: 2021 Jun 25.
Typ publikacji:
Journal Article; Research Support, Non-U.S. Gov't
Język:
English
Imprint Name(s):
Original Publication: [Amsterdam] : Elsevier/North-Holland Biomedical Press, 1980-
MeSH Terms:
Virus Internalization*/drug effects
Neutralization Tests/*methods
SARS-CoV-2/*physiology
Angiotensin-Converting Enzyme 2/pharmacology ; Animals ; Antibodies, Neutralizing/pharmacology ; Antiviral Agents/pharmacology ; Cell Line ; Genes, Reporter ; Green Fluorescent Proteins/genetics ; Green Fluorescent Proteins/metabolism ; Humans ; Lentivirus/genetics ; Luciferases, Firefly/genetics ; Luciferases, Firefly/metabolism ; Recombinant Proteins/pharmacology ; SARS-CoV-2/drug effects ; SARS-CoV-2/genetics ; SARS-CoV-2/immunology ; Spike Glycoprotein, Coronavirus/genetics ; Spike Glycoprotein, Coronavirus/metabolism ; Virion
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Contributed Indexing:
Keywords: Pseudovirus; SARS-CoV-2; Susceptible cell lines; Viral entry inhibitors
Substance Nomenclature:
0 (Antibodies, Neutralizing)
0 (Antiviral Agents)
0 (Recombinant Proteins)
0 (Spike Glycoprotein, Coronavirus)
0 (spike protein, SARS-CoV-2)
147336-22-9 (Green Fluorescent Proteins)
EC 1.13.12.7 (Luciferases, Firefly)
EC 3.4.17.23 (ACE2 protein, human)
EC 3.4.17.23 (Angiotensin-Converting Enzyme 2)
Entry Date(s):
Date Created: 20210628 Date Completed: 20210723 Latest Revision: 20221217
Update Code:
20240105
PubMed Central ID:
PMC8233049
DOI:
10.1016/j.jviromet.2021.114221
PMID:
34182038
Czasopismo naukowe
SARS-CoV-2 is the culprit causing Coronavirus Disease 2019 (COVID-19). For the study of SARS-CoV-2 infection in a BSL-2 laboratory, a SARS-CoV-2 pseudovirus particle (SARS2pp) production and infection system was constructed by using a lentiviral vector bearing dual-reporter genes eGFP and firefly luciferase (Luc2) for easy observation and analysis. Comparison of SARS2pp different production conditions revealed that the pseudovirus titer could be greatly improved by: 1) removing the last 19 amino acids of the spike protein and replacing the signal peptide with the mouse Igk signal sequence; 2) expressing the spike protein using CMV promoter other than CAG (a hybrid promoter consisting of a CMV enhancer, beta-actin promoter, splice donor, and a beta-globin splice acceptor); 3) screening better optimized spike protein sequences for SARS2pp production; and 4) adding 1 % BSA in the SARS2pp production medium. For infection, this SARS2pp system showed a good linear relationship between MOI 2-0.0002 and then was successfully used to evaluate SARS-CoV-2 infection inhibitors including recombinant human ACE2 proteins and SARS-CoV-2 neutralizing antibodies. The kidney, liver and small intestine-derived cell lines were also found to show different susceptibility to SARSpp and SARS2pp. Given its robustness and good performance, it is believed that this pseudovirus particle production and infection system will greatly promote future research for SARS-CoV-2 entry mechanisms and inhibitors and can be easily applied to study new emerging SARS-CoV-2 variants.
(Copyright © 2021 Elsevier B.V. All rights reserved.)

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