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

https://doi.org/10.1016/j.jviromet.2021.114221Get rights and content

Highlights

  • The SARS2pp described here was based on a lentiviral vector bearing dual-reporter genes eGFP and Luc2.

  • Under optimized conditions, the SARS2pp was successfully produced with high titers above 105-106 TU/mL in most cases.

  • This SARS2pp infection system showed a good linear relationship between MOI 2-0.0002.

  • The kidney, liver, and small intestine-derived cell lines showed different susceptibility to SARSpp and SARS2pp.

Abstract

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.

Abbreviations

SARS2pp
SARS-CoV-2 pseudovirus particles
SARSpp
SARS-CoV pseudovirus particles
VSVpp
vesicular stomatitis virus pseudovirus particles
S
SARS-CoV-2 full-length spike protein

Keywords

SARS-CoV-2
Pseudovirus
Viral entry inhibitors
Susceptible cell lines

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1

These authors contributed equally to this work.

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