tetano
Editor, Senior Moderator
J Virol
. 2021 Mar 16;JVI.00044-21.
doi: 10.1128/JVI.00044-21. Online ahead of print.
Deletion of the SARS-CoV-2 Spike Cytoplasmic Tail Increases Infectivity in Pseudovirus Neutralization Assays
Jingyou Yu[SUP] 1 [/SUP], Zhenfeng Li[SUP] 1 [/SUP], Xuan He[SUP] 1 [/SUP], Makda S Gebre[SUP] 1 2 [/SUP], Esther A Bondzie[SUP] 1 [/SUP], Huahua Wan[SUP] 1 [/SUP], Catherine Jacob-Dolan[SUP] 1 2 3 [/SUP], David R Martinez[SUP] 4 [/SUP], Joseph P Nkolola[SUP] 1 [/SUP], Ralph S Baric[SUP] 4 [/SUP], Dan H Barouch[SUP] 5 2 3 [/SUP]
Affiliations
Abstract
Pseudotyped viruses are valuable tools for studying virulent or lethal viral pathogens that need to be handled in biosafety level 3 (BSL-3) or higher facilities. With the explosive spread of the coronavirus disease 2019 (COVID-19) pandemic, the establishment of a BSL-2 adapted SARS-CoV-2 pseudovirus neutralization assay is needed to facilitate the development of countermeasures. Here we describe an approach to generate a single-round lentiviral vector-based SARS-CoV-2 pseudovirus, which produced a signal more than 2 logs above background. Specifically, a SARS-CoV-2 spike variant with a cytoplasmic tail deletion of 13 amino acids, termed S?CT13, conferred enhanced spike incorporation into pseudovirions and increased viral entry into cells as compared with full-length spike (S). We further compared S and S?CT13 in terms of their sensitivity to vaccine sera, purified convalescent IgG, hACE2-mIgG, and the virus entry inhibitor BafA1. We developed a S?CT13-based pseudovirus neutralization assay and defined key assay characteristics, including linearity, limit of detection, and intra- and intermediate-assay precision. Our data demonstrate that the S?CT13-based pseudovirus shows enhanced infectivity in target cells, which will facilitate the assessment of humoral immunity to SARS-CoV-2 infection, antibody therapeutics, and vaccination. This pseudovirus neutralization assay can also be readily adapted to SARS-CoV-2 variants that emerge.IMPORTANCESARS-CoV-2 is the etiologic agent of the COVID-19 pandemic. The development of a high throughput pseudovirus neutralization assay is critical for the development of vaccines and immune-based therapeutics. In this study, we show that deletion of the cytoplasmic tail of the SARS-CoV-2 spike leads to pseudoviruses with enhanced infectivity. This S?CT13-based pseudovirus neutralization assay should be broadly useful for the field.
. 2021 Mar 16;JVI.00044-21.
doi: 10.1128/JVI.00044-21. Online ahead of print.
Deletion of the SARS-CoV-2 Spike Cytoplasmic Tail Increases Infectivity in Pseudovirus Neutralization Assays
Jingyou Yu[SUP] 1 [/SUP], Zhenfeng Li[SUP] 1 [/SUP], Xuan He[SUP] 1 [/SUP], Makda S Gebre[SUP] 1 2 [/SUP], Esther A Bondzie[SUP] 1 [/SUP], Huahua Wan[SUP] 1 [/SUP], Catherine Jacob-Dolan[SUP] 1 2 3 [/SUP], David R Martinez[SUP] 4 [/SUP], Joseph P Nkolola[SUP] 1 [/SUP], Ralph S Baric[SUP] 4 [/SUP], Dan H Barouch[SUP] 5 2 3 [/SUP]
Affiliations
- PMID: 33727331
- DOI: 10.1128/JVI.00044-21
Abstract
Pseudotyped viruses are valuable tools for studying virulent or lethal viral pathogens that need to be handled in biosafety level 3 (BSL-3) or higher facilities. With the explosive spread of the coronavirus disease 2019 (COVID-19) pandemic, the establishment of a BSL-2 adapted SARS-CoV-2 pseudovirus neutralization assay is needed to facilitate the development of countermeasures. Here we describe an approach to generate a single-round lentiviral vector-based SARS-CoV-2 pseudovirus, which produced a signal more than 2 logs above background. Specifically, a SARS-CoV-2 spike variant with a cytoplasmic tail deletion of 13 amino acids, termed S?CT13, conferred enhanced spike incorporation into pseudovirions and increased viral entry into cells as compared with full-length spike (S). We further compared S and S?CT13 in terms of their sensitivity to vaccine sera, purified convalescent IgG, hACE2-mIgG, and the virus entry inhibitor BafA1. We developed a S?CT13-based pseudovirus neutralization assay and defined key assay characteristics, including linearity, limit of detection, and intra- and intermediate-assay precision. Our data demonstrate that the S?CT13-based pseudovirus shows enhanced infectivity in target cells, which will facilitate the assessment of humoral immunity to SARS-CoV-2 infection, antibody therapeutics, and vaccination. This pseudovirus neutralization assay can also be readily adapted to SARS-CoV-2 variants that emerge.IMPORTANCESARS-CoV-2 is the etiologic agent of the COVID-19 pandemic. The development of a high throughput pseudovirus neutralization assay is critical for the development of vaccines and immune-based therapeutics. In this study, we show that deletion of the cytoplasmic tail of the SARS-CoV-2 spike leads to pseudoviruses with enhanced infectivity. This S?CT13-based pseudovirus neutralization assay should be broadly useful for the field.