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Construction of SARS-CoV-2 spike-pseudotyped retroviral vector inducing syncytia formation

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is handled in biosafety level 3 (BSL-3) facilities, whereas the antiviral screening of pseudotype virus is conducted in BSL-2 facilities. In this study, we developed a SARS-CoV-2 spike-pseudotyped virus based on a semi-replication-competen...

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Autores principales: Lee, Se Yeong, Kim, Do Woo, Jung, Yong Tae
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Springer US 2022
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8942147/
https://www.ncbi.nlm.nih.gov/pubmed/35322356
http://dx.doi.org/10.1007/s11262-022-01890-z
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author Lee, Se Yeong
Kim, Do Woo
Jung, Yong Tae
author_facet Lee, Se Yeong
Kim, Do Woo
Jung, Yong Tae
author_sort Lee, Se Yeong
collection PubMed
description Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is handled in biosafety level 3 (BSL-3) facilities, whereas the antiviral screening of pseudotype virus is conducted in BSL-2 facilities. In this study, we developed a SARS-CoV-2 spike-pseudotyped virus based on a semi-replication-competent retroviral (s-RCR) vector system. The s-RCR vector system was divided into two packageable vectors, each with gag-pol and env genes. For env vector construction, SARS-CoV-2 SΔ19 env was inserted into the pCLXSN-IRES-EGFP retroviral vector to generate pCLXSN-SΔ19 env-EGFP. When pCLXSN-gag-pol and pCLXSN-SΔ19env-EGFP were co-transfected into HEK293 T cells to generate an s-RCR virus, titers of the s-RCR virus were consistently low in this transient transfection system (1 × 10(4) TU/mL). However, a three-fold higher amounts of MLV-based SARS-CoV-2 pseudotyped viruses (3 × 10(4) TU/mL) were released from stable producer cells, and the spike proteins induced syncytia formation in HEK293-hACE2 cells. Furthermore, s-RCR stocks collected from stable producer cells induced more substantial syncytia formation in the Vero E6-TMPRSS2 cell line than in the Vero E6 cell line. Therefore, a combination of the s-RCR vector and the two cell lines (HEK293-hACE2 or Vero E6-TMPRSS2) that induce syncytia formation can be useful for the rapid screening of novel fusion inhibitor drugs.
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spelling pubmed-89421472022-03-24 Construction of SARS-CoV-2 spike-pseudotyped retroviral vector inducing syncytia formation Lee, Se Yeong Kim, Do Woo Jung, Yong Tae Virus Genes Original Paper Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is handled in biosafety level 3 (BSL-3) facilities, whereas the antiviral screening of pseudotype virus is conducted in BSL-2 facilities. In this study, we developed a SARS-CoV-2 spike-pseudotyped virus based on a semi-replication-competent retroviral (s-RCR) vector system. The s-RCR vector system was divided into two packageable vectors, each with gag-pol and env genes. For env vector construction, SARS-CoV-2 SΔ19 env was inserted into the pCLXSN-IRES-EGFP retroviral vector to generate pCLXSN-SΔ19 env-EGFP. When pCLXSN-gag-pol and pCLXSN-SΔ19env-EGFP were co-transfected into HEK293 T cells to generate an s-RCR virus, titers of the s-RCR virus were consistently low in this transient transfection system (1 × 10(4) TU/mL). However, a three-fold higher amounts of MLV-based SARS-CoV-2 pseudotyped viruses (3 × 10(4) TU/mL) were released from stable producer cells, and the spike proteins induced syncytia formation in HEK293-hACE2 cells. Furthermore, s-RCR stocks collected from stable producer cells induced more substantial syncytia formation in the Vero E6-TMPRSS2 cell line than in the Vero E6 cell line. Therefore, a combination of the s-RCR vector and the two cell lines (HEK293-hACE2 or Vero E6-TMPRSS2) that induce syncytia formation can be useful for the rapid screening of novel fusion inhibitor drugs. Springer US 2022-03-23 2022 /pmc/articles/PMC8942147/ /pubmed/35322356 http://dx.doi.org/10.1007/s11262-022-01890-z Text en © The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature 2022 This article is made available via the PMC Open Access Subset for unrestricted research re-use and secondary analysis in any form or by any means with acknowledgement of the original source. These permissions are granted for the duration of the World Health Organization (WHO) declaration of COVID-19 as a global pandemic.
spellingShingle Original Paper
Lee, Se Yeong
Kim, Do Woo
Jung, Yong Tae
Construction of SARS-CoV-2 spike-pseudotyped retroviral vector inducing syncytia formation
title Construction of SARS-CoV-2 spike-pseudotyped retroviral vector inducing syncytia formation
title_full Construction of SARS-CoV-2 spike-pseudotyped retroviral vector inducing syncytia formation
title_fullStr Construction of SARS-CoV-2 spike-pseudotyped retroviral vector inducing syncytia formation
title_full_unstemmed Construction of SARS-CoV-2 spike-pseudotyped retroviral vector inducing syncytia formation
title_short Construction of SARS-CoV-2 spike-pseudotyped retroviral vector inducing syncytia formation
title_sort construction of sars-cov-2 spike-pseudotyped retroviral vector inducing syncytia formation
topic Original Paper
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8942147/
https://www.ncbi.nlm.nih.gov/pubmed/35322356
http://dx.doi.org/10.1007/s11262-022-01890-z
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