Generation and characterization of SARS-CoV-2 pseudoviruses

Abstract

BACKGROUND: Pseudovirus technology is a versatile and valuable tool for both fundamental and applied virological research. Pseudotyped viruses provide the same cell entry mechanism as SARS-CoV-2 and are widely used to investigate the virus entry mechanism, cell tropism, and virus neutralization assays.

AIM: The aim of the work is to obtain pseudotyped SARS-CoV-2 viruses and evaluate their transducing activity.

MATERIALS AND METHODS: Using genetic engineering methods, a genetic construct carrying the SARS-CoV-2 glycoprotein S gene was obtained, as well as the pLenti-Luc-GFP reporter plasmid encoding the green fluorescent protein (GFP) and firefly luciferase genes. Pseudovirus particles were generated by transfection of eukaryotic cells. The transducing activity of pseudoviral particles displaying SARS-CoV-2 glycoprotein S on their surface was studied using HEK293, HEK293-hACE2, and HEK293-hACE2-TMPRSS2 (t) cell cultures.

RESULTS: Based on the second-generation lentiviral platform, pseudoviruses were obtained that exhibit SARS-CoV-2 S glycoprotein on their surface. It was found that the pseudoviruses penetrate more efficiently into HEK293-hACE2-TMPRSS2 cells than into HEK293-hACE2. Pseudoviruses have been shown to be sensitive to neutralization by recombinant monoclonal antibodies that interact with the receptor-binding domain (RBD) of the SARS-CoV-2 S glycoprotein.

CONCLUSIONS: The pseudoviruses can be used both to search for antiviral drugs that would be able to block the penetration of SARS-CoV-2 into the target cell, and to evaluate the effectiveness of the developed monoclonal antibodies and vaccines against SARS-CoV-2.

Keywords

SARS-CoV-2 / pseudoviruses / neutralization assay / S glycoprotein

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