# Development and Application of a Pseudovirus-Based Assay for Modelling SARS-CoV-2 Spike Protein Mediated Drug Screening

## TL;DR

This paper describes a new lab-based method using pseudoviruses to test potential drugs against SARS-CoV-2, focusing on how the virus enters human cells.

## Contribution

The study introduces a novel pseudovirus-based assay using a modified cell line for high-efficiency screening of SARS-CoV-2 entry inhibitors.

## Key findings

- The H1299-hACE2 cell line showed significantly higher infection rates with spike-pseudotyped lentiviruses compared to parental cells.
- The assay was used to test derivatives of a potential spike protein inhibitor with moderate antiviral activity.
- The system supports the study of multiple SARS-CoV-2 variants in a unified experimental setting.

## Abstract

Requirements for novel effective antiviral agents against SARS-CoV-2 emphasizes the importance of robust in vitro screening platforms. We developed a test system based on spike-pseudotyped lentiviruses, carrying either luc+ or EGFP reporter genes as a payload, and a human non-small cell lung carcinoma (NSCLC) cell line, overexpressing ACE2 (H1299-hACE2). The cell origin makes our system resemble lung epithelium infection. Transmission electron microscopy confirmed that the spike glycoproteins on the pseudotyped lentiviral particles resemble native SARS-CoV-2 spike glycoproteins, thus validating their use in inhibitor screening. H1299-hACE2 cells showed significantly higher infection rate (p < 0.005) with spike-pseudotyped lentiviruses compared to parental H1299 cells, as determined by luciferase and fluorescence assays. The susceptibility of the stable H1299-hACE2 cell line to a broad panel of SARS-CoV-2 variants (Wuhan, Beta, Delta, Omicron) was assessed here for the first time in a unified experimental setting. Infection of H1299-hACE2 cells with SARS-CoV-2 induced cell fusion and syncytium formation with subsequent cell death. The developed pseudovirus-based assay was further used for assessment of the antiviral properties of derivatives of 1,7,7-trimethyl-[2.2.1]-bicycloheptane-potential spike protein inhibitors, which possess moderate activity against lentiviral particles. The H1299-hACE2/spike-pseudotyped lentivirus assay is, therefore, a reliable, high-efficiency platform for screening spike-mediated entry inhibitors. The cell line obtained during the development of the platform can be used to isolate and study new variants of SARS-CoV-2.