Procedures for the Identification of SARS-CoV-2 Entry Inhibitors as Potential Antivirals using MLV-Based Pseudoviruses

The SARS-CoV-2 virus is an emergent, zoonotic pathogen that has caused a huge burden of disease and mortality around the world since its emergence in 2019. Safe and accessible platforms to evaluate viral entry and potential antiviral compounds remain essential for ongoing drug discovery against emerging variants. This article describes a highly optimized protocol for generating murine leukemia virus (MLV)-based pseudotyped particles expressing the SARS-CoV-2 spike protein and carrying a luciferase reporter gene, enabling the study of viral entry under biosafety level 2 conditions. The methods described detail the use of optimized cell lines, high transfection efficiency using cost-effective reagents, production of highly effective pseudoviruses, and titration of infectivity to determine the minimum viral input for screening assays. Representative results demonstrate robust luciferase activity in infected cells compared to non-infected controls, while control spike-less and VSV-G-pseudoviruses confirm assay specificity. Furthermore, the system supports compound screening by quantifying inhibitory effects on viral entry and assessing cytotoxicity using an MTT viability assay. Overall, this reproducible and scalable protocol provides a reliable platform for medium to high-throughput screening of novel viral entry inhibitors, contributing to the identification of antiviral candidates and advancing research on SARS-CoV-2 entry mechanisms.