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

Summary

This article describes the methodology for generating SARS-CoV-2 pseudotyped particles using a luciferase reporter system and its use to identify entry inhibitors. These protocols are demonstrated using two different entry-inhibitors, a recombinant human ACE2-Fc fusion protein and the small-molecule compound arbidol.

Abstract

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.

Introduction

The emergence of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) during late 2019 led to a global pandemic that resulted in millions of deaths worldwide and continues to pose a threat through new variants with increased transmissibility and immune escape potential¹. As an enveloped RNA virus from the Coronaviridae family, SARS-CoV-2 utilizes its trimeric spike (S) glycoprotein to mediate host cell entry by binding to the angiotensin-converting enzyme 2 (ACE2) receptor and undergoing proteolytic activation by TMPRSS2 and other host proteases^2,3. While several treatment optio....

Protocol

1. High efficiency transfection of HEK293T-derived cells using branched polyethyleneimine (PEI)

NOTE: Perform all steps in a biosafety cabinet using standard aseptic technique and BSL-2 procedures. Use HEK293T-derived cells for transfection and generation of pseudotyped viruses. In this Method cells are transfected in cell culture-treated 6 well plates. The PEI stock solution is prepared as previously described by Yang and colleagues (2017)¹². Before proceeding to do the triple transfection to generate the pseudovirus, it is advisable to optimize the best PEI-plasmid DNA proportion to ensure a high tra....

Results

Several transfection methods have been reported to generate PVs, including calcium phosphate¹⁵, lipid-based methods¹⁶, and PEI, both linear and branched. This article shows that transfection of HEK293T-derived cells with branched PEI allows for high-efficiency transfection (Figure 1), usually comparable to transfection methods using commercially available lipid-based transfection agents (data not shown). Representative results corresponding to .......

Discussion

This versatility of the MLV system for generating pseudotypes for other viruses has been reported by other important viruses, such as Ebola virus¹⁸, MERS-CoV⁸, SARS-CoV¹⁹, La Crosse virus²⁰, Hantavirus²⁰, Visna virus²¹, Arenavirus²², Influenza virus²³, and others. These pseudotypes have been very useful for an array of applications .......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
0.2 µm Low protein-binding syringe filters	Thermo Scientific	720-1320	Syringe Filters (0.2 μm,13 mm, PES, sterile)
0.25% Trypsin-EDTA 1X solution	Gibco	25200-056	Trypsin-EDTA (0.25%), phenol red
0.45 µm PVDF Bottle-Top Filter Units	Avantor	514-1045	Bottle-Top Vacuum Filtration Systems (PVDF, 250 ml, 50 mm, 0,45 µm)
1.5 mL Sterile Microcentrifuge Tubes	Eppendorf	30123611	Safe-Lock Tubes
100 X Penicillin Streptomycin solution	Corning	30-002-CI	Penicillin-Streptomycin Solution, 100x
5 X Luciferase assay lysis buffer	Promega	E2650	part of the Promega Bright-Glo Luciferase Assay System
50 mL Sterile Conical Tubes	Corning	352070	50 mL High Clarity PP Centrifuge Tube, Conical Bottom, Sterile
6-well Tissue Culture Plates	Thermo Scientific	140675	Cell-Culture Treated Multidishes
96-well Tissue Culture Plates	Thermo Scientific	168055	Nunc™ MicroWell™ 96-Well, Nunclon Delta-Treated, Flat-Bottom Microplate
Arbidol hydrochloride	Sigma-Aldrich	SML0860
Branched polyethylenimine, 25 kDa	Sigma-Aldrich	408727
Confocal laser microscope (FV 3000)	Olympus Co.	SKU: FV3000
Dimethyl sulfoxide (DMSO)	Sigma-Aldrich	D8418
DMEM (Dulbecco's Modified Eagle Medium) With: High Glucose, L-glutamine, Phenol Red, Sodium Pyruvate)	Gibco	11995-065
Fetal Bovine Serum (FBS)	Gibco	26140-079	Heat-inactivated (56°C, 30 min)
HEK293T-derived cells (Lenti-X 293T Cell Line)	Takara Bio	632180	Highly transfectable subclone of HEK293 cell line and supports high levels of viral protein expression
HEPES Buffer	Gibco	15630-080
Highly susceptible cells for infection (293T-ACE2.TMPRSS2 (mCherry) Cell Line)	BEI Resources	NR-55293	HEK293T cell line modified to stably express ACE2 and TMPRSS2
Hydrochloric acid	Sigma-Aldrich	H1758-500ML
Inverted phase contrast microscope	Olympus Co.	SKU: 2404040005
Isopropanol	Sigma-Aldrich	I9516-500ML
Luciferase substrate	Promega	E2650	part of the Bright-Glo Luciferase Assay System
Microplate reader with luminescence detection	Agilent BioTek	BTH1MG
MTT [3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide]	Sigma-Aldrich	M2128
Opaque White 96-well Plates	Corning	3917	96-well Solid White Flat Bottom Polystyrene TC-treated Microplates
Parafilm	Bemis	PM999
pcDNA3-EGFP plasmid	Addgene	13031	pcDNA3-EGFP was a gift from Doug Golenbock (Addgene plasmid # 13031 ; http://n2t.net/addgene:13031 ; RRID:Addgene_13031)
pCMV-MLVgagpol plasmid	-	-	Kindly donated by Dr. Gary Whittaker
Phosphate-buffered saline without Ca2+ and Mg2+ (PBS)	Corning	21-040-CV	Phosphate-Buffered Saline, 1X without calcium and magnesium
Promega PureYield Plasmid Maxiprep System	Promega	A2393
pTG-Luc plasmid	-	-	Kindly donated by Dr. Gary Whittaker
pUNO1-SARS-CoV-2 Spike (Wuhan) plasmid	Invivogen	p1-spike
pVSV-G plasmid	Addgene	138479	pVSV-G was a gift from Akitsu Hotta (Addgene plasmid # 138479 ; http://n2t.net/addgene:138479 ; RRID:Addgene_138479)
Recombinant Human ACE2-Fc fusion protein	Invivogen	fc-hace2
Reduced serum medium (Opti-MEM)	Gibco	31985-070	Opti-MEM Reduced Serum Medium