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 options have been developed to manage COVID-19 symptoms, the continued evolution and rapid development of viral variants highlight the urgent need for safe and scalable systems to study viral entry and evaluate new antiviral compounds targeting this early step in infection⁴.

The SARS-CoV-2 virus remains classified as a biosafety level 3 agent⁵ in some territories due to its high transmission potential. Therefore, any replicative laboratory task requires a high-containment BSL-3 facility, hindering the ability of most labs in the field to do scientific research with this virus. Pseudotyped virus-based systems, also known as pseudoviruses (PVs), offer a practical alternative that allows for viral entry studies in biosafety level 2 (BSL-2) laboratories, expanding accessibility for global research efforts⁶. The pseudotyped particles bearing the SARS-CoV-2 spike protein are non-replicating, making them a safe surrogate to evaluate virus entry and screen for small-molecule inhibitors⁷. These particles can be engineered to carry a luciferase or fluorescent reporter gene, enabling rapid, quantitative detection of infection by measuring reporter signal in infected cells. Systems based on murine leukemia virus (MLV) or lentivirus backbones have been widely used for studying the entry of SARS-CoV, MERS-CoV, SARS-CoV-2, Ebola virus, among others^8,9,10,11.

In these protocols, we describe the generation of MLV-based PVs using a three-plasmid system, based on the work reported by Millet and Whittaker⁸. The plasmids used include a packaging plasmid encoding the MLV core genes gag-pol (pCMV-MLVgagpol), a transfer vector encoding the reporter gene luciferase (pTG-Luc), and a plasmid expressing the SARS-CoV-2 spike glycoprotein corresponding to the original Wuhan-Hu-1 variant. Additionally, pseudoviruses are also generated using the G-glycoprotein of the vesicular stomatitis virus (VSV-G) to be used later as a counter-screening of potential hits. Pseudoviruses are produced in HEK293-derived cells and used to infect highly susceptible cells stably expressing hACE2 and TMPRSS2. Transfection efficiency is first evaluated using a green fluorescent protein (GFP)-expressing plasmid. Then, using optimized transfection conditions, PVs are generated by co-transfecting these three plasmids, followed by the titration of the resulting viruses. Using the appropriate amount of PVs, an assay is performed in 96-well plates to screen for compounds able to inhibit the entry of the viruses. This protocol utilizes branched polyethylenimine (PEI) as a cost-effective transfection reagent, enabling easier implementation of the procedures.

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 transfection efficiency (more than 70-80%). This optimization should be done by transfection of a green fluorescent protein (GFP)-expressing plasmid and then checking the proportion of fluorescent cells using a fluorescence microscope. For this optimization, keep plasmid DNA constant at 2.5 µg per well and test several amounts of PEI, in the range of 1-20 µL. Here are the best conditions for these lots of cells and PEI, consisting of 5 µL of PEI stock solution (1 mg/mL) and 2.5 µg of GFP-expressing plasmid per well in a 6-well plate.

1. Seed 7.5 x 10⁵ HEK293-derived cells per well in a 6-well tissue culture plate, in a total volume of 2 mL per well of complete medium (DMEM-C: Dulbecco's Modified Eagle's Medium supplemented with 10% heat-inactivated fetal bovine serum (FBS), 20 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), 100 IU/mL penicillin, and 100 µg/mL streptomycin) 24 h before transfection.
   NOTE: When seeding cells, avoid moving the tissue culture plate in circular motions, since this may cause cell aggregation at the center of wells, resulting in irregular cell distribution and subsequently lower transfection efficiency. One 6-well plate is enough for one experiment, including one well as a cell control (non-transfected), and the rest of the plate for testing several proportions of PEI-plasmid DNA.
2. Incubate the cells at 37 °C with 5% CO₂ overnight. Next day, ensure that the cells are approximately 50-60% confluent.
3. On the day of transfection, prepare the PEI-plasmid DNA complexes in sterile, 1.5 mL microcentrifuge tubes.
   1. For each well, prepare a PEI solution tube containing 5 µL of PEI stock (1 mg/mL) in 50 µL of reduced serum medium, and one DNA solution tube containing 2.5 µg of the GFP-expressing plasmid, in 50 µL of the same medium.
   2. If several wells are to be transfected, solutions may be scaled up accordingly. Mix each tube briefly by vortex and spin down for 5 sec. Incubate tubes at room temperature (RT) for 10 min.
4. During the time of this incubation, carefully remove the medium from each well of the 6-well plate, avoiding disruption of the cell monolayer. Replace carefully with 1 mL/well of complete transfection medium (DMEM-T, same as DMEM-C, but without antibiotics).
5. Prepare the transfection complex. After the 10 min incubation, add the 50 µl of the DNA solution tube to the PEI solution tube. Mix quickly by pipetting up and down six times and incubate at RT for exactly 3 min. Immediately proceed to add complex dropwise to cells in each corresponding well.
6. Seal the borders of the plate with parafilm and centrifuge at 1,000 x g for 30 min at RT, without brake, using a swing rotor and plate bucket.
7. After centrifugation, remove parafilm gently and incubate the plate at 37 °C, 5% CO₂ for 3 h.
8. After incubation, gently remove the transfection medium from each well and replace it slowly with 2 mL of pre-warmed DMEM-C per well.
9. Incubate transfected cells at 37 °C with 5% CO₂ for 48 h.
10. Assess transfection efficiency by GFP expression using fluorescence microscopy. The frequency of fluorescent cells should be higher than 70-80% (Figure 1). This is critical for the efficiency of the next method in section 2.

2. Generation of SARS-CoV-2 spike- and VSV-G pseudotyped pseudoviral particles

NOTE: This Method uses the same procedure as section 1, except that instead of using the GFP plasmid, cells will be co-transfected with the three plasmids required to generate pseudotyped viruses, using the optimal transfection conditions to assure high transfection efficiency. The protocol also uses HEK293T-derived cells in 6-well plates, sufficient for the following three required conditions: a) non transfected cells (one well), b) cells transfected only with the MLV packaging plasmid and the luciferase reporter transfer vector (one well; spike-less pseudoviruses) and c) cells transfected with these two plasmids and the spike-expressing plasmid to generate SARS-CoV-2 pseudotyped viruses (four wells). The spike-expressing plasmid used here codes for the spike of the SARS-CoV-2, Wuhan strain, and contains sequence modifications to allow for an efficient presentation of the protein in PVs. The spike-less pseudovirus will be required as a control later during infections, to validate the spike-dependent entry in permissive cells. Similarly, VSV-G-PVs are generated using the same experimental design to be used later as a counter-screening of potential inhibitory compounds. If a larger volume of pseudovirus is required, the protocol may be scaled up to two 6-well plates, which may also be practical for the plate centrifugation step.

1. The day before transfection, seed HEK293T-derived cells into two 6-well plates, as described in section 1. Use one plate for the generation of spike-PVs and the other for the VSV-G-PVs.
2. The day of transfection, prepare the transfection mixes as shown in Table 1, following step 1.3.
   NOTE: PEI solution is prepared as previously reported¹². Plasmids pCMV-MLVgagpol and pTG-Luc were obtained from Millet and Whittaker⁸. Plasmid pVSV-G was obtained from Gee et al 2020¹³. All plasmids should be prepared in advance as high-quality preps, free of endotoxins. It is recommended to use commercially available maxiprep kits, with a final step of sterile filtration through low-binding, 0.2 µm syringe filter.
3. Mix each tube as described in section 1 and incubate at RT for 10 min.
4. During this incubation, carefully remove the medium from each well of the 6-well plates, avoiding disruption of the cell monolayer. Slowly replace with 1 mL of pre-warmed DMEM-T medium to each well, ensuring the medium runs down the wall of the well and not directly onto the cells.
5. Prepare transfection complexes as described in section 1 and transfect cells using the previously described protocol.
6. After the 48-h incubation post-transfection, collect the supernatants, transfer them to 50 mL sterile conical centrifuge tubes, and centrifuge at 290 x g for 7 min at 4 °C. Collect the cell-free supernatant, and filter it using a polyvinylidene fluoride (PVDF), sterile 0.45 µm top bottle filter unit. Prepare aliquots of 1 mL and store them at -80 °C.
   ​NOTE: Pseudotyped particles are stable at -80 °C for several months. Avoid repeated freeze-thaw cycles to preserve infectivity.

	For SARS-CoV-2 spike-PVs (per well)	For VSV-G-PVs (per well)	For spike-less PVs (per well)
PEI solution:
PEI (1 mg/ml)1	5 µL	5 µL	5 µL
Reduced serum medium	45 µL	45 µL	45 µL
Plasmid DNA solution:2
pCMV-MLVgagpol	0.79 µg	0.79 µg	0.79 µg
pTG-luc	0.92 µg	0.92 µg	0.92 µg
pUNO1-spike	0.79 µg	-	-
pVSV-G	-	0.79 µg	-
Reduced serum medium	to 50 µL	to 50 µL	To 50 µL

Table 1: Preparation of transfection mixes for the generation of PVs.

3. Titration of SARS-CoV-2 and VSV-G pseudotyped particles

NOTE: This step is essential to confirm that the produced pseudotyped particles are infectious and to determine the minimum effective volume required for downstream applications such as compound screening. The titration should be done for every new batch of pseudovirus. To this purpose, cells highly susceptible to infection with SARS-CoV-2 (HSI cells) are infected with decreasing amounts of the pseudotyped viruses. The entry of the pseudoviruses, mediated by the spike-human ACE2 interaction, or by VSV-G with other cellular receptors, is monitored 72 h later by the detection of the luciferase activity in a luminescence assay.

1. The day before the transduction, seed 2.5 x 10⁴ HSI cells per well in a 96-well tissue culture plate, in 50 µL of complete medium (DMEM-C). Prepare three wells for each amount of pseudovirus to test, and include wells for infection, also with spike-less pseudoviruses, and for non-infected cells. Incubate the plate at 37 °C with 5% CO₂ overnight.
2. The day of the transduction, thaw one aliquot of each pseudovirus batch to test on ice and gently mix by inversion. Include also one aliquot of the spike-less control. Add directly to each well several amounts of the pseudovirus preparation (100, 50, 25, 12.5, 6.2, 3.1, 1.5, and 0.8 µL). Adjust the volumes of all wells to 150 µL with DMEM-C.
3. Seal the plate with parafilm and centrifuge at 2,500 × g for 45 min at RT, with no brake.
4. Remove the parafilm seal and incubate the plate for 72 h at 37 °C, 5% CO₂.
5. After incubation, prepare for the luciferase assay following the instructions of the manufacturer. Thaw luciferase substrate (stored at -80 °C) and 5x luciferase assay lysis buffer (stored at -20 °C) and equilibrate at RT (10-15 min).
6. Dilute the luciferase assay lysis buffer to 1x with sterile water. Prepare at least 3 mL.
7. Slowly and carefully aspirate and discard the supernatant of each well using a multichannel pipette.
8. Add 30 µL of 1x lysis buffer to each well using a multichannel pipette. Place the plate in a plate shaker for 10 min at 150 rpm, at RT. Verify complete lysis under an inverted microscope (all cells should be lysed and not distinguishable upon visual examination).
   NOTE: Prepare the microplate reader in advance to perform luminescence detection immediately after substrate addition to ensure consistency and avoid signal decay.
9. Transfer the lysates to an opaque, white 96-well plate.
10. Add 50 µL of luciferase substrate to each well and mix briefly by moving the plate in a circular motion. Place the plate in the luminometer and measure luminescence.
11. Analyze the data obtained.
    1. Verify that controls give the expected luminescence values. Ensure that the non-infected cells give very low background luminescence values.
       NOTE: Cells infected with spike-less pseudovirus should give values at the level of non-infected cells, and cells infected with decreasing amounts of spike-presenting pseudovirus should show high, dose-dependent values of luminescence (Figure 2). The luminescence values generated by the VSV-G-PVs are usually much higher than those of spike-PVs, about one or two orders of magnitude.
    2. For subsequent screenings, select the amount of pseudovirus that generates a signal at least one hundred to one thousand times that of the non-infected cells.

4. Screening of compounds for pseudoviral entry inhibition using spike-PVs

1. Seed 2.5 x 10⁴ HSI cells per well in 50 µL of complete medium (DMEM-C) in a 96-well plate, as described in section 3. Incubate overnight at 37 °C, 5% CO₂.
   1. Consider a plate design that includes columns 1 to 11 for compounds plus pseudovirus, and column 12 for controls. In column 12, allocate four wells for virus control (pseudovirus only) and four wells for non-infected, non-treated cells.
2. The next day, prepare serial dilutions (1:3) of compounds to be tested, starting at 50 µM (final concentrations during infections: 50, 16.6, 5.5, 1.8, 0.6, 0.2, and 0.06 µM), in DMEM-C containing 0.5% DMSO.
   1. For convenience, prepare dilutions in a separate 96-well "dilution plate", where serial dilutions are first made and then transferred (50 µL) to the plate containing the cells.
   2. If the number of compounds is very large, in the order of hundreds or more, reduce this first dilution series to about 4 points, using a single well per dilution point, and consider a prescreening. Then, re-test the potential hits using 7-point serial dilutions, 1:3, with three replicates, starting at the same concentration of 50 µM.
3. Once dilutions of compounds are ready, gently aspirate the medium of cells using a multichannel pipette and add 50 µL of compound dilutions to the corresponding wells. Incubate for 1 h at 37 °C, 5% CO₂. For controls in column 12, add DMEM-C only.
4. Without removing the compounds, add to each well the predetermined amount of pseudotyped SARS-CoV-2 pseudovirus, in a volume of 50 µL of complete medium, containing the compounds at the same concentrations. Seal the plates with parafilm.
5. Centrifuge the plate at 2,500 × g for 45 min at RT without brake, then remove parafilm and incubate at 37 °C, 5% CO₂ for 72 h.
6. Proceed with luciferase-based infectivity quantification as described in section 3.
7. Perform data analysis.
   1. Export luminescence values into a spreadsheet to format the data for subsequent import into data analysis and graphing software.
   2. Prepare bar graphs plotting the mean of each concentration point, including the standard error of the mean bars. Plot also the values obtained from positive (PVs alone) and negative controls (non-infected cells).
      NOTE: The results of the assay are considered valid if 1) non-infected cells show very low values, 2) PVs controls show high values, 3) positive control of inhibition (i.e., arbidol or hACE2-Fc) shows inhibition of infection, in a dose-dependent manner (Figure 3A, B).
   3. Then, observe the values produced by unknown compounds to assess if there is inhibition and if there is a dose-dependent effect. If so, then subject these compounds to further confirmatory analyses.
      NOTE: For further confirmation of potential hits, follow the next steps. First, retest the potential hits compounds as suggested in step 4.2. If the dose response effect is confirmed, then perform a counter screening using the same dilution series but infecting with the VSV-G-PVs. This test is important to check if the inhibition of entry is specific for the spike-ACE2 interaction, and not due to interference at any other step later during the production of the luciferase in the infected cells (Figure 3A, B). Here, the positive compound should not show inhibitory activity against the VSV-G-PVs. If this is the case, then the same series of concentrations should be tested for cytotoxicity using a cell viability assay (MTT assay, shown in section 5). As a summary, a potential hit compound should show inhibition of SARS-CoV-2-spike-PVs, and no or less inhibition of VSV-G-PVs, and no, or at least a much weaker inhibition of the viability of the cells. Adjusting a dose-response curve using the luminescence data will allow the estimation of the half-maximal inhibitory concentration (IC₅₀) for the inhibition of the PVs, and a half-maximal cytotoxic concentration (CC₅₀) from the viability assay. The ratio CC₅₀ / IC₅₀ is used to calculate the selectivity index (SI), which is an important indicator of the potential usefulness of the positive compound. For the analysis of the dose-response curves and the estimation of the IC₅₀ and CC₅₀ very detailed procedures have been described¹⁴.

5. MTT-based cell viability assay

1. Seed 2.5 x 10⁴ HSI cells per well in 50 µL of DMEM-C in a 96-well plate. Incubate the plate overnight at 37 °C, 5% CO₂. Ensure the cells are about 50% confluency the next day. Prepare enough wells, considering all different concentrations of the compound to be evaluated, untreated cells, and blank wells containing only medium, in triplicate. Test the potential hit compounds at the same concentrations evaluated in the PV entry inhibition assay.
2. After incubation, carefully remove the medium and replace it with 100 µL of complete medium containing different concentrations of the compound to evaluate, in 0.5% DMSO.
3. Centrifuge the plate at 2,500 × g for 45 min at RT without brake and incubate for 72 h at 37 °C, 5% CO₂. This step is included to handle the cells exactly as done in the previous section, except for the addition of the PVs.
4. Prepare a 0.5 mg/mL MTT solution using DMEM. Sterilize by filtering through 0.2 µm and keep in the dark.
   NOTE: 3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) is light sensitive. It should be kept frozen, in the dark.
5. Carefully remove the medium from each well and replace it with 100 µL of MTT solution. Incubate for 2 h at 37 °C, 5% CO₂.
6. During this incubation, prepare a solubilization solution containing 40 mM HCl in isopropyl alcohol.
7. Carefully remove the MTT solution using a multichannel pipette, without disturbing the formazan crystals at the bottom of each well.
8. Add 100 µL of the solubilization solution to each well. Wrap the plate in aluminum foil and place it on a plate shaker at 150 rpm for 15 min.
9. Record absorbance at 570 nm and 630 nm.
10. Perform data analysis.
    1. Using the raw values of optical density, subtract the values obtained at 630 nm from those obtained at 570 nm for each well. Then average the corrected values of the untreated cell wells. Consider these untreated cells 100% viable.
    2. Then normalize treated cells' values against the untreated cells' wells, using the formula:
       Viability (%) =
11. Plot viability percentages as bars representing the mean of replicates ±± standard error of the mean. Perform further statistical analysis to compare groups using procedures implemented in a data analysis and graphing software, comparing viabilities at different concentrations with the untreated cells control.
12. Compare means of each concentration point to that of the DMSO-only treated cells using nonparametric Kruskal-Wallis with Dunn post-test (differences are considered significant at p-values < 0.05). For plotting a dose-response curve and estimating the CC₅₀ out of OD values, check the procedure reported elsewere¹⁴.

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