Name: Production of a SARS-CoV-2 Virus-Like-Particle System to Investigate Viral Life Cycles In Vitro (Video) | JoVE Journal
Uploaded: 2025-06-06T15:57:00.000+00:00
Duration: 9 min 26 s
Description: Learn to produce SARS-CoV-2 virus-like particles using HEK-293T cells for studying viral assembly and drug screening without biosafety level 3 requirements.

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5 minutes

This work was supported by the Startup fund program at Beijing University of Chinese Medicine (BUCM) (90011451310011). We extend our gratitude to all the member of Dr. Ma lab at BUCM for the invaluable discussion and assistance with the experiments.

1. Generation of SC2-VLPs
<ol>
	<li>Seed ~3.0 &#215; 106 HEK-293T cells in a 10 cm diameter tissue culture plate with DMEM complete medium, supplemented with 10% v/v FBS and 1% penicillin-streptomycin. 
	NOTE: To ensure high transfection efficiency and optimal SC2-VLP production, HEK-293T cells should be maintained at low passage numbers ~10.</li>
	<li>Culture the HEK-293T cell at 37 &#176;C, 5% CO2 for about 24 h, and check the cell confluency under the microscope. Proceed if ~70% confluent.</li>
	<li>Dilute 60 &#181;L of PEI from 1 mg/mL stock to 200 &#181;L with serum-freemedium.</li>
	<li>Add 6.7 &#181;g of N plasmid, 10 &#181;g of Luc-T20 plasmid, 0.016 &#181;g of S plasmid, and 3.3 &#181;g of M-IRES-E plasmid into 200 &#181;L of serum-free medium (see Supplemental File 1).</li>
	<li>Gently add the diluted PEI from step 1.3 into the solution containing plasmids coding for viral structure proteins from step 1.4, and incubate at room temperature for 10 min. This is the transfection solution.</li>
	<li>Carefully drop the transfection solution from step 1.5 onto HEK-293T cells, and gently swirl the tissue culture plate for thorough mixing.</li>
	<li>Exchange the cell culture medium 6 h post infection with the DMEM complete medium, and incubate the transfected HEK-293T cells at 37 &#176;C, 5% CO2 for 48 h.</li>
	<li>Collect the supernatant of the infected HEK-293T cells, which contains the SC2-VLPs, and then filter the supernatant through a 0.45 &#181;m syringe filter to remove cell debris. This is the SC2-VLP medium. 
	NOTE: We attempted to produce SC2-VLPs in HeLa, Vero E6, and Caco2 cell lines, but no measurable viral titer was obtained, indicating the protocol&#39;s specificity for HEK-293T cells.</li>
</ol>
2. Examination of SC2-VLP effectiveness
NOTE: The HEK-293T cell line stably expressing ACE2 and TMPRSS2 was established using a lentiviral transduction approach14,15. Protein expression of both ACE2 and TMPRSS2 was confirmed by western blot analysis, following a protocol similar to that described in Step 3 (SC2-VLP composition analysis).
<ol>
	<li>Seed 4.0 &#215; 104 HEK-293T cells with stable expression of ACE2 and TMPRSS2 in a 96-well plate and add 50 &#181;L of SC2-VLP medium from step 1.8.</li>
	<li>Incubate the 96-well tissue culture plate at 37 &#176;C with 5% CO2 for 24 h.</li>
	<li>Remove the medium from the 96-well plate, and wash once with 100 &#181;L of PBS buffer at 37 &#176;C, containing 137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, and 1.8 mM KH2PO4.</li>
	<li>Lyse the HEK-293T ACE2/TMPRSS2 cells with 20 &#181;L of passive lysis buffer, gently rocking the sample for 15 min on an orbital shaker at room temperature. 
	NOTE: The lysis buffer contains 25 mM Tris-phosphate (pH 7.8), 2 mM DTT, 2 mM 1,2-diaminocyclohexane-N,N,N&#8242;,N&#8242;-tetraacetic acid, 1.25 mg/mL lysozyme, 2.5 mg/mL BSA, 10% glycerol, and 1% Triton X-100).</li>
	<li>Spin the 96-well plate at 4,000 &#215; g for 15 min at 4 &#176;C in a refrigerated microplate centrifuge, and then immediately transfer the plate to an ice bath.</li>
	<li>Take 100 &#181;L of reconstituted luciferase assay buffer to an opaque white 96-well plate, and add 20 &#181;L of lysate from step 2.5. Mix them briefly by pipetting up and down 2-3x.</li>
	<li>Measure the luminescence using a plate reader with the following parameters: Detection mode: Luminescence; Wavelength range: Full spectrum; Plate format: 96-well standard opaque plate; Integration time: 200 ms.</li>
</ol>
3. Examination of the SC2-VLP composition
<ol>
	<li>Add 1.36 mL of PEG 8000 solution, containing 50% PEG 8000 and 2.2% NaCl, to 10 mL of SC2-VLP medium from step 1.8.</li>
	<li>Keep the mixture on an orbital shaker, and slowly mix the solution at 4 &#176;C overnight.</li>
	<li>Centrifuge the solution at 4 &#176;C, 2,000 &#215; g for 30 min, and collect the SC2-VLP pellet for western blotting analysis16. 
	NOTE: For the western blotting analysis, all information regarding the antibodies is provided in the Table of Materials.</li>
</ol>
4. Subcellular localization analysis of S and its mutants in SC2-VLP-producing cells
<ol>
	<li>Seed ~3.0 &#215; 106 HEK-293T cells evenly in the glass-bottom culture dish with 15 mm diameter, and then allow the cells to adhere and grow until ~70% confluency before transfection.</li>
	<li>Repeat the transfection procedure as described in steps 1.3 through 1.7 and modify only the plasmid quantities as follows: N plasmid: 1.3 &#181;g, Luc-T20 plasmid: 2 &#181;g, S plasmid: 0.0032 &#181;g, and M-IRES-E plasmid: 0.66 &#181;g.</li>
	<li>Gently wash the culture dish twice with 1 mL of ice-cold PBS, and then add 1 mL of 4% paraformaldehyde (PFA) fixation solution at room temperature (RT) for 15 min.</li>
	<li>Wash the cells for 2 x 5 min with 1 mL of PBS at RT, and then permeabilize the cells by adding 1 mL of 0.25% Triton X-100 at RT for 10 min.</li>
	<li>Wash the cells for 2 x 5 min with 1 mL of PBS at RT, and then add 1 mL of 5% bovine serum albumin (BSA) at RT for 1 h to block nonspecific antibody interactions.</li>
	<li>Add ~200 &#181;L of primary antibody solution (enough to cover the glass bottom) and incubate at 4 &#176;C overnight. 
	NOTE: The primary antibody solutions are prepared by diluting in 5% BSA dissolved in PBS at the following dilution ratios: the mouse anti-S antibody at 1:200, the rabbit anti-Sec61&#946; antibody at 1:200, the rabbit anti-GM130 antibody at 1:200, and the rabbit anti-ERGIC53 antibody at 1:200. The 5% BSA/PBS solution served as both the diluent and blocking buffer to minimize nonspecific binding during subsequent immunofluorescence staining procedures.</li>
	<li>Remove the primary antibody solution, and then wash the cells for 3 x 5 min with 1 mL of PBS at RT.</li>
	<li>Add fluorescence-conjugated secondary antibody solution at RT for 1 h, and then wash the cells for 3 x 5 min with 1 mL of PBS at RT. 
	NOTE All subsequent steps must be performed in the dark or under minimal light exposure to prevent photobleaching of fluorescent conjugates and preserve signal integrity. Two types of fluorescence-conjugated secondary antibodies, which are derived from mouse or rabbit, are employed to stain either the S protein or cell organelle marker proteins.</li>
	<li>Stain nuclei with 2.5 &#181;g/mL Hoechst solution at RT for 5 min, and then wash the cells for 3 x 5 min with 1 mL of PBS at RT.</li>
	<li>Observe S protein or organelle staining and acquire images using a confocal microscope with the following parameters: PMT mode set to VBF (no averaging, line sequential scan) with auto-confocal aperture. For channel 1 (FITC), use the 488 nm laser at 25% power with 500 - 548 nm emission, HV 525 V, gain 1x, and offset 5%. For channel 2 (Alexa Fluor 594), use the 594 nm laser at 25% power with 610 - 670 nm emission, HV 500 V, gain 1x, and offset 5%. For channel 3 (DAPI), use the 405 nm laser at 25% power with 430 - 470 nm emission, HV 490 V, gain 1x, and offset 5%.</li>
</ol>

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</ol>

The authors have no conflicts of interest to disclose.

A simple and effective method to model the SARS-CoV-2 life cycle in cell culture systems, without the constraints of BSL-3 laboratories, represents a pivotal advancement for anti-SARS-CoV-2 research. The SC2-VLP method fulfills this need, offering a robust and accessible platform. In this study, we provide a detailed protocol for the SC2-VLP method, outlining critical experimental steps for producing SC2-VLPs. Additionally, we highlight its versatility and potential applications in advancing our understanding of SARS-CoV...

The COVID-19 pandemic has emerged as one of the most devastating global health crises in modern history, resulting in millions of deaths worldwide1. The virus responsible, SARS-CoV-2, follows a complex life cycle that includes key stages such as infection, genome replication, assembly, and egress. The infection process begins when the viral spike protein (S) binds to the host cell receptor, Angiotensin-Converting Enzyme 2 (ACE2), facilitating the release of the viral genome into the host cell2,3. The viral RNA-dependent RNA polymerase (RdRp) then catalyzes the replication of the genomic RNA. This RNA, in complex with the nucleocapsid protein (N), forms a stable structure that is recognized by the membrane protein (M). The M protein plays a central role in viral assembly by recruiting the RNA-N complex, S, and the envelope protein (E)4,5. After assembly, the virion completes its egress through a noncanonical lysosome-mediated trafficking pathway6.
In response to the pandemic, substantial global resources have been mobilized to develop vaccines, neutralizing antibodies, and antiviral drugs. The evaluation of these interventions has been essential in advancing SARS-CoV-2 research7. However, studying the live virus presents significant logistical challenges, as experiments involving the virus must be conducted in Biosafety Level 3 (BSL-3) laboratories. The limited availability of BSL-3 facilities has constrained the pace of research aimed at understanding and combating SARS-CoV-2.
To address these challenges, two major systems-virus-like particle (VLP) and lentiviral pseudotyping-have been widely adopted in SARS-CoV-2 research, both of which do not require BSL-3 containment8. The VLP system involves the co-transfection of cells with genes encoding viral structural proteins, including M, S, E, and N, which together generate virus-like particles. These particles mimic the structural and functional properties of the virus, making them a valuable tool for studying key processes in the SARS-CoV-2 life cycle, and even an effective antigen for vaccine development9,10,11.
Conversely, the lentiviral pseudotyping system involves replacing the Vesicular Stomatitis Virus (VSV) G protein in lentivirus with SARS-CoV-2 S protein, enabling the production of lentiviral particles that can incorporate reporter genes such as luciferase or GFP. This system is particularly useful for investigating neutralizing antibodies that block the S-ACE2 interaction12. However, lentiviral pseudotyping does not mirror SARS-CoV-2 viral assembly or egress due to the use of HIV structural proteins, which mediate particle release at the plasma membrane.
To overcome these limitations, Syed et al. recently identified the SARS-CoV-2 packaging signal within its RNA genome, which demonstrates the N protein&#39;s specificity toward viral genome recognition13. By fusing this packaging signal to reporter genes, it is possible to efficiently incorporate these genes into SARS-CoV-2 virus-like particles (SC2-VLPs)13. This strategy not only replicates the SARS-CoV-2 assembly and egress processes but also allows for the sensitive measurement of infection steps. In this study, we introduce the experimental methodology for using the SC2-VLP system and highlight key considerations for conducting this approach.

<table><tbody><tr><td>Bovine Serum Albumin (BSA)</td><td>Cell Signaling Technology</td><td>9998S</td><td></td></tr><tr><td>Confocal Laser Scanning Microscope</td><td>Olympus</td><td>FV3000</td><td></td></tr><tr><td>DMEM</td><td>Corning</td><td>10-013-CV</td><td></td></tr><tr><td>Fetal Bovine Serum (FBS)</td><td>Thermofisher</td><td>A5670402</td><td></td></tr><tr><td>Goat Anti-Mouse IgG H&amp;amp;L (Alexa Fluor 488) antibody</td><td>Invitrogen</td><td>A11001</td><td>dilution ratio: IF 1:1000</td></tr><tr><td>Goat Anti-Rabbit IgG H&amp;amp;L (Alexa Fluor 594) antibody</td><td>Abcam</td><td>ab150080</td><td>dilution ratio: IF 1:1000</td></tr><tr><td>HEK-293T cell line</td><td>National Infrastructure of Cell Line Resource (NICR)</td><td>NICR-293T-001</td><td>To ensure high transfection efficiency and optimal SC2-VLP production, HEK-293T cells should be maintained at low passage numbers (&amp;le; P10).</td></tr><tr><td>Hoechst 33342</td><td>Invitrogen</td><td>H1399</td><td>Working concentration: 2.5 &amp;mu;g/mL</td></tr><tr><td>Luciferase Reporter Assay System</td><td>Promega</td><td>E1500</td><td></td></tr><tr><td>Luc-T20</td><td>Addgene</td><td>177941</td><td></td></tr><tr><td>Mouse monoclonal anti-GAPDH</td><td>Proteintech</td><td>60004-1-Ig</td><td>dilution ratio: WB 1:20,000</td></tr><tr><td>Mouse monoclonal anti-S RBD</td><td>Abclonal</td><td>A23771</td><td>dilution ratio: IF 1:200</td></tr><tr><td>OptiMEM</td><td>Thermofisher</td><td>31985070</td><td>serum-free medium for transfection</td></tr><tr><td>PEG3350</td><td>Sigma-Aldrich</td><td>P3635</td><td></td></tr><tr><td>PEG8000</td><td>Sigma-Aldrich</td><td>P2139</td><td></td></tr><tr><td>Penicillin-Streptomycin</td><td>Thermofisher</td><td>15140122</td><td></td></tr><tr><td>Polyethyleneimine (PEI)</td><td>Thermofisher</td><td>43896.01</td><td></td></tr><tr><td>Promega passive lysis buffer</td><td>Promega</td><td>E1941</td><td></td></tr><tr><td>Rabbit polyclonal anti-ACE2</td><td>Proteintech</td><td>21115-1-AP</td><td>dilution ratio: WB 1:1000</td></tr><tr><td>Rabbit polyclonal anti-ERGIC53</td><td>Proteintech</td><td>13364-1-AP</td><td>dilution ratio: IF 1:200</td></tr><tr><td>Rabbit polyclonal anti-GM130</td><td>Proteintech</td><td>11308-1-AP</td><td>dilution ratio: IF 1:200</td></tr><tr><td>Rabbit polyclonal anti-S</td><td>Abcam</td><td>ab272504</td><td>dilution ratio: WB 1:1000</td></tr><tr><td>Rabbit polyclonal anti-Sec61&amp;beta;</td><td>Proteintech</td><td>15087-1-AP</td><td>dilution ratio: IF 1:200</td></tr><tr><td>Rabbit polyclonal anti-TMPRSS2</td><td>Abcam</td><td>ab109131</td><td>dilution ratio: WB 1:1000</td></tr><tr><td>SARS-CoV-2 M-IRES-E plasmid</td><td>Addgene</td><td>177938</td><td></td></tr><tr><td>SARS-CoV-2 N plasmid</td><td>Addgene</td><td>177959</td><td></td></tr><tr><td>SARS-CoV-2 Nucleoprotein Rabbit mAb</td><td>Abclonal</td><td>A21042</td><td>dilution ratio: WB 1:4000</td></tr><tr><td>SARS-CoV-2 S plasmid</td><td>Addgene</td><td>177960</td><td></td></tr><tr><td>Secondary Antibody, HRP, Goat anti-Mouse IgG (H+L)</td><td>Invitrogen</td><td>31460</td><td>dilution ratio: WB 1:5000</td></tr><tr><td>Secondary Antibody, HRP, Goat anti-Rabbit IgG (H+L)</td><td>Invitrogen</td><td>31430</td><td>dilution ratio: WB 1:5000</td></tr><tr><td>SpectraMax i3x plate reader&amp;nbsp;</td><td>Molecular Devices</td><td>SpectraMax i3x</td><td></td></tr></tbody></table>

production of a sars-cov-2 virus-like-particle system to investigate viral life cycles in vitro

Production of a SARS-CoV-2 Virus-Like-Particle System to Investigate Viral Life Cycles In Vitro

The severe acute respiratory syndrome-coronavirus 2 (SARS-CoV-2) virus-like particle (SC2-VLP) method offers a powerful and accessible tool for studying the SARS-CoV-2 life cycle without the need for biosafety level 3 (BSL-3) laboratories. This system effectively mimics critical stages of the viral life cycle, including assembly, genome packaging, and egress, using a luciferase reporter fused to the T20 signal for sensitive and precise detection of viral particle production. SC2-VLPs are generated by co-expressing SARS-CoV-2 structural proteins, including membrane (M), nucleocapsid (N), envelop (E), and spike (S), along with the RNA packaging signal in HEK-293T cells. Unlike traditional virus-like particle systems, the SC2-VLP method ensures accurate quantification and greater fidelity to the natural viral life cycle. Furthermore, compared to lentiviral pseudotyping methods, which are limited to studying viral entry through the incorporation of S protein into HIV-based lentiviral particles, the SC2-VLP system provides a more comprehensive platform for exploring multiple stages of SARS-CoV-2 biology. While this method bypasses the risks of handling live virus and expands accessibility. The SC2-VLP method represents a significant advancement in antiviral research and the development of therapeutic strategies against SARS-CoV-2.

The egress and assembly steps of the SARS-CoV-2 life cycle have been less studied than the infection and replication steps17,18. Before the SC2-VLP method was developed, these processes could only be investigated using live SARS-CoV-2, confining research to BSL-3 laboratories13. The SC2-VLP workflow, shown in Figure 1, outlines the experimental protocol and illustrates the processes involved in these steps of ...

We present an optimized in vitro protocol for producing SARS-CoV-2 virus-like particles that closely mimic the authentic virus. This approach enables the investigation of viral infection, assembly, and egress mechanisms without the constraints of requiring a biosafety level 3 laboratory.

The severe acute respiratory syndrome-coronavirus 2 (SARS-CoV-2) virus-like particle (SC2-VLP) method offers a powerful and accessible tool for studying ...

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JoVE Journal

Biochemistry

Source: Jamieson, T. R., et al. Detection of SARS-CoV-2 Neutralizing Antibodies using High-Throughput Fluorescent Imaging of Pseudovirus Infection. J. Vis. Exp. (2021)This video demonstrates the procedure for infecting mammalian cells with green fluorescent protein (GFP)-encoding pseudovirus, monitoring viral replication and spread by fluorescence microscopy, and collecting virus-containing supernatant for downstream applications.

Production and Collection of Pseudovirus Particles Using Mammalian Cell Culture

Source: Scarborough, R. J., et al. Evaluation of the Efficacy And Toxicity of RNAs Targeting HIV-1 Production for Use in Gene or Drug Therapy. J. Vis. Exp. (2016).This video demonstrates a reverse transcriptase (RT) assay to assess the effect of a test RNA on virus-like particle (VLP) production. Cells are co-transfected with plasmids, and RT activity is measured as a quantitative indicator of VLP output. A decrease in RT activity reflects suppression of viral gene expression by the test RNA.

Reverse Transcriptase Activity as a Marker for Virus-Like Particle Production

This video demonstrates the generation of influenza virus-like particles (VLPs) in a mammalian system. Upon expressing three eukaryotic expression vectors — encoding different viral structural proteins — in suitable mammalian host cells, the expressed viral proteins self-assemble into VLPs on the host plasma membrane and release via budding from the cell surface.

A Technique for the Generation of Influenza Virus-Like Particles via a Mammalian System

Work with infectious Ebola viruses is restricted to biosafety level 4 laboratories. Tetracistronic minigenome-containing replication and transcription-competent virus like particles (trVLPs) represent a lifecycle modeling system that allows us to safely model multiple infectious cycles under biosafety level 2 conditions, relying exclusively on Ebola virus components.

Modeling The Lifecycle Of Ebola Virus Under Biosafety Level 2 Conditions With Virus-like Particles Containing Tetracistronic Minigenomes

Here, we present a protocol for synthesizing virus-like particles using either baculovirus or mammalian expression systems, and ultracentrifugation purification. This highly customizable approach is used to identify viral antigens as vaccine targets in a safe and flexible manner.

Expression and Purification of Virus-like Particles for Vaccination

Here, we present a protocol to generate pseudotyped particles in a BSL-2 setting incorporating the spike protein of the highly pathogenic viruses Middle East respiratory syndrome and severe acute respiratory syndrome coronaviruses. These pseudotyped particles contain a luciferase reporter gene allowing quantification of virus entry into target host cells.

Production of Pseudotyped Particles to Study Highly Pathogenic Coronaviruses in a Biosafety Level 2 Setting

The protocol described here outlines a fast and effective method for measuring neutralizing antibodies against the SARS-CoV-2 spike protein by evaluating the ability of convalescent serum samples to inhibit infection by an enhanced green fluorescent protein-labeled vesicular stomatitis virus pseudotyped with spike glycoprotein.

Detection of SARS-CoV-2 Neutralizing Antibodies using High-Throughput Fluorescent Imaging of Pseudovirus Infection

This protocol describes the dynamics of viral infections using luciferase- and fluorescence-expressing recombinant (r)SARS-CoV-2 and an in vivo imaging systems (IVIS) in K18 hACE2 transgenic mice to overcome the need of secondary approaches required to study SARS-CoV-2 infections in vivo.

Live Imaging and Quantification of Viral Infection in K18 hACE2 Transgenic Mice Using Reporter-Expressing Recombinant SARS-CoV-2

Pseudotyped viruses (PVs) are replication-defective virions that are used to study host-virus interactions under safer conditions than handling authentic viruses. Presented here is a detailed protocol that shows how SARS-CoV-2 PVs can be used to test the neutralizing ability of patients' serum after COVID-19 vaccination.

Pseudotyped Viruses As a Molecular Tool to Monitor Humoral Immune Responses Against SARS-CoV-2 Via Neutralization Assay

Here, we describe a newly developed multiplex fluorescent immunoassay that uses a dual-reporter flow cytometric system to concurrently detect two unique spike protein epitopes on intact severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) viral particles that had been captured by angiotensin-converting enzyme-2-coupled magnetic microspheres.

Profiling of Surface Protein Epitopes on Viral Particles by Multiplex Dual-Reporter Strategy

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.

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

Learn to produce SARS-CoV-2 virus-like particles using HEK-293T cells for studying viral assembly and drug screening without biosafety level 3 requirements.

Production of a SARS-CoV-2 Virus-Like-Particle System to Investigate Viral Life Cycles In Vitro

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