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

Summary

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.

Abstract

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.

Introduction

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 cell^2,3. 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 development^9,10,11.

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'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.

Protocol

1. Generation of SC2-VLPs

1. Seed ~3.0 × 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.
2. Culture the HEK-293T cell at 37 °C, 5% CO₂ for about 24 h, and check the cell confluency under the microscope. Proceed if ~70% confluent.
3. Dilute 60 µL of PEI from 1 mg/mL stock to 200 µL with serum-freemedium.
4. Add 6.7 µg of N plasmid, 10 µg of Luc-T20 plasmid, 0.016 µg of S plasmid, and 3.3 µg of M-IRES-E plasmid into 200 µL of serum-free medium (see Supplemental File 1).
5. 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.
6. Carefully drop the transfection solution from step 1.5 onto HEK-293T cells, and gently swirl the tissue culture plate for thorough mixing.
7. Exchange the cell culture medium 6 h post infection with the DMEM complete medium, and incubate the transfected HEK-293T cells at 37 °C, 5% CO₂ for 48 h.
8. Collect the supernatant of the infected HEK-293T cells, which contains the SC2-VLPs, and then filter the supernatant through a 0.45 µm 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's specificity for HEK-293T cells.

2. Examination of SC2-VLP effectiveness

NOTE: The HEK-293T cell line stably expressing ACE2 and TMPRSS2 was established using a lentiviral transduction approach^14,15. 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).

1. Seed 4.0 × 10⁴ HEK-293T cells with stable expression of ACE2 and TMPRSS2 in a 96-well plate and add 50 µL of SC2-VLP medium from step 1.8.
2. Incubate the 96-well tissue culture plate at 37 °C with 5% CO₂ for 24 h.
3. Remove the medium from the 96-well plate, and wash once with 100 µL of PBS buffer at 37 °C, containing 137 mM NaCl, 2.7 mM KCl, 10 mM Na₂HPO₄, and 1.8 mM KH₂PO₄.
4. Lyse the HEK-293T ACE2/TMPRSS2 cells with 20 µL 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′,N′-tetraacetic acid, 1.25 mg/mL lysozyme, 2.5 mg/mL BSA, 10% glycerol, and 1% Triton X-100).
5. Spin the 96-well plate at 4,000 × g for 15 min at 4 °C in a refrigerated microplate centrifuge, and then immediately transfer the plate to an ice bath.
6. Take 100 µL of reconstituted luciferase assay buffer to an opaque white 96-well plate, and add 20 µL of lysate from step 2.5. Mix them briefly by pipetting up and down 2-3x.
7. 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.

3. Examination of the SC2-VLP composition

1. 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.
2. Keep the mixture on an orbital shaker, and slowly mix the solution at 4 °C overnight.
3. Centrifuge the solution at 4 °C, 2,000 × 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.

4. Subcellular localization analysis of S and its mutants in SC2-VLP-producing cells

1. Seed ~3.0 × 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.
2. 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 µg, Luc-T20 plasmid: 2 µg, S plasmid: 0.0032 µg, and M-IRES-E plasmid: 0.66 µg.
3. 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.
4. 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.
5. 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.
6. Add ~200 µL of primary antibody solution (enough to cover the glass bottom) and incubate at 4 °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-Sec61β 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.
7. Remove the primary antibody solution, and then wash the cells for 3 x 5 min with 1 mL of PBS at RT.
8. 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.
9. Stain nuclei with 2.5 µg/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.
10. 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%.

Results

The egress and assembly steps of the SARS-CoV-2 life cycle have been less studied than the infection and replication steps^17,18. 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 ...

Discussion

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...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Bovine Serum Albumin (BSA)	Cell Signaling Technology	9998S
Confocal Laser Scanning Microscope	Olympus	FV3000
DMEM	Corning	10-013-CV
Fetal Bovine Serum (FBS)	Thermofisher	A5670402
Goat Anti-Mouse IgG H&L (Alexa Fluor 488) antibody	Invitrogen	A11001	dilution ratio: IF 1:1000
Goat Anti-Rabbit IgG H&L (Alexa Fluor 594) antibody	Abcam	ab150080	dilution ratio: IF 1:1000
HEK-293T cell line	National Infrastructure of Cell Line Resource (NICR)	NICR-293T-001	To ensure high transfection efficiency and optimal SC2-VLP production, HEK-293T cells should be maintained at low passage numbers (≤ P10).
Hoechst 33342	Invitrogen	H1399	Working concentration: 2.5 μg/mL
Luciferase Reporter Assay System	Promega	E1500
Luc-T20	Addgene	177941
Mouse monoclonal anti-GAPDH	Proteintech	60004-1-Ig	dilution ratio: WB 1:20,000
Mouse monoclonal anti-S RBD	Abclonal	A23771	dilution ratio: IF 1:200
OptiMEM	Thermofisher	31985070	serum-free medium for transfection
PEG3350	Sigma-Aldrich	P3635
PEG8000	Sigma-Aldrich	P2139
Penicillin-Streptomycin	Thermofisher	15140122
Polyethyleneimine (PEI)	Thermofisher	43896.01
Promega passive lysis buffer	Promega	E1941
Rabbit polyclonal anti-ACE2	Proteintech	21115-1-AP	dilution ratio: WB 1:1000
Rabbit polyclonal anti-ERGIC53	Proteintech	13364-1-AP	dilution ratio: IF 1:200
Rabbit polyclonal anti-GM130	Proteintech	11308-1-AP	dilution ratio: IF 1:200
Rabbit polyclonal anti-S	Abcam	ab272504	dilution ratio: WB 1:1000
Rabbit polyclonal anti-Sec61β	Proteintech	15087-1-AP	dilution ratio: IF 1:200
Rabbit polyclonal anti-TMPRSS2	Abcam	ab109131	dilution ratio: WB 1:1000
SARS-CoV-2 M-IRES-E plasmid	Addgene	177938
SARS-CoV-2 N plasmid	Addgene	177959
SARS-CoV-2 Nucleoprotein Rabbit mAb	Abclonal	A21042	dilution ratio: WB 1:4000
SARS-CoV-2 S plasmid	Addgene	177960
Secondary Antibody, HRP, Goat anti-Mouse IgG (H+L)	Invitrogen	31460	dilution ratio: WB 1:5000
Secondary Antibody, HRP, Goat anti-Rabbit IgG (H+L)	Invitrogen	31430	dilution ratio: WB 1:5000
SpectraMax i3x plate reader	Molecular Devices	SpectraMax i3x