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. Author manuscript; available in PMC: 2023 Jun 6.
Published in final edited form as: Methods Mol Biol. 2022;2524:235–248. doi: 10.1007/978-1-0716-2453-1_18

Bioluminescent and Fluorescent Reporter-Expressing Recombinant SARS-CoV-2

Desarey Morales Vasquez 1, Kevin Chiem 1, Chengjin Ye 1, Luis Martinez-Sobrido 1,2
PMCID: PMC10243362  NIHMSID: NIHMS1901127  PMID: 35821476

Abstract

Reporter-expressing recombinant severe acute respiratory syndrome coronavirus 2 (rSARS-CoV-2) represents an excellent tool to understand the biology of and ease studying viral infections in vitro and in vivo. The broad range of applications of reporter-expressing recombinant viruses is due to the facilitated expression of fluorescence or bioluminescence readouts. In this chapter, we describe a detailed protocol on the generation of rSARS-CoV-2 expressing Venus, mCherry, and NLuc that represents a valid surrogate to track viral infections.

Keywords: Recombinant severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), Coronavirus, Coronavirus disease 2019 (COVID-19), Bioluminescence (BL), Fluorescence (FL), NanoLuc (NLuc), Venus, mCherry, Reporter genes, Reporter virus

1. Introduction

In December 2019, there were a reported, increased number of patients with pneumonia of unknown etiology in Wuhan, China [1, 2]. The Coronavirus Research Group (CSG) of the International Committee for the classification of newly identified viruses found this etiological agent to be related to severe acute respiratory syndrome coronavirus (SARS-CoV) and therefore designated it as severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) [3]. Additionally, the World Health Organization (WHO) named the disease caused by SARS-CoV-2 as coronavirus disease 2019 (COVID-19) [3]. The emergence of SARS-CoV-2 was followed by a period of evolutionary stability, but, since late 2020/early 2021, its evolution has been characterized by the emergence of mutant strains named variants of concern (VoC) or variants of interest (VoI) that have impacted the virus characteristics and the still ongoing COVID-19 pandemic [4, 5]. Since its emergence, there has been a limited number of prophylactic (vaccines) and/or therapeutic (drugs) options available for the treatment of COVID-19. To date, only one United States (US) Food and Drug Administration (FDA) therapeutic antiviral (remdesivir) and one Emergency Use Authorization (EUA) drug (baricitinib), in combination with remdesivir, have been approved for the treatment of SARS-CoV-2 infection. The US FDA has also given EUA for monoclonal antibodies REGEN-COV (casirivimab and imdevimab, administered together), sotrovimab, bamlanivimab, and etesevimab [6–10]. Recently, the US FDA has approved the Pfizer-BioNTech vaccine for the prevention of COVID-19, and the Moderna and Janssen COVID-19 vaccines are available under EUA [11–13].

SARS-CoV-2 is an enveloped virus consisting of a positive-sense, single-stranded RNA genome of approximately 30 kb (see Fig. 1) [14]. Translation of two overlapping open reading frames (ORFs) produces polypeptides that are cleaved into 16 non-structural proteins (nsps), and the different subgenomic RNAs in the genome encode 4 structural proteins, envelope (E), spike (S), nucleocapsid (N), and membrane (M), and 6 accessory ORF proteins, 3a, 6, 7a, 7b, 8, and 10 (see Fig. 1a) [15]. The lipid bilayer surface of SARS-CoV-2 is decorated by the E, M, and S proteins, and inside the virion is the viral RNA that is encapsulated by the N protein (see Fig. 1b). The structural proteins are essential components of the virion and are involved in viral entry, assembly, virion production, and viral budding [16–19]. The nsps are mainly involved in viral RNA synthesis and processing, while the ORF accessory proteins 3a, 6, 7a, 7b, 8, and 10 (see Fig. 1a) play important roles, among others, in regulating stress response, innate immunity, autophagy, and apoptosis [20–24].

Fig. 1.

Fig. 1

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Genomic organization and virion structure of SARS-CoV-2. (a) Schematic representation of SARS-CoV-2 genome organization: SARS-CoV-2 has a genome of approximately 30,000 nucleotides made up of 5′ and 3′ non-coding untranslated regions (UTR), ORF1a polyprotein (blue), ORF1b polyprotein (orange), and structural spike (S), envelop (E), membrane (M), and nucleocapsid (N) proteins, along with the accessory open reading frame (ORF) proteins 3a, 6, 7a, 7b, 8, and 10 (green). ORF1a polyprotein is processed into 11 non-structural proteins (nsps, blue). ORF1b is generated through a ribosomal shift and cleaved into nsp12-nsp16 (orange). (b) Schematic representation of SARS-CoV-2 virion: SARS-CoV-2 has a lipid bilayer made of the E, M, and S proteins on the surface. Inside the virion is the positive-sense single-stranded RNA viral genome that is encapsulated by the viral N protein

Due to the large size of the CoV genome, there are critical hindrances for the development of reverse genetic systems that are similar to those used for other smaller RNA viruses [25]. Several reverse genetic approaches for CoVs have been previously described, including the use of multiple plasmid systems, infectious clones, and yeast-based synthetic genomics [26–28]. The bacterial artificial chromosome (BAC)-based SARS-CoV-2 reverse genetics system is based on the E. coli pBeloBAC11 vector that is capable of maintaining DNA fragments of >300 kb [29]. Successful cloning with high efficiency, easy manipulation, and stability of cloned DNA has been shown for large RNA viruses, including other CoVs [29–32]. Previously, we have described the feasibility of generating replication-competent recombinant (r)SARS-CoV-2 using a BAC-based reverse genetics system for the USA-WA1/2020 SARS-CoV-2 strain [32]. The full-length copy of SARSCoV-2 USA-WA1/2020 was assembled downstream of the cytomegalovirus (CMV) promoter into the pBeloBAC11 plasmid by assembling together five synthetic cDNA fragments (1 Fig. 2a) [32]. The viral genome is followed by the hepatitis delta virus ribozyme (Rz) to produce synthetic RNAs with an accurate 3′ end and the bovine growth hormone (bGH) polyadenylation signal for sequence termination (see Fig. 2a) [30, 32]. Cells transfected with the BAC SARS-CoV-2 USA-WA1/2020 initiate viral RNA production from the CMV promoter in the nuclei of transfected cells by the cellular RNA polymerase II (see Fig. 2b) [32]. Further amplification steps in the cytoplasm are driven by the viral polymerase [32].

Fig. 2.

Fig. 2

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BAC-based SARS-CoV-2 reverse genetics. (a) Schematic representation of the BAC to rescue rSARS-CoV-2: the SARS-CoV-2 full-length genome is flanked at the 5′ end by the cytomegalovirus (CMV) polymerase II-driven promoter and at the 3′ end by the hepatitis delta ribozyme (Rz) and bovine growth hormone (bGH) polyadenylation signal within the pBeloBAC11 plasmid. (b) Schematic representation of the experimental approach to generate rSARS-CoV-2: Vero E6 cells (1.2 × 106 cells/well, 6-well plates, triplicates) are transfected, using LPF2000, with pBeloBAC11-SARS-CoV-2 overnight at 37 °C in the CO2 incubator. At 24 h post-transfection (day 2), media are replaced for post-infection media. Four days post-transfection (day 4), Vero E6 cells are scaled up into T75 flasks, and after 72 h, tissue culture supernatants are collected to infect fresh monolayers of Vero E6 cells to evaluate the presence of the recombinant virus by cytopathic effect (CPE) and immunofluorescence. In the case of fluorescent viruses, fluorescent microscopy can assess the presence of the rSARS-CoV-2 (Venus and mCherry). In the case of NLuc-expressing rSARS-CoV-2, successful viral rescue can be determined in the tissue culture supernatant using the Nano-Glo luciferase substrate and a luciferase plate reader

Reporter genes have advanced the ability to study viral infections, pathogenesis, and disease [33–36]. The most common reporter genes used are fluorescent or bioluminescent proteins due to their stability, detectability, and sensitivity [37, 38]. Fluorescent proteins have been shown to be a better option to identify infected cells and track viral infections in vitro and ex vivo [39–44]. On the other hand, bioluminescent proteins have been shown to be optimal for quantification studies in vitro and for whole animal imaging using in vivo imaging systems (IVIS) [39–44]. Using our previously described BAC-based reverse genetics, we generated fluorescent monomeric mCherry and Venus and bioluminescent reporter (NLuc)-expressing rSARS-CoV-2 by replacing the viral ORF7a with mCherry (rSARS-CoV-2/mCherry), Venus (rSARS-CoV-2/Venus), or NLuc (rSARS-CoV-2/NLuc) genes (see Fig. 3) [32, 35]. We have also shown these reporter viruses replicate to the same extent as a rSARS-CoV-2 wild type (WT) that does not express a reporter gene (see Fig. 4).

Fig. 3.

Fig. 3

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Reporter-expressing rSARS-CoV-2. (a) Schematic representation of reporter-expressing rSARS-CoV-2: the rSARS-CoV-2 expressing Venus, mCherry, or NLuc is generated by inserting the reporter gene instead of the viral ORF7a protein. (b) Expression of reporters for fluorescence: Vero E6 cells (1.2 × 106 cells/well, 6-well plates, triplicates) were mock-infected or infected (multiplicity of infection, MOI 0.01) with wild-type (WT), Venus-, or mCherry-expressing rSARS-CoV-2. At 48 h post-infection, cells were visualized for confirming Venus (top) or mCherry (bottom) expression for fluorescence (FL). Representative images (20× magnification) are shown. Scale bar, 100 μm. (c) NLuc expression: Vero E6 cells were mock-infected or infected (MOI 0.01) with WT and NLuc-expressing rSARS-CoV-2. At 48 h post-infection, NLuc expression in tissue culture supernatants was analyzed using the Nano-Glo luciferase substrate (Promega) and the Synergy LX microplate reader (BioTek). (This figure has been modified from Chiem K. et al. [35])

Fig. 4.

Fig. 4

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Plaque assay and viral growth kinetics. (a) Plaque assay: Vero E6 cells (1.2 × 106 cells/well, 6-well plates) were infected with ~25 plaque-forming units (PFU) of WT (left), Venus (middle), and mCherry (right) rSARS-CoV-2. After 72 h, cells were incubated in 10% (v/v) neutral buffered formalin at 4 °C for 16 h for fixation and viral inactivation. Viral plaques were detected using the anti-SARS-CoV N protein monoclonal antibody 1C7C7. (b) Growth kinetics: Vero E6 cells (1.2 × 106 cells/well, 6-well plates, triplicates) were infected (MOI of 0.01) with WT or the reporter-expressing mCherry and Venus rSARS-CoV-2. At the indicated times post-infection, tissue culture supernatants were collected, and viral titers were determined by plaque assay and immunostaining. (This figure has been modified from Chiem K. et al. [35])

Replication-competent reporter-expressing rSARS-CoV-2 represents an excellent option to understand the biology and to facilitate the study of SARS-CoV-2 in vitro and in vivo. Traditional plaque reduction neutralization test (PRNT) and microneutralization test (MNT) assays are labor-intensive and require the use of secondary reagents (e.g., antibodies) to detect the presence of virus in infected cells [45, 46]. The use of replication-competent reporter-expressing viruses facilitates and decreases the timeline to confirm the presence of virus in infected cells, allowing to track viral infection [33, 36, 39, 47–49], testing vaccine efficacy [50, 51], identification of neutralizing antibodies and/or antiviral compounds [33–36], and conducting high-throughput screenings (see Fig. 5) [46, 52–54]. The broad range of applications of reporter-expressing recombinant viruses is due to the facilitated expression of fluorescent (Venus or mCherry) or bioluminescent (NLuc) readouts.

Fig. 5.

Fig. 5

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Applications of reporter viruses. Reporter-expressing rSARS-CoV-2 has broad applications for in vitro and in vivo (not shown) studies. For in vitro studies, reporter-expressing rSARS-CoV-2 can be used to track viral infections via fluorescence imaging, to test vaccine efficacy using reporter-based microneutralization assays using sera from vaccinated individuals, to identify neutralizing antibodies and compounds with antiviral activity using similar reporter-based microneutralization assays, and to interrogate large libraries of biological compounds using high-throughput screenings (HTS) to identify those with antiviral activity

In this chapter, we describe a protocol to generate replicating-competent rSARS-CoV-2 expressing fluorescent (Venus and mCherry) or bioluminescent (NLuc) reporters. We exemplify the characterization of these reporter-expressing recombinant viruses to track viral infections in cultured cells as well as their potential applications to facilitate SARS-CoV-2 studies in vitro.

2. Materials

2.1. Reagents and Labware

  1. 2% (w/v) agar.

  2. 2.5% (w/v) bovine serum albumin (BSA-PBS).

  3. Dulbecco’s modified Eagle medium (DMEM).

  4. DMEM/F-12 powder.

  5. 100× penicillin-streptomycin l-glutamine (PSG).

  6. Fetal bovine serum (FBS).

  7. Cell culture grade water.

  8. 5% (w/v) sodium bicarbonate (NaHCO3) solution.

  9. 1% (w/v) diethylaminoethyl (DEAE)-dextran.

  10. OptiMEM reduced serum medium.

  11. 10% (v/v) neutral buffered formalin.

  12. 0.5% (v/v) Triton X-100-PBS solution.

  13. Mouse anti-SARS-CoV nucleocapsid (N) protein monoclonal antibody 1C7C7 generated at the Center for Therapeutic Antibody Development at the Icahn School of Medicine at Mount Sinai (ISMMS) (Millipore Sigma, catalog number ZMS1075).

  14. Lipofectamine 2000 (LPF2000) as a transfection reagent.

  15. Nano-Glo luciferase substrate kit (Promega, catalog number N1110).

  16. VECTASTAIN ABC-HRP Kit, peroxidase (Vector Laboratories, catalog number PK-4000).

  17. DAB substrate kit, peroxidase (HRP), with nickel, (3,3-′-diaminobenzidine) (Vector Laboratories, catalog number SK-4100).

  18. 6-well plate for cell culture.

  19. 96-well microplate for cell culture.

  20. Polystyrene tissue culture flask (T75 flask).

  21. Polypropylene sterile conical tubes, 15-mL and 50-mL.

  22. Serological pipettes, 5-mL, 10-mL, and 25-mL.

  23. Universal pipette tips, 20-μL, 200-μL, and 1,000-μL.

  24. Microcentrifuge tube, 1.5-mL.

  25. Cryogenic vials, 2-mL.

  26. Polystyrene reservoirs, 50-mL.

  27. Humidified 5% (v/v) CO2 incubator.

  28. Cell maintenance media (DMEM supplemented with 10% (v/v) FBS and 1% (v/v) PSG).

  29. Post-infection media (DMEM supplemented with 2% (v/v) FBS and 1% (v/v) PSG).

  30. DMEM/F-12/agar mixture (DMEM-F12 with 1% (w/v) DEAE-dextran, 2% (w/v) agar, and 5% (w/v) NaHCO3).

2.2. Cells

  1. African green monkey kidney epithelial cells (Vero E6, CRL-1586) obtained from American Type Culture Collection (ATCC) (Bethesda, MD).

2.3. Virus

  1. rSARS-CoV-2 is generated based on the backbone of the USA-WA1/2020 strain (accession no. MN985325) using the previously described BAC-based reverse genetics system (see Note 1) [32].

2.4. Software

  1. Gen5 Software (BioTek, Ver. 3.11).

  2. GraphPad Prism (GraphPad Software Inc., Ver. 8.0).

2.5. Instrumentation

  1. EVOS M5000 imaging system (Thermo Fisher Scientific).

  2. Synergy LX microplate reader (BioTek).

3. Methods

3.1. Generation of rSARS-CoV-2 (See Fig. 2b)

  1. (OptiMEM-LPF2000) In a 1.5-mL microcentrifuge tube, add 250 μL of OptiMEM media with 8 μg of LPF2000 (2 μg LPF2000/μg BAC) per transfection. Incubate the OptiMEM-LPF2000 mixture for 5–10 min at room temperature (RT). During this incubation time, prepare the OptiMEM-BAC mixture.

  2. (OptiMEM-BAC) In a separate tube, mix 4 μg of the pBeloBAC11-SARS-CoV-2 -Venus, -mCherry, or -NLuc, and empty pBeloBAC11 as negative control, in a total volume of 50 μL of OptiMEM media (see Notes 2 and 3).

  3. Add 250 μL of OptiMEM-LPF2000 into the OptiMEM-BAC tubes, and incubate for ~30 min at RT.

  4. During the incubation, prepare the Vero E6 cells for transfection: i.e., warm up PBS, trypsin/EDTA solution, and cell maintenance media to 37 °C. Wash Vero E6 cells twice with 10 mL of PBS. Trypsinize the cells using 2 mL of trypsin/EDTA solution. Let the cells detach by incubating for ~5 min at 37 °C in the CO2 incubator. Resuspend the cells in 8–10 mL of cell maintenance media. Place the cell suspension in a 15-mL tube, and centrifuge the cells for 5 min at 500 × g. Remove the supernatant and resuspend the cells in 10 mL of fresh cell maintenance media. Count the cells using a hemocytometer. Adjust the cell concentration in the tube to ~1–2 × 106 cells/mL, and dispense 1 mL into individual tubes (see Notes 4 and 5).

  5. After 30-min incubation, add the incubated OptiMEM-LPF2000 and OptiMEM-BAC mixture to ~1–2 × 106 cells, and incubate ~5 min at RT.

  6. After incubation, transfer the OptiMEM-LPF2000 and OptiMEM-BAC cell mixture into individual wells in a fresh 6-well plate.

  7. Incubate the transfected cells in the 6-well plate at 37 °C for 14 h.

  8. After 14-h transfection, change the transfection media with the post-infection media (DMEM supplemented with 2% (v/v) FBS).

  9. Two days after changing the media, split the cells, and seed them into T75 flasks.

  10. After 72 h, collect the tissue culture supernatants, label as P0, and stored them at −80 °C.

  11. Before titration of viral stocks, aliquot the viral stocks in cryogenic vials for long-term storage (see Note 6).

  12. Confirm viral rescues by infecting fresh monolayers of Vero E6 cells (1.2 × 106 cells/well, 6-well plates, triplicates) using the supernatant P0 from step 10 and assessing the presence of cytopathic effect (CPE), Venus or mCherry fluorescence expression using fluorescent microscope, or NLuc expression for bioluminescence via luciferase activity (see Note 7).

3.2. Viral Titration by Plaque Assay

  1. Once viral rescue is confirmed, make a virus stock (P1) by infecting fresh Vero E6 cells (T75 flask format, 8.4 × 106 cells) with the supernatant from P0.

  2. After a 72-h infection, collect the tissue culture supernatant,and centrifuge for 10 min at 2,000 × g, 4 °C, to remove the cellular debris (see Note 8).

  3. Harvest and titrate supernatants of P1 for further in vitro and/or in vivo experiments.

  4. Determine viral titers by plaque assay (in plaque forming units (PFU) per milliliter) in Vero E6 cells (1.2 × 106 cells/well, 6-well plates, triplicates) (see Note 9).

  5. Infect confluent monolayers of Vero E6 cells (1.2 × 106 cells/well, 6-well plates, triplicates) with tenfold serial dilutions of reporter-expressing rSARS-CoV-2 for 1 h at 37 °C in the CO2 incubator.

  6. After viral absorption, overlay cells with DMEM/F-12/agar mixture, and incubate at 37 °C in the CO2 incubator for 72 h.

  7. Then submerge cells in 10% (v/v) neutral buffered formalin at 4 °C for 16 h for fixation and viral inactivation, followed by removing the agar overlay.

  8. To observe Venus and mCherry fluorescence expression, add PBS to each well, and image plates under a fluorescence microscope (EVOS M5000 imaging system) (see Fig. 3b).

  9. To assess NLuc expression, collect tissue culture supernatants, and measure the bioluminescence using the Nano-Glo luciferase assay kit and a Synergy LX microplate reader (see Fig. 3c). Take 25 μL of the tissue culture supernatant, and mix with 25 μL of Nano-Glo reagent, and read the bioluminescence intensities in the Synergy LX microplate reader following the manufacturer’s recommendations.

  10. (Perform immunostaining) To that end, permeabilize the cells with 0.5% (v/v) Triton X-100-PBS for 10 min at RT, and then block with 2.5% (v/v) BSA-PBS for 1 h at RT, and incubate at 37 °C for 1 h with the anti-SARS-CoV N protein monoclonal antibody 1C7C7 (see Notes 10 and 11).

  11. Develop plaques for visualization using the Vectastain ABC kit and DAB HRP substrate kit, following the manufacturer’s recommendations (see Fig. 4a).

3.3. Viral Growth Kinetics

  1. Infect confluent monolayers of Vero E6 cells (1.2 × 106 cells/well, 6-well plates, triplicates) at a multiplicity of infection (MOI) of 0.01 with rSARS-CoV-2/Venus, rSARS-CoV-2/mCherry, or rSARS-CoV-2/NLuc and rSARS-CoV-2/WT as control.

  2. After 1-h viral adsorption at 37 °C, replace the media with the post-infection media, and place the plates in the incubator.

  3. Determine viral titers in the tissue culture supernatants at different times post-infection (e.g., 12, 24, 48, 72, and 96 h post-infection) by plaque assay and immunostaining, using the anti-SARS-CoV N protein monoclonal antibody 1C7C7, as described above (see Fig. 4b) (see Note 12).

  4. Determine mean values and standard deviations (SD) using GraphPad Prism software (Ver. 8.0).

4. Notes

  1. Manipulation of SARS-CoV-2 should only be carried out in a biosafety level 3 (BSL3) facility with an appropriate engineering system designed to produce negative air pressure and enhance the safety of laboratory workers. All individuals working with SARS-CoV-2 must have undergone proper biosafety training to work at BSL3 laboratories and be cleared proficient to carry out procedures requiring working with the virus. Cell culture procedures are carried out in BSL2 and moved to BSL3 when ready for viral rescue and/or infection.

  2. pBeloBAC11 is produced using DH10B electrocompetent E. coli cells. To increase chances of a successful viral rescue, good preparations of the pBeloBAC11 are critical. We recommend determining BAC concentrations and purity by assessing the 260:280 nm ration for optimal viral rescue.

  3. We recommend transfecting Vero E6 cells with the pBeloBAC11 plasmids in triplicates to increase the chances of successful viral rescue.

  4. It is important to maintain and have fresh Vero E6 cells for a successful viral rescue.

  5. Additional cell lines beside Vero E6 cells can be used for rescuing rSARS-CoV-2. We have previously used similar approaches to those described in this protocol with Vero E6 cells to generate rSARS-CoV-2 in HEK293T cells expressing hACE2.

  6. We recommend making small-volume aliquots to prevent multiple thaw cycles, which may reduce virus titers. We usually aliquot the virus stock in 500-μL aliquots/tube to avoid repeated freeze and thaw.

  7. For reporter viruses, successful viral rescue can be determined by fluorescent microscopy (Venus and mCherry) or by assessing NLuc expression in the tissue culture supernatant in a luciferase plate reader.

  8. We recommend confirming the sequence of the reporter-expressing rSARS-CoV-2 from the generated virus stock using next-generation sequencing [35].

  9. Vero E6 cells can yield up to ~106–107 PFU/mL of rSARS-CoV-2.

  10. Cells can be blocked overnight at 4 °C.

  11. Other monoclonal or polyclonal antibodies against the N or other viral proteins can be used for immunostaining in the plaque assays.

  12. In the case of fluorescent viruses, viral titers can also be determined by fluorescent focus units (FFU)/mL. In the case of NLuc virus, viral titers can be determined by median tissue culture infectious dose (TCID50).

Acknowledgments

We would like to thank the members at Texas Biomedical Research Institute for their efforts in keeping them fully operational during the COVID-19 pandemic and the Institutional Biosafety Committee at Texas Biomedical Research Institute for reviewing our protocols in a time-efficient manner. SARS-CoV-2 research in the Martinez-Sobrido’s laboratory is currently supported by the NIAID/NIH grants RO1AI161363–01, RO1AI161175–01A1, and R43AI165089–01; the Department of Defense (DoD) grants W81XWH2110095 and W81XWH2110103; the San Antonio Partnership for Precision Therapeutic; the Texas Biomedical Research Institute Forum; the University of Texas Health Science Center at San Antonio; the San Antonio Medical Foundation; and the Center for Research on Influenza Pathogenesis and Transmission (CRIPT), a NIAID-funded Center of Excellence for Influenza Research and Response (CEIRR, contract # 75N93021C00014).

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