Generation and Characterization of recombinant SARS-CoV-2 expressing reporter genes

Abstract

The emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the pathogen responsible of coronavirus disease 2019 (COVID-19), has devastated public health services and economies worldwide. Despite global efforts to contain the COVID-19 pandemic, SARS-CoV-2 is now found in over 200 countries and has caused an upward death toll of over 1 million human lives as of November 2020. To date, only one Food and Drug Administration (FDA)-approved therapeutic drug (Remdesivir) and a monoclonal antibody, MAb (Bamlanivimab) are available for the treatment of SARS-CoV-2. As with other viruses, studying SARS-CoV-2 requires the use of secondary approaches to detect the presence of the virus in infected cells. To overcome this limitation, we have generated replication-competent recombinant (r)SARS-CoV-2 expressing fluorescent (Venus or mCherry) or bioluminescent (Nluc) reporter genes. Vero E6 cells infected with reporter-expressing rSARS-CoV-2 can be easily detected via fluorescence or luciferase expression and display a good correlation between reporter gene expression and viral replication. Moreover, rSARS-CoV-2 expressing reporter genes have comparable plaque sizes and growth kinetics to those of wild-type virus, rSARS-CoV-2/WT. We used these reporter-expressing rSARS-CoV-2 to demonstrate their feasibility to identify neutralizing antibodies (NAbs) or antiviral drugs. Our results demonstrate that reporter-expressing rSARS-CoV-2 represent an excellent option to identify therapeutics for the treatment of SARS-CoV-2, where reporter gene expression can be used as valid surrogates to track viral infection. Moreover, the ability to manipulate the viral genome opens the feasibility of generating viruses expressing foreign genes for their use as vaccines for the treatment of SARS-CoV-2 infection.IMPORTANCE Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the pathogen that causes coronavirus disease 2019 (COVID-19), has significantly impacted the human health and economic status worldwide. There is an urgent need to identify effective prophylactics and therapeutics for the treatment of SARS-CoV-2 infection and associated COVID-19 disease. The use of fluorescent- or luciferase-expressing reporter expressing viruses has significantly advanced viral research. Here, we generated recombinant (r)SARS-CoV-2 expressing fluorescent (Venus and mCherry) or luciferase (Nluc) reporter genes and demonstrate that they represent an excellent option to track viral infections in vitro. Importantly, reporter-expressing rSARS-CoV-2 display similar growth kinetics and plaque phenotype that their wild-type counterpart (rSARS-CoV-2/WT), demonstrating their feasibility to identify drugs and/or neutralizing antibodies (NAbs) for the therapeutic treatment of SARS-CoV-2. Henceforth, these reporter-expressing rSARS-CoV-2 can be used to interrogate large libraries of compounds and/or monoclonal antibodies (MAb), in high-throughput screening settings, to identify those with therapeutic potential against SARS-CoV-2.

FIG 1

rSARS-CoV-2 expressing reporter genes. (A) Schematic representation of rSARS-CoV-2 expressing Venus (green box), mCherry (red box), and Nluc (blue box) reporter genes instead of the viral ORF 7a. The molecular sizes of the three reporter genes are indicated. The locations of other viral proteins and untranslated regions (UTR) are also shown. (B) Genetic characterization of reporter-expressing rSARS-CoV-2. Vero E6 cells were mock infected or infected (MOI, 0.01) with WT or reporter-expressing rSARS-CoV-2. At 72 h postinfection, total RNA collected from cells was used to amplify, using RT-PCR, the viral NP, the ORF 7a region, and the different reporter genes (Venus, mCherry, or Nluc). Primers used for this RT-PCR analysis are shown on the left. The molecular sizes (base pairs) of the RT-PCR amplified products are shown on the right.

FIG 2

Characterization of reporter-expressing rSARS-CoV-2. (A) Fluorescence expression. Vero E6 cells were mock infected or infected (MOI of 0.01) with WT and Venus- or mCherry-expressing rSARS-CoV-2. At 48 h postinfection, cells were fixed and permeabilized, visualized for Venus (left) or mCherry (right) expression, and immunostained with a SARS-CoV NP MAb (1C7). DAPI was used for nuclear staining. Merged images for Venus (left) or mCherry (right), viral NP, and DAPI are presented. Representative images are shown. Magnification, ×20. Bar, 100 μm. (B) Nluc expression. Vero E6 cells were mock infected or infected (MOI of 0.01) with WT and Nluc-expressing rSARS-CoV-2. At 48 h postinfection, Nluc expression in tissue culture supernatants was analyzed using a Synergy LX microplate reader (BioTek). (C) Western blotting. Vero E6 cells were mock infected or infected (MOI 0.01) with WT and Venus (left)-, mCherry (center)-, or Nluc (right)-expressing rSARS-CoV-2. At 48 h postinfection, viral NP and reporter gene protein expression levels were analyzed using specific antibodies. An antibody against beta-actin was used as an internal control. The sizes of molecular markers are shown on the right of each blot.

FIG 3

Viral growth kinetics and plaque phenotype. (A) Fluorescence expression. Vero E6 cells were infected (MOI of 0.01) with WT (left), Venus-expressing (center), and mCherry-expressing (right) rSARS-CoV-2. At 12, 24, 48, 72, and 96 h postinfection. fluorescence protein expression was determined using a fluorescence microscope. Representative images are shown. Magnification, ×20. Bar, 100 μm. (B) Nluc expression. Vero E6 cells were mock infected or infected (MOI of 0.01) with WT and Nluc-expressing rSARS-CoV-2. At the indicated times postinfection, Nluc expression in the tissue culture supernatants was analyzed using a Synergy LX microplate reader (BioTek). (C) Growth kinetics. Vero E6 cells were infected (MOI of 0.01) with WT or reporter-expressing rSARS-CoV-2. At 12, 24, 48, 72, and 96 h postinfection, the presence of infectious virus in the tissue culture supernatants was determined using a plaque assay. (D) Plaque phenotype. Vero E6 cells were infected with ∼25 PFU of WT (left), Venus-expressing (middle), and mCherry-expressing (right) rSARS-CoV-2. At 72 h postinfection, plaques were observed under a fluorescence microscope to detect Venus or mCherry expression. In the case of rSARS-CoV-2/WT-infected cells, images correspond to fluorescence filters to detect Venus (left) or mCherry (right) expression. Thereafter, viral plaques were detected using the 1C7 SARS-CoV NP MAb. A selected number (n = 6) of plaques were used to determine the percentage of viral plaques expressing fluorescent proteins (Venus or mCherry). Magnification, ×4. Bar, 750 μm.

FIG 4

Reporter-based microneutralization assay for the identification of antivirals. Vero E6 cells (96-well plate format, ∼4 × 10⁴ cells/well, triplicates) were infected with 100 PFU of Venus (A and B), mCherry (C and D), Nluc (E), or WT (F) rSARS-CoV-2. After 1 h viral absorption, postinfection medium containing 3-fold serial dilutions of remdesivir (starting concentration, 50 μM) was added to the cells. At 24 h postinfection, cells were fixed and visualized for Venus (B) and mCherry (D) expression using a fluorescence microscope. In the case of cells infected with rSARS-CoV-2 expressing Nluc, luciferase expression in the tissue culture supernatant was determined at 48 h postinfection using a luciferase assay and a Synergy LX microplate reader (BioTek) (E). For the detection of rSARS-CoV-2/WT, the amount of virus was determined by plaque assay using the SARS-CoV NP MAb 1C7 (F). The amount of viral infection for Venus-expressing, mCherry-expressing, or WT rSARS-CoV-2 (after IFA) was determined using fluorescent images of each well and quantified using Cell Profiler (Broad Institute) cell image analysis software. Nluc activity was quantified using Gen5 data analysis software (BioTek). The 50% effective concentration (EC₅₀) of remdesivir was determined using GraphPad Prism. Dashed lines indicate 50% viral inhibition. Data are means and SD from triplicate wells. Representative images are shown. Magnification, ×10. Bar, 300 μm.

FIG 5

Reporter-based microneutralization assay for the identification of NAbs: Vero E6 cells (96-well plate format, ∼4 × 10⁴ cells/well, triplicates) were infected with 100 PFU of Venus (A and B), mCherry (C and D), Nluc (E) or WT (F) rSARS-CoV-2. After 1 h viral absorption, postinfection medium containing 3-fold serial dilutions (starting concentration, 500 ng) of a SARS-CoV-2 NAb (1212C2) was added to the cells. At 24 h postinfection, cells were fixed and visualized for Venus (B) and mCherry (D) expression using a fluorescence microscope. In the case of cells infected with rSARS-CoV-2 expressing Nluc, luciferase expression in the tissue culture supernatant was determined at 48 h postinfection using a luciferase assay and a Synergy LX microplate reader (BioTek) (E). For the detection of rSARS-CoV-2/WT, the amount of virus was determined by plaque assay using the SARS-CoV NP MAb 1C7 (F). The amount of viral infection for Venus-expressing, mCherry-expressing, and WT rSARS-CoV-2 (after IFA) was determined using fluorescent images of each well and quantified using Cell Profiler (Broad Institute). Nluc was quantified using Gen5 data analysis software (BioTek). The 50% neutralizing titer (NT₅₀) of 1212C2 was determined using GraphPad Prism. Dashed lines indicate 50% viral neutralization. Data are means and SD from triplicate wells. Representative images are shown. Magnification, ×10. Bar, 300 μm.

FIG 6

Genetic stability of fluorescence-expressing rSARS-CoV-2. (A) Plaque assay. Fluorescence-expressing rSARS-CoV-2 constructs were passaged up to 5 times in Vero E6 cells, and infectious-virus-containing tissue culture supernatants from passages 3 to 5 (P3 to P5) were assessed for Venus or mCherry expression at 72 h postinfection, before immunostaining with the SARS-CoV NP MAb 1C7. The percentage of reporter-expressing viruses was determined from ∼40 to 50 viral plaques per passage. Representative images of immunostaining and fluorescence obtained from each P3 to P5 viral plaque are shown. Magnification, ×4. Bar, 750 μm. (B) Sequence analysis. Reporter-expressing rSARS-CoV-2 nonreference allele frequencies from virus stock (P3) and after two consecutive passages in Vero cells (P4 and P5) were determined using next-generation sequencing, using modified rSARS-CoV-2/WT reference genomes. Nonreference alleles that were below 1% of reads are not shown, and those greater than 20% are indicated in red.