Generation of Recombinant SARS-CoV-2 Using a Bacterial Artificial Chromosome

Abstract

SARS-CoV-2, the causative agent of COVID-19, has been responsible for a million deaths worldwide as of September 2020. At the time of this writing, there are no available US FDA-approved therapeutics for the treatment of SARS-CoV-2 infection. Here, we describe a detailed protocol to generate recombinant (r)SARS-CoV-2 using reverse-genetics approaches based on the use of a bacterial artificial chromosome (BAC). This method will allow the production of mutant rSARS-CoV-2-which is necessary for understanding the function of viral proteins, viral pathogenesis and/or transmission, and interactions at the virus-host interface-and attenuated SARS-CoV-2 to facilitate the discovery of effective countermeasures to control the ongoing SARS-CoV-2 pandemic. © 2020 Wiley Periodicals LLC. Basic Protocol: Generation of recombinant SARS-CoV-2 using a bacterial artificial chromosome Support Protocol: Validation and characterization of rSARS-CoV-2.

Keywords: COVID-19; SARS-CoV-2; bacterial artificial chromosome; coronavirus; infectious clone; reverse genetics; virus rescue.

Figure 1

Virion structure and genomic organization of SARS‐CoV‐2. (A) Schematic representation of SARS‐CoV‐2 virion: The lipid bilayer surface of SARS‐CoV‐2 is decorated by the envelope (E), membrane protein (M), and spike (S) glycoproteins. S is the viral protein responsible for attachment and entry. Inside the virion is the positive‐sense, single‐stranded RNA viral genome that is encapsulated by the viral nucleocapsid (N) protein. (B) Schematic representation of SARS‐CoV‐2 genome organization: SARS‐CoV‐2 has a genome of ∼29,930 nucleotides. At the end of the viral genome are located the 5′ and 3′ untranslated non‐coding regions (NCR). The SARS‐CoV‐2 genome encodes for the viral ORF1a (orange) and ORF1b (blue). ORF1a polyprotein is cleaved into leader protein (SAR2), nsp2, nsp4, nsp4, 3C‐like proteinase (SAR3), nsp6, nsp7, nsp8, nsp9, nsp10, and nsp11. ORF1b polyprotein is generated through a ribosomal frameshift, and cleaved into the RNA‐dependent RNA polymerase, RdRp (nsp12), helicase, 3′‐to‐5′ exonuclease (SAR4), endoRNase, and 2′‐O‐ribose methyltransferase (SAR5). After the ORF1ab, SARS‐CoV‐2 genome encodes for the viral structural S, E, M and N proteins, along with the accessory proteins 3a, 6, 7a, 7b, 8, and 10.

Figure 2

Schematic representation of the pBAC plasmid to rescue rSARS‐CoV‐2: The full‐length cDNA of the SARS‐CoV‐2 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. The entire ∼30,815‐bp construct was inserted into the pBeloBAC11 plasmid using Pcil and HindIII restriction sites. The pBeloBAC11 plasmid is an E. coli vector commonly used to generate BACs because it can support the insertion of large DNA fragments as a single copy in cells. The pBeloBAC11 contains a chloramphenicol resistance (Cm^R) gene as a selective marker. Viral proteins are those previously described in Figure 1.

Figure 3

Generation of rSARS‐CoV‐2: Vero E6 cells (6‐well plate format, 1.2 × 10⁶ cells/well, triplicates) are transfected, using LPF2000, with 2 µg of pBeloBAC11‐SARS‐CoV‐2 (Fig. 2) overnight in a tissue culture incubator at 37°C with 5% CO₂. At 6‐8 hr post‐transfection, the transfection medium is replaced by post‐infection medium. At day 4 post‐transfection, Vero cells are scaled up into T‐75 flasks. After an additional 48 hr, CPE should be detected, and tissue culture supernatants are collected to evaluate the presence of recombinant virus (Fig. 4).

Figure 4

Detection and in vitro characterization of rSARS‐CoV‐2. (A) CPE: At 48 hr after scaling up transfected Vero E6 cells into T‐75 flasks (Fig. 3), CPE can be already observed. Representative images of mock‐infected, SARS‐CoV‐2‐infected, and transfected Vero E6 are shown. Scale bars are 400 µm. (B) IFA: Vero E6 cells (12‐well plate format, 0.5 × 10⁶ cells/well, triplicates) were infected with tissue culture supernatants from transfected Vero E6 cells. Mock‐infected and SARS‐CoV‐2‐infected Vero E6 cells were included as internal controls. At 24 hr post‐infection, cells were fixed with 10% neutral buffered formalin. After fixation for 16 hr, cells were permeabilized with 0.5% Triton X‐100 for 10 min. Cells were then washed three times with 1× PBS and incubated with 1 μg/ml of an anti‐SARS NP MAb 1C7 at 37°C. After 1 hr incubation with the primary NP 1C7 MAb, cells are washed three times with 1× PBS and incubated with an anti‐mouse FITC‐conjugated secondary antibody at 37°C. After 1 hr, cells are washed three times with 1× PBS and observed under a fluorescent microscope. DAPI was used to stain the nucleus. Scale bars are 100 µm. (C) Plaque assay: Confluent monolayers of Vero E6 cells (6‐well plate format, 1.2 × 10⁶ cells/well, triplicates) were infected with SARS‐CoV‐2 or rSARS‐CoV‐2 at 37°C for 1 hr and overlaid with agar. After 72 hr in a 37°C incubator with 5% CO₂, cells were fixed in 10% neutral buffered formalin for 16 hr before agar was removed. Next, cells were permeabilized with 0.5% Triton X‐100 for 10 min and prepared for immunostaining as previously described using the anti‐NP MAb (1C7) and vector kits (Vectastain ABC kit and DAB HRP substrate kit; Vector Laboratories). (D) Viral growth kinetics: Vero E6 cells (12‐well plate format, 0.5 × 10⁶ cells/well, triplicates) were infected (MOI of 0.01) with SARS‐CoV‐2 (black) or rSARS‐CoV‐2 (red) and placed in a 37°C incubator with 5% CO₂ for 4 days. At 12, 24, 48, 72, and 96 hr post‐infection, viral titers in supernatants were determined by plaque assay (PFU/ml). Error bars indicate the standard deviations from three separate experiments. The dashed black line indicates the limit of detection (10 PFU/ml).