Establishment of a reverse genetics system for SARS-CoV-2 using circular polymerase extension reaction

Abstract

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has been identified as the causative agent of coronavirus disease 2019 (COVID-19). Although multiple mutations have been observed in SARS-CoV-2, functional analysis of each mutation of SARS-CoV-2 has been limited by the lack of convenient mutagenesis methods. In this study, we establish a PCR-based, bacterium-free method to generate SARS-CoV-2 infectious clones. Recombinant SARS-CoV-2 could be rescued at high titer with high accuracy after assembling 10 SARS-CoV-2 cDNA fragments by circular polymerase extension reaction (CPER) and transfection of the resulting circular genome into susceptible cells. The construction of infectious clones for reporter viruses and mutant viruses could be completed in two simple steps: introduction of reporter genes or mutations into the desirable DNA fragments (∼5,000 base pairs) by PCR and assembly of the DNA fragments by CPER. This reverse genetics system may potentially advance further understanding of SARS-CoV-2.

Keywords: CPER; SARS-CoV-2; infectious clone; mutagenesis; reverse genetics.

Graphical abstract

Figure 1

Establishment of CPER-based reverse genetics for SARS-CoV-2 (A) Schematic representation of a CPER approach for the generation of recombinant SARS-CoV-2. A total of 9 fragments covering the full-length of the SARS-CoV-2 genome were amplified and then assembled with a UTR linker fragment by CPER. The resulting CPER products were transfected into the susceptible cells. (B) HEK293-3P6C33 cells were transfected with the CPER product, and the bright-field image was acquired at 7 dpt (left). As a negative control, the CPER product obtained without fragment F9/10 was transfected into cells, and the image was obtained at 7 dpt (right). (C) Genetic markers (2 silent mutations, A7486T and T7489A) in the recombinant SARS-CoV-2 genome. (D) Comparison of the growth kinetics of the recombinant SARS-CoV-2 with those of the original isolate. VeroE6/TMPRSS2 cells were infected with the viruses (MOI = 0.001 or 0.01), and infectious titers were determined from 12 to 48 hpi. (E) Northern blot analyses of subgenomic RNAs. RNAs extracted from cells infected with the parental virus and the recombinant SARS-CoV-2 were subjected to northern blot analyses.

Figure 2

Characterization of SARS-CoV-2 recombinants possessing reporter genes and mutations (A) Gene structure of recombinant SARS-CoV-2 carrying the sfGFP or HiBiT gene. (B) Growth kinetics of wild-type SARS-CoV-2 (WT) and SARS-CoV-2 carrying sfGFP (GFP). VeroE6/TMPRSS2 cells were infected with the viruses (MOI = 0.001), and infectious titers were determined at the indicated time points. (C) Fluorescent signal in VeroE6/TMPRSS2 cells infected with the WT virus and GFP virus was observed for 36 hpi. (D) Growth kinetics of the WT virus and recombinant virus possessing the HiBiT gene in ORF6 (ORF6-HiBiT). Titers in the culture supernatants of VeroE6/TMPRSS2 cells, infected with the viruses (MOI = 0.01), were measured for 48 h. (E) Luciferase activities in VeroE6/TMPRSS2 cells infected with the WT virus and ORF6-HiBiT virus were determined from 12 to 48 hpi. (F) Infectious titers of the WT virus or mutant virus, harboring a substitution of D614 to G in the spike protein (D614G), were determined at the indicated time points.