Reverse genetic systems of SARS-CoV-2 for antiviral research

Abstract

Reverse genetic systems are widely used to engineer recombinant viruses with desired mutations. In response to the COVID-19 pandemic, four types of reverse genetic systems have been developed for SARS-CoV-2: (i) a full-length infectious clone that can be used to prepare recombinant SARS-CoV-2 at biosafety level 3 (BSL3), (ii) a trans-complementation system that can be used to produce single-round infectious SARS-CoV-2 at BSL2, (iii) an attenuated SARS-CoV-2 vaccine candidate (with deletions of viral accessory genes) that may be developed for veterinary use as well as for antiviral screening at BSL2, and (iv) replicon systems with deletions of viral structural genes that can be used at BSL2. Each of these genetic systems has its advantages and disadvantages that can be used to address different questions for basic and translational research. Due to the long genomic size and bacteria-toxic sequences of SARS-CoV-2, several experimental approaches have been established to rescue recombinant viruses and replicons, including (i) in vitro DNA ligation, (ii) bacterial artificial chromosome (BAC) system, (iii) yeast artificial chromosome (YAC) system, and (iv) circular polymerase extension reaction (CPER). This review summarizes the current status of SARS-CoV-2 genetic systems and their applications for studying viral replication, pathogenesis, vaccines, and therapeutics.

Fig. 1

Diagrams for an infectious cDNA clone, a trans complementary genetic system, and a live attenuated SARS-CoV-2. (A) An in vitro ligation approach for making recombinant SARS-CoV-2. The genome structure of SARS-CoV-2 with the approach of in vitro ligation is shown. Approximate genome locations of the cohesive overhangs are indicated. Length is not to scale. The full-length cDNA of SARS-CoV-2 was directionally assembled using in vitro ligation. The full-length genomic cDNA is flanked by a T7 promoter (T7) on the 5′ end and a polyA tail on the 3′ end. The assembled full-length cDNA was in vitro transcribed to genomic RNA which is electroporated into Vero E6 cells to rescue recombinant SARS-CoV-2. (B) A trans complementation system of SARS-CoV-2. The genome structures of full-length mNG SARS-CoV-2 and trans complement ΔORF3-E mNG SARS-CoV-2 are shown. Wild-type (WT) and a mutant transcriptional regulatory sequence (TRS) are shown. Electroporation of in vitro transcribed RNA leads to its replication and translation followed by assembly with the help of ORF3 and E protein expressed in trans in the ORF3-E Vero E6 cells. ΔORF3-E mNG SARS-CoV-2 can lead to a single round of infection in Vero E6 cells. (C) Attenuated Δ3678 SARS-CoV-2. The genome structure of SARS-CoV-2 with WT TRS, Δ3678 SARS-CoV-2 with mutant TRS sequence and deletion of ORF3, 6, 7, and 8 are shown. T7, T7 promoter; L, leader sequence; TRS, transcription regulatory sequences; ORF, open-reading frame; E, envelope glycoprotein gene; M, membrane glycoprotein gene; N, nucleocapsid gene; pA, poly-A tails. Figures adapted and modified from (Liu et al., 2022c; Xie et al., 2020a; Zhang et al., 2021a).

Fig. 2

BAC-derived SARS-CoV-2 reverse genetic system. The genome structure of SARS-CoV-2 followed by genome fragments with indicated restriction sites used for cloning the entire viral genome in pBeloBAC11 plasmid is shown. Length is not to scale. The Complete viral genome was covered by six fragments. The 5′ end of viral genome was flanked by the CMV promoter and the 3′ end by HDVr and BGH. Transfection of SARS-CoV-2 BAC plasmid into Vero E6 cells leads to the rescue of infectious SARS-CoV-2. ORF, open-reading frame; E, envelope glycoprotein gene; M, membrane glycoprotein gene; N, nucleocapsid gene; CMV, Cytomegalovirus promoter; HDVr, hepatitis delta virus ribozyme; BGH, bovine growth hormone termination and polyadenylation sequences. Figure adapted and modified from (Ye et al., 2020).

Fig. 3

YAC-derived SARS-CoV-2 reverse genetic system. The genome structure of SARS-CoV-2 followed by schematic representation of twelve overlapping DNA fragments used to clone SARS-CoV-2 into YAC vector pVC604. Length is not to scale. Fragments 1 and 12 had overlapping sequences for the YAC vector pVC604. T7 promoter was fused to the 5′ end of the viral genome and a cleavage site (PacI) was added after the poly(A) sequence at the 3′ end. Transformation of SARS-CoV-2 genome fragments along with linearized pVC604 vector in yeast leads to whole-genome assembly in vector through homologous end recombination. Purified YAC plasmid DNA is linearized using PacI and subjected to in vitro transcription. The resulting RNAs were electroporated into BHK1 cells which are plated on Vero E6 cells, leading to the rescue of infectious SARS-CoV-2. T7, T7 promoter; L, leader sequence; ORF, open-reading frame; E, envelope glycoprotein gene; M, membrane glycoprotein gene; N, nucleocapsid gene; pA, poly-A tails. Figure adapted and modified from (Thi Nhu Thao et al., 2020).

Fig. 4

CPER derived SARS-CoV-2 reverse genetic system. The complete genome of SARS-CoV-2 is covered by six overlapping DNA fragments. A linker fragment containing a CMV promoter is engineered upstream of the 5′ UTR. A sequence representing pA, HDVr, and SV40 pA signal is engineered downstream of the 3′ UTR. Linker and SARS-CoV-2 genome fragments are assembled in the CPER reaction. CPER reaction products are transfected in HEK 293T cells to rescue infectious SARS-CoV-2. ORF, open-reading frame; E, envelope glycoprotein gene; M, membrane glycoprotein gene; N, nucleocapsid gene; CMV, Cytomegalovirus promoter; pA, polyA tail; HDVr, hepatitis delta virus ribozyme; SV40 pA, SV40 polyA; UTR, untranslated region. Figure adapted and modified from (Amarilla et al., 2021).