Reverse genetics systems for SARS-CoV-2: Development and applications

Abstract

The recent emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) caused serious harm to human health and struck a blow to global economic development. Research on SARS-CoV-2 has greatly benefited from the use of reverse genetics systems, which have been established to artificially manipulate the viral genome, generating recombinant and reporter infectious viruses or biosafety level 2 (BSL-2)-adapted non-infectious replicons with desired modifications. These tools have been instrumental in studying the molecular biological characteristics of the virus, investigating antiviral therapeutics, and facilitating the development of attenuated vaccine candidates. Here, we review the construction strategies, development, and applications of reverse genetics systems for SARS-CoV-2, which may be applied to other CoVs as well.

Keywords: Infectious clones; Live attenuated vaccines; Replicons; Reverse genetics systems; SARS-CoV-2.

Fig. 1

Generic procedure for the construction of a reverse genetics system. The viral genome is divided into several fragments that are amplified from RT-PCR or chemically synthesized DNA. These cDNA fragments are stably incorporated into vectors with a bacteriophage promoter or eukaryotic promoter. In RNA-launched systems, cDNA fragments are cloned into plasmids with T7 promoters and used to assemble the full-length cDNA by in vitro ligation, or directly achieved by one-step assembly in plasmids. Then, the ligated products or linearized DNA serve as templates for in vitro transcription to obtain capped infectious genomic RNA, which will generate infectious viruses by electroporation of RNA transcripts into susceptible cells. In DNA-launched systems, direct transfection of cytomegalovirus (CMV)-driven plasmids containing the full-length cDNA allows initiation of RNA polymerase II-dependent in vivo transcription in host cells, leading to the successful rescue of the virus.

Fig. 2

Schematic representation of in vitro ligation–based reverse genetics systems for constructing a SARS-CoV-2 cDNA clone. The viral genome of SARS-CoV-2 strain USA-WA1/2020 is split into seven contiguous fragments (F1 to F7). A T7 promoter and poly(A) tail are added at the 5′ and 3′ ends, respectively. Each fragment is flanked by unique class IIS restriction endonuclease sites (BsaI or Esp3I) to generate unique cohesive ends and used to assemble into full-length cDNA by in vitro ligation. The obtained full-length cDNA allows synthesis of full-length SARS-CoV-2 RNA transcripts by in vitro transcription, which are electroporated into Vero E6 cells together with SARS-CoV-2 N mRNA to rescue infectious viruses.

Fig. 3

Schematic representation of TAR cloning–based reverse genetics systems for constructing a SARS-CoV-2 cDNA clone. The SARS-CoV-2 genome is divided into several overlapping cDNA fragments (F1 to F12). The first fragment contains overlapping sequences of the TAR vector pCC1BAC-His3 and a T7 promoter at the 5′ end, while the last fragment includes overlapping sequences of the TAR vector and a restriction enzyme cleavage site (EagI) after the poly(A) tail downstream of the 3′ end. Afterwards, the amplified fragments are simultaneously transformed into S. cerevisiae together with the linearized TAR vector to achieve one-step assembly in yeast. After the purification and linearization of yeast DNA, infectious viruses are generated using in vitro transcription followed by RNA electroporation.

Fig. 4

Schematic representation of BAC-based reverse genetics systems for constructing a SARS-CoV-2 cDNA clone. The entire viral genome of SARS-CoV-2 strain USA-WA1/2020 is cloned into the pBeloBAC11 plasmid. Repetitive restriction sites BstBI and MluI within the genome are removed by artificially introducing silent mutations into the S and M genes, which also serve as genetic markers to distinguish rescued viruses from the natural isolate. The intermediate plasmid pBeloBAC-F1 is constructed under the control of the eukaryotic cytomegalovirus (CMV) promoter and is flanked at the 3′ end by a poly(A) tail, the hepatitis delta virus ribozyme (HDVRz), the bovine growth hormone (BGH) or simian virus 40 (SV40) termination and polyadenylation sequences. The full-length cDNA clone is assembled by sequential cloning of other chemically synthesized fragments (F2 to F5) into the intermediate plasmids using the indicated restriction enzyme sites. The BAC plasmid containing the entire viral genome is directly transfected into Vero E6 cells for recovery of recombinant SARS-CoV-2.

Fig. 5

Schematic representation of PCR amplicon-based reverse genetics systems for constructing a SARS-CoV-2 cDNA clone. Several cDNA fragments encompassing the entire SARS-CoV-2 genome are amplified with a high-fidelity DNA polymerase. Left side: In a circular polymerase extension reaction (CPER) approach, ten overlapping cDNA fragments of SARS-CoV-2 strain Hu/DP/Kng/19–020 are used to generate a circular full-length cDNA in a single CPER reaction, together with a linker fragment harboring the hepatitis delta virus ribozyme (HDVRz), the bovine growth hormone (BGH) or simian virus 40 (SV40) termination and polyadenylation sequences, and the cytomegalovirus (CMV) promoter. The CPER reaction products are then transfected into permissive cells for recovery of infectious viruses. Right side: Alternatively, using the infectious subgenomic amplicon (ISA) technology, infectious viruses are rescued after direct transfection of eight overlapping cDNA fragments of SARS-CoV-2 European strain with the CMV promoter and HDVRz/SV40pA sequences added to the first and last fragment during PCR amplification, respectively.

Fig. 6

Schematic representation of SARS-CoV-2 replicons with different deletion and insertion strategies. (A) A SARS-CoV-2 replicon is established by deleting the genomic region spanning from the spike (S) gene to ORF8 (generating ΔS-ORF8), and replacing it with reporter genes to produce transient reporter replicons under the control of transcription regulatory sequence (TRS) of the deleted S. (B) The ΔS-ORF8 region is replaced by a fusion cassette of reporter genes and selection markers (sms), which are separated by cleavage of the foot-and-mouth disease virus (FMDV) 2A autoprotease sequence. Expression of sms allows for selection of stable cell lines harboring reporter replicons. (C) Reporter genes are engineered to replace the S gene (ΔS). (D) A replicon is established by replacement of the S gene with reporter genes fused to sms via a porcine teschovirus 1 (PTV-1) 2A proteolytic cleavage site. (E) To generate transient or stable replicons, the S gene is replaced by reporter genes, and the envelope (E) and membrane (M) genes are replaced with sms.