Coronavirus reverse genetic systems: infectious clones and replicons

Abstract

Coronaviruses (CoVs) infect humans and many animal species, and are associated with respiratory, enteric, hepatic, and central nervous system diseases. The large size of the CoV genome and the instability of some CoV replicase gene sequences during its propagation in bacteria, represent serious obstacles for the development of reverse genetic systems similar to those used for smaller positive sense RNA viruses. To overcome these limitations, several alternatives to more conventional plasmid-based approaches have been established in the last 13 years. In this report, we briefly review and discuss the different reverse genetic systems developed for CoVs, paying special attention to the severe acute respiratory syndrome CoV (SARS-CoV).

Keywords: Coronavirus; Infectious clones; Replicons; Reverse genetics.

Fig. 1

Assembly of a SARS-CoV full-length cDNA clone as a BAC. After selection of appropriate restriction sites in the genome of the SARS-CoV Urbani strain (top of the figure), the intermediate plasmid pBAC-SARS-CoV 5′–3′ was generated and used as the backbone to assemble the full-length cDNA clone (pBAC-SARS-CoV^FL) by sequential cloning of five overlapping cDNA fragments (SARS-1 to SARS-5) covering the entire viral genome. The viral genes (ORF 1a, ORF 1b, S, 3a, 3b, E, M, 6, 7a, 7b, 8a, 8b, 9b and N), relevant restriction sites and their genomic position (in brackets), the CMV promoter, the poly(A) tail (pA), the HDV ribozyme (Rz), and the BGH termination and polyadenylation sequences (BGH) are indicated. L, leader; UTR, untranslated region.

Fig. 2

Systematic assembly of a SARS-CoV full-length cDNA clone by in vitro ligation. A full-length cDNA of the SARS-CoV Urbani strain (SARS-CoV^FL) was assembled by in vitro ligation of six contiguous cDNA fragments (SARS-1 to SARS-6) spanning the entire viral genome, which were flanked by native (in black) or engineered (in red) unique BglI restriction endonuclease sites. The assembled full-length cDNA contained a T7 RNA polymerase promoter (T7) at the 5′-end and a poly(A) tail (pA) at the 3′-end, allowing for in vitro transcription of full-length, capped polyadenylated transcripts. The viral genes and relevant restriction sites are indicated.

Fig. 3

Assembly of a SARS-CoV full-length cDNA clone in the vaccinia virus genome. A panel of five contiguous cDNA fragments (SARS-1 to SARS-5) spanning the entire viral genome of the SARS-CoV HKU-39849 strain was cloned in vaccinia virus DNA (V) by in vitro ligation to produce two recombinant vaccinia viruses containing nucleotides 1–20,288 (vSARS-CoV 5′) and 20,272–29,727 (vSARS-CoV 3′) of the SARS-CoV genome, respectively. SARS-1 fragment contained a T7 RNA polymerase promoter (T7) at the 5′-end and SARS-5 fragment a poly(A) tail (pA) at the 3′-end. DNAs from both recombinant vaccinia viruses were then cleaved with SfiI and BglI, and in vitro ligated to create a full-length SARS-CoV cDNA (vSARS-CoV^FL) template for in vitro transcription of SARS-CoV infectious RNA transcripts. The viral genes and relevant restriction sites (native in black and engineered in red) used for the assembly of the infectious clone are indicated.