Reverse genetics with a full-length infectious cDNA of severe acute respiratory syndrome coronavirus

Abstract

A previously undescribed coronavirus (CoV) is the etiologic agent responsible for severe acute respiratory syndrome (SARS). Using a panel of contiguous cDNAs that span the entire genome, we have assembled a full-length cDNA of the SARS-CoV Urbani strain, and have rescued molecularly cloned SARS viruses (infectious clone SARS-CoV) that contained the expected marker mutations inserted into the component clones. Recombinant viruses replicated as efficiently as WT virus and both were inhibited by treatment with the cysteine proteinase inhibitor (2S,3S)-transepoxysuccinyl-L-leucylamido-3-methylbutane ethyl ester. In addition, subgenomic transcripts were initiated from the consensus sequence ACGAAC in both the WT and infectious clone SARS-CoV. Availability of a SARS-CoV full-length cDNA provides a template for manipulation of the viral genome, allowing for the rapid and rational development and testing of candidate vaccines and therapeutics against this important human pathogen.

Fig. 1.

Assembly of a full-length SARS-CoV cDNA. (A) Structure of the SARS-CoV genome (8, 9). The SARS Urbani ≈29,727-bp genome contains 10 or more ORFs, which are expressed from full-length and subgenomic-length mRNAs (8). (B) Several BglI-interconnecting junctions were inserted between the component clones to allow assembly of a full-length cDNA. Lowercase letters represent WT sequence and numbers represent nucleotide positions in genome. (C) “No see'm” Aar I repair of SARS sibling clones. Asterisks represent sites of mutation. Numbers in bold represent portions of sibling clones that were assembled into a consensus SARS F subclone.

Fig. 2.

SARS-CoV full-length transcripts are infectious. Full-length transcripts, in the presence or absence of SARS N transcripts, were electroporated into BHK cells and overlaid with susceptible VeroE6 cells. A portion of the cells was examined by fluorescent Ab staining. (A) Cultures transfected with SARS-CoV full-length transcripts. (B) SARS full-length transcripts plus N transcripts. (C) Uninfected control. (D) Virus growth was determined for the first 96 h after transfection by plaque assay; icSARS transcripts alone (▪) and icSARS plus N transcripts, trials 1 (⋄) and 2 (▵). (E) icSARS-CoV and WT SARS-CoV plaques in Vero E6 cells.

Fig. 3.

icSARS-CoV marker mutations. cDNAs spanning the different BglI junctions were amplified from WT and icSARS-CoV-infected cells. The fragments were purified, and a portion was digested with BglI and separated in 1.2% agarose gels. (A) icSARS-CoV (lanes 2, 3, 6, 7, 10, and 11) and WT SARS-CoV (lanes 4, 5, 8, 9, 12, and 13). Lanes 2–5, a 1,668-nt amplicon (nucleotide positions 1007–2675) digested with BglI (lanes 3 and 5); lanes 6–9, a 799-nt amplicon spanning the SARS-CoV B/C junction (positions 8381–9180) digested with BglI (lanes 7 and 9); lanes 10–13, a 544-nt amplicon (positions 11,721–12,265) spanning the SARS-CoV C/D junction digested with BglI (lanes 11 and 13). Lanes 1 and 14, a 1-kb ladder. (B) WT SARS (lanes 4, 5, 8, and 9); icSARS-CoV (lanes 2, 3, 6, and 7). Lanes 2–5, a 652-nt amplicon spanning the SARS-CoV D/E junction digested with BglI (lanes 3 and 5); lanes 6–9, a 1,594-nt amplicon (positions 23,665–25,259) spanning the SARS-CoV E/F junction digested with BglI (lanes 7 and 9). Lanes 1 and 10, a 1-kb ladder. (C) The 1,594-nt SARS E/F junction-containing amplicon was subcloned and sequenced, demonstrating the mutations introduced within the icSARS-CoV.

Fig. 4.

Phenotype comparisons between WT and icSARS-CoV. Cultures of cells were infected with an multiplicity of infection of 0.1. At 1 h, cultures were treated with E64-d at a concentration of 500 μg/ml, and virus titers were determined by plaque assay in VeroE6 cells (C). (A) Growth of Urbani (▪), icSARS (□), and the Tor-2 (▴) and Tor-7 (▵) CoVs. (B) Leader-containing transcripts encoding various icSARS-CoV subgenomic ORFs. *, predicted ORFs classified by using standard CoV nomenclature (11). (C) Growth of icSARS and Urbani SARS-CoV in the presence or absence of E64-d. Hatched bars, 24-h titers; black bars, 48-h titers. (D) Urbani SARS-CoV-infected cells at 72 h after infection.