A plasmid DNA-launched SARS-CoV-2 reverse genetics system and coronavirus toolkit for COVID-19 research (original) (raw)
Fig 1
An openly available plasmid launched SARS-CoV-2 RG system.
(A) Schematic of the design and construction of a pCC1-4K-SARS-CoV-2-Wuhan-Hu-1 icDNA clone. Synthetic DNA fragments 1, 2, and 3 based on the SARS-CoV-2-Wuhan-Hu1 sequence were cloned into the pCC1 plasmids, and fragments 4 and 5 were cloned into high copy plasmid pUC57Kan by the gene synthesis company (Genscript). Fragments were designed to contain specific restriction cloning sites _San_Dl, _Pac_I, _Mlu_l, _Bsu36_I, and _Bam_HI for cloning purposes. Sequences encoding for mCherry, ZsGreen, and NLuc markers were cloned in-frame to the C-terminus of the ORF7a protein via an FMDV 2A linker. (B) A summary of the passage (P) history of the extensively propagated wt 1 and wt 2 plasmid preps (expanded 6 times on solid agar and 5 times in liquid culture prior to being “grown up” in liquid culture for DNA extraction). (C) Summary plots of the number of reads mapping to the wt rescue plasmid and to the rescued viral genome. (D) Detection of SARS-CoV-2 N antigen in Vero E6 cells infected with RG-rescued SARS-CoV-2-Wuhan-Hu-1. Cells were not infected (Mock) or infected at MOI 1.0 for 48 h and then fixed and stained with mouse monoclonal anti-N antibody and counterstained with DAPI. Cells were imaged using a confocal microscope. Scale bar = 50 μm. (E) Titre of the mCherry virus (plaque forming units on Vero E6 cells) following propagation in Vero E6 cells (P3). (F) Detection of infected Vero E6 cells using the RG-rescued SARS-CoV-2-mCherry. Cells were infected for 48 h at MOI 1.0, then fixed, stained with anti-N antibody, and imaged as in (D). Scale bar = 50 μm. (G) Quantification of mCherry and N expression in Vero E6 cells infected with SARS-CoV-2-mCherry at an MOI of 0.1 for 48 h. Fixed and permeabilised cells were stained for N protein and with Hoechst 33342 (2 μg/ml). Cells were imaged using the Celigo Imaging Cytometer to identify the proportion of infected cells positive for mCherry and/or N protein. (H) Fixed plaque assays were scanned using a Celigo Imaging Cytometer (Nexcelom Bioscience) using the red channel to visualise mCherry. The cells were then subsequently stained with Coomassie staining solution and imaged again. (I) Detection of viral replication in infected Vero E6 cells using the RG-rescued SARS-CoV-2-NLuc. Cells were not infected (Mock) or infected at MOI 1.0 for 24 h, then lysed. NLuc activity was measured in the lysate using a luminometer. (J) Fluorescent plaques of the SARS-CoV-2-ZsGreen virus were visualised using the green channel as in panel H. (K) A schematic of the passage (P) history of the SARS-CoV-2 mCherry virus used to assess reporter stability. Each filled circle represents 1 day of propagation. (L) Plaque assays of passages 3 to 5 were scanned (Celigo) as in (H). The percentage of plaques visible following Coomassie staining that were mCherry-positive in the linear range of the dilution series (total plaque number in the range of 22 to 55 for each replicate) are plotted for each lineage at each passage. (M) Typical images of fluorescent plaques used for the quantification in (L) are shown. (N) A summary of the variation generated during the passage of the mCherry virus summarised in panel K. The variation is detailed in S1 Table. (O) The percentage of the viral swarm displaying all variants that exceed 25% of the swarm at any time point is shown. The low-level deletion of the furin cleavage site is also highlighted for interest. The data underlying Fig 1E, 1G, 1I and 1L may be found in S1 Data. FMDV, foot-and-mouth disease virus; icDNA, infectious cDNA; MOI, multiplicity of infection; NLuc, Nanoluciferase; RG, reverse genetics; SARS-CoV-2, Severe Acute Respiratory Syndrome Coronavirus 2; wt, wild-type.