DesignConstruct 1
Construct 2
Construct 3Construct 4
Construct 5
Construct 6rSARS-CoV-2rSARS-CoV-2-GFPTAR cloning
Positive out of
10 screened clonesYACs for
subsequent rescue
10
10
910
10
9Clones 1.1 and 1.2
Clones 2.1 and 2.2
Clones 3.1 and 3.2Clones 4.1 and 4.2
Clones 5.1 and 5.2
Clones 6.1 and 6.2aVirus rescue and characterisation
Electroporation Co-cultivation with
susceptible cell lineVeroE6 cellsCollection of
supernatantb48 h.p.e
72 h.p.e4 d.p.e5 d.p.eTransfer of P.0 supernatant to new VeroE6 cells
to generate P.1 virusElectroporated cells
BHK SARS-NBHK-YAC1.1 1.2 1.1
2.1 2.2 2.1
3.1 3.2 3.1
4.1 4.2 4.1
5.1 5.2 5.1
6.1 6.2 6.1Electroporated
cells Clone RescuedPresence of
plaques caused
by P.0 virusCPE and/or GFP
signals caused by
P.0 virusBHK1.1 Yes Yes Yes
1.2 Yes Yes Yes
2.1 Yes Yes Yes
2.2 Yes Yes Yes
3.1 Yes Yes Yes
3.2 Yes Yes Yes
4.1 Yes Yes Yes
4.2 No No No
5.1 No No No
5.2 Yes No Yes
6.1 No No No
6.2 Yes No YesBHK-SARS-N1.1 Yes Yes Yes
2.1 Yes Yes Yes
3.1 Yes Yes Yes
4.1 Yes Yes Yes
5.1 Yes No Yes
6.1 No No NoExtended Data Fig. 3 | Workf low for the reconstruction and rescue of
rSARS-CoV-2 and rSARS-CoV-2-GFP. a, Overview of the constructs and clones.
Six constructs were initially designed on the basis of three different 5′-UTR
regions. These regions comprised a modified sequence of the 5′-UTR region of
S A R S - C oV-2 ( 5 ′-ATAUUAGG) in which nucleotides 3–5 (UA A) of SARS-CoV-2
were changed to AUU to match nucleotides 3–5 of bat SARS-related CoV
(constructs 1 and 4); a SARS-CoV-2 5′-terminus in which the first 124 nucleotides
were changed to the corresponding 5′-terminal sequence of SARS-CoV
(constructs 2 and 5); and the reported sequence of the SARS-CoV-2
(MN996528.1) (constructs 3 and 6). After transformation in yeast, ten colonies
were randomly picked for each of the six constructs and all of the junctions
bridging the overlapping fragments were verified by multiplex PCR. For each
construct, two clones (x.1 and x.2) were randomly selected and YAC DNAs were
isolated (12 clones in total). b, Rescue of rSARS-CoV-2 and rSARS-CoV-2-GFP
clones. RNAs were generated from YAC DNAs by in vitro transcription and
electroporated together with an mRNA encoding the SARS-CoV-2 N protein
either into BHK-21 cells (12 clones) or BHK-SARS-N cells (cells expressing the
SARS-CoV N protein) (6 clones). Electroporated cells were then co-cultured
with susceptible Vero E6 cells to rescue the recombinant viruses. Passage 0
(P.0) supernatants were collected at different time points after electroporation
(from 2 to 5 days after electroporation) and transferred to Vero E6 cells to
generate passage 1 (P.1) virus stocks, and in parallel to demonstrate the
presence of infectious virus in plaque assays (for virus clones that do not
encode GFP) or f luorescence microscopy (for GFP-encoding virus clones).
h.p.e, hours post-electroporation; d.p.e, days post-electroporation; CPE,
cytopathogenic effects.