T7mmessage mmachine t7 transcription kit

The present work
with infectious SARS-CoV-2 was approved by the Institutional Biosafety
Committee (IBC#21–22) and carried out in a fully certified
Biosafety level-3 laboratory at Oklahoma State University. To generate
recombinant Nsp5^S144A, Nsp5^S144M, Nsp5^E166Q, Nsp5^H172Q, and Nsp5^H172Y mutant viruses,
corresponding nucleotide substitutions were introduced into the SARS-CoV-2
infectious cDNA subclone plasmid using a Q5 site-directed mutagenesis
kit (NEB, E0554S) and then verified by sequencing of the plasmid.
Virus recovery was conducted as described previously.^{30 (link)} Briefly, viral cDNA fragments were ligated in an equal
molar ratio to assemble a full-length genomic cDNA with T4 DNA ligase
(NEB, M0202L). The ligated full-length cDNA and a SARS-CoV-2-N plasmid
were used for in vitro transcription using the T7
mMESSAGE mMACHINE T7 transcription kit (ThermoFisher, AM1344). The
transcribed viral RNA and N gene sgRNA were subsequently electroporated
into Vero-TA cells. These cells were maintained in DMEM containing
2% FBS at 37 °C. Culture supernatants were collected at the time
when the cytopathic effect was evident. All harvested viral stocks
were titrated in Vero-TA cells and subjected to sequencing of the
Nsp5 coding region to validate the genotypes.