2.15 Comparison of the infectivity of WT and D614G mutant of
pSARS-CoV-2
To generate pSARS-CoV-2 harboring D614G mutation on spike protein, a
single nucleotide A-to-G substitution was introduced by site-directed
mutagenesis into SARS-CoV-2 spike plasmid using the primer sequence
5′-GTG GCC GTG CTG TAC CAG GGC GTG AAT TGC ACC GAG GTG -3′. WT and D614G
pSARS-CoV2 viruses were produced as described above. The culture
supernatants containing viral particles were filtered on 0.45 µm pore
filter and concentrated by ultracentrifugation at 28,000 rpm for 2 h at
4°C in a Beckman SW28 rotor and an Optima XE-100 Ultracentrifuge
(Beckman Coulter). The virus pellets were resuspended in PBS buffer and
then viral titers were determined by qRT-PCR method using Lentivirus
Titration Kit (LV900, ABM). The equivalent amount of WT and D614G
pSARS-CoV2 viruses were used to infect host cells for pSARS-CoV-2 entry
assay.