SARS-CoV-2 pseudovirus (PsV) was produced as described
by Schmidt et al.56 (link) Plasmids containing
the SARS-CoV-2 spike gene [pSARS-CoV1-Strunc, pSARS-CoV2-Strunc (K417N/
E484K/N501Y mutations)], pCRV1NHG GagPol, and pNanoLuc2AEGFP were
used to produce the pseudoviral particle and were kindly provided
by Drs. Theodora Hatziioannou and Paul Bieniasz, Rockefeller University.
The plasmids were used to transfect 293T cells (ATCC, Manassas, VA)
monolayers prepared in six-well plates. Briefly, the DNA/lipofectamine
2000 (Thermo Fisher Scientific, Waltham, MA) mixtures were added to
293T cell monolayers and incubated for 6 h at 37 °C, 5% CO2, and 98% humidity. The cell monolayers were then washed twice
with D-PBS (Thermo Fisher Scientific) and finally incubated for 48
h at 37 °C, 5% CO2, 98% humidity in Dulbecco’s
modified Eagle’s medium (DMEM) (Thermo Fisher Scientific) with
10% FBS (Thermo Fisher Scientific) and Penicillin + Streptomycin (Thermo
Fisher Scientific, Waltham, MA). After the 48 h incubation, the cell
supernatants were collected, filtered (using a 0.22 μm pore
size PVDF filter), aliquoted, and stored at −80 °C. The
pseudoviral titer was determined using a cell-based pseudoviral entry
assay57 (link) and the TurboLuc Luciferase One-Step
Glow Assay Kit (Thermo Fisher Scientific).
by Schmidt et al.56 (link) Plasmids containing
the SARS-CoV-2 spike gene [pSARS-CoV1-Strunc, pSARS-CoV2-Strunc (K417N/
E484K/N501Y mutations)], pCRV1NHG GagPol, and pNanoLuc2AEGFP were
used to produce the pseudoviral particle and were kindly provided
by Drs. Theodora Hatziioannou and Paul Bieniasz, Rockefeller University.
The plasmids were used to transfect 293T cells (ATCC, Manassas, VA)
monolayers prepared in six-well plates. Briefly, the DNA/lipofectamine
2000 (Thermo Fisher Scientific, Waltham, MA) mixtures were added to
293T cell monolayers and incubated for 6 h at 37 °C, 5% CO2, and 98% humidity. The cell monolayers were then washed twice
with D-PBS (Thermo Fisher Scientific) and finally incubated for 48
h at 37 °C, 5% CO2, 98% humidity in Dulbecco’s
modified Eagle’s medium (DMEM) (Thermo Fisher Scientific) with
10% FBS (Thermo Fisher Scientific) and Penicillin + Streptomycin (Thermo
Fisher Scientific, Waltham, MA). After the 48 h incubation, the cell
supernatants were collected, filtered (using a 0.22 μm pore
size PVDF filter), aliquoted, and stored at −80 °C. The
pseudoviral titer was determined using a cell-based pseudoviral entry
assay57 (link) and the TurboLuc Luciferase One-Step
Glow Assay Kit (Thermo Fisher Scientific).