Skip to main content
. 2020 Nov 17;11(46):4201–4223. doi: 10.18632/oncotarget.27799

Figure 6. Establishment of a SARS-CoV-2 pseudovirus that expresses SPIKE protein variants on the envelope of a lentiviral core, infection of human airway epithelial cells or lung cancer cells, and demonstration of MEKi attenuation of infectivity on primary human cells.

Figure 6

(A) Western blot to demonstrate FLAG-tagged S protein of SARS-CoV-2 pseudoviruses. We generated a pseudotyped SARS-CoV-2 virus which has a lentiviral core but with the SARS-CoV-2 D614 or G614 spike (S) protein on its envelope. VSV-G lentivirus was used as a negative control. Lenti- X™ p24 Rapid Titration ELISA Kit (TaKaRa) was used to determine virus titer. Equal number of lentiviral particles were analyzed on an SDS-PAGE gel followed by Western blot to detect FLAG tagged S protein. The results suggest more G614 than D614 S protein on each viral particle. (B) Fluorescence microscopic evaluation of SARS-CoV-2 pseudovirus infection of Beas 2B cells. SARS-CoV-2 pseudoviruses (5 × 106) or VSV-G lentivirus (2 × 105) were used to spin-infect Beas 2B cells in a 12-well plate. Fluorescence microscopic images were taken 18 h after infection. (C) Flow cytometry analysis of SARS-CoV-2 pseudovirus infection of Beas 2B cells. SARS-CoV-2 pseudoviruses (5 × 106) or VSV-G lentivirus (2 × 105) were used to spin-infect Beas 2B cells in a 12-well plate. Flow cytometry analysis of ZsGreen+ cells was carried out 48 h after infection. (D) Fluorescence microscopic evaluation of SARS-CoV-2 pseudovirus infection of Calu-3 cells. SARS-CoV-2 pseudoviruses (5 × 106) or VSV-G lentivirus (2 × 105) were used to spin-infect Calu-3 cells in a 12-well plate. Fluorescence microscopic images were taken 18 h after infection. (E) Flow cytometry analysis of SARS-CoV-2 pseudovirus infection of Calu-3 cells. SARS-CoV-2 pseudoviruses (5 × 106) or VSV-G lentivirus (2 × 105) were used to spin-infect Calu-3 cells in a 12-well plate. Flow cytometry analysis of ZsGreen+ cells was carried out 48 h after infection. (F) Inhibition of SARS-CoV-2 pseudovirus cell entry of human primary small airway epithelial cells. The cells were grown on 12-well plates till 80% confluence, pre-treated with the inhibitors at the indicated concentrations for 24 hr, spun-infected with the pseudoviruses followed by another 24 hr incubation with the inhibitors. All MEKi significantly blocked pseudovirus cell entry of the primary human cells while having no effect on the pantropic VSV-G lentivirus cell entry. Overall cell survival was more than 75% as determined by DAPI- in flow cytometry. The percentage of ZsGreen+ live cells was analyzed by using the FlowJo software version 10 (FlowJo, LLC, Ashland, OR).