Characterization of SARS-CoV-2 spike pseudotyped virus.

A: Schematic showing the strategy for generating SARS-CoV-2 Spike-pseudovirus. 2nd generation lentiviral helper plasmid psPAX was co-transfected with the reporter plasmid pHRmCherry and SARS-CoV-2 Spike protein-encoding plasmid pTwist Spike in HEK-293T cells to generate Spike pseudotyped virus particles. pHRmCherry reporter plasmid was used to score for infected cells by mCherry expression. B: Western blot showing bands of different molecular weights as detected by the anti-Strep-tag antibody which recognizes the C-term 2X Strep-tag on the Spike proteins incorporated into the pseudovirus particles. C: Comparison of infection by Spike-pseudotyped or bald pseudotyped (lacking spike) virus particles in AGS cells. Quantification shows that Spike-pseudotyped viruses infect AGS cells at various dilutions while bald-pseudoviruses do not infect at same dilutions. Number of repeats for each condition = 2 for each dilution. D: Transduction of AGS cells by Spike-pseudotyped viruses compared to an alternatively pseudotyped virus. (i) Quantification of infection at indicated MOI shows that VSV-G pseudotyped viruses are capable of transducing AGS cells at a higher efficiency. Number of repeats is 3 for Spike-pseudotyped viruses and 2 for VSV-G pseudotype. Data is plotted as mean +/- SD. (ii) Infection by VSV-G pseudotyped viruses is also susceptible to BafA1. Number of repeats = 2 for each condition. E: Specificity of Spike protein-ACE-2 dependent pseudovirus entry was tested in a competition assay in the presence of excess purified RBD of Spike. Quantification of percentage transduction shows a reduction in transduction efficiency with both monomeric and trimeric Spike RBD in AGS cells. Number of repeats = 2 for each condition. F: Characterization of transduction efficiency in AGS cells of Spike-pseudovirus at varying MOIs and varying incubation times. Quantification of percentage transduced mCherry positive cells depicts a steady increase in transduction as a function of MOI and incubation times. Number of repeats = 2 for each condition. The data is plotted as mean +/- SD. G: (i) Treatment of AGS cells across different concentrations of AN96 shows no significant reduction in transduction efficiency of Spike-pseudotyped viruses compared to the pooled controls from 0.6%, 0.24%, 0.12%, 0.048% and 0.024% DMSO treatments. Number of repeats = 15 for pooled controls and 3 for each concentration of AN96. (ii) Treatment of AGS cells with 5μM of ML141 shows no significant reduction in transduction efficiency compared to control (p-value = 0.55). Number of repeats = 3,2 for control (0% DMSO) and ML141 respectively. Data representation in C, D, E, G is as described in Fig 3.

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