Hydroxychloroquine-mediated inhibition of SARS-CoV-2 entry is attenuated by TMPRSS2
Fig 3
Antiviral effect of hydroxychloroquine is dependent on TMPRSS2 expression.
(A) Cell-surface staining was performed by detecting the flag tag at the C-terminal of TMPRSS2 to validate the overexpression of TMPRSS2. A stable cell line of 293T-ACE2 cells was generated to express TMPRSS2 (orange line). 293T-ACE2 cells were transiently transfected with a vector control (black line), or TMPRSS2 (red line). The 293T/ACE2/TMPRSS2 stable cell line had much lower expression of TMPRSS2 compared to 293T-ACE2 transiently transfected with TMPRSS2 plasmids, and thus were referred as TMPRSS2 Lo and TMPRSS2 Hi, respectively. Shown is a representation of flow cytometry data from two independent experiments. (B) 293-ACE2 cells with different levels of TMPRSS2 were treated with hydroxychloroquine or DMSO before virus inoculation. The results are presented as a percentage of infection of DMSO-treated cells (≈ 250,000 RLU for all three pseudotypes from TMPRSS-expressing cells.) Shown are representative plots of the mean value (±SD) of triplicate samples from the viral entry inhibition assay. The bar graph is a summary of IC50 from three independent experiments. Each point represents the IC50 calculated from one experiment. Unpaired Student’s t-test was used to assess the statistical significance of the difference between IC50 on mock transfected cells (TMPRSS2-negative) and TMPRSS2-positive cells. (**: P < 0.01. *: P < 0.05. n.s.: P > 0.05.) (C) Shown is a representative western blot (of two independent experiments performed) reflecting expression level of TMPRSS2 in different cell lines. H1299, H1975 and Calu-3: human non-small cell lung cancer cells; Vero: kidney epithelial cells. β-Actin served as loading controls. (D) Lentiviruses pseudotyped with SARS-CoV-1, SARS-CoV-2 S, and VSV G proteins were used for infection. Stable cells lines of H1299 and H1975 over-expressing ACE2 were generated to allow efficient infection. Drug treatment, virus inoculation, and luciferase measurement were the same with the procedures in (B). Shown are the mean value (±SD) of triplicate samples from three independent experiments.
