Hydroxychloroquine-mediated inhibition of SARS-CoV-2 entry is attenuated by TMPRSS2
Fig 5
Furin cleavage in the virion producer cell correlates with TMPRSS2 dependence.
(A-B) The infectivity of SARS-CoV-1 and SARS-CoV-2 and their mutants on 293-ACE2 cells was compared with or without overexpression of TMPRSS2. The infection assay was performed as described in Fig 2A. Retroviruses were pseudotyped with S proteins of SARS-CoV-2 wildtype (WT), D614G S-protein variant, SARS-CoV-2 with furin-site knockout (FKO), and (B) SARS-CoV-1 WT (1-WT), and SARS-CoV-1 with the furin site derived from SARS-CoV-2 (1-FS). Pseudovirus titers were adjusted so that they were equivalent in the absence (left panel) or presence (right panel) of TMPRSS2. Shown is a representative plot of the mean (±SD) of duplicate samples from at least two independent experiments. (C) Western blot analysis of S protein cleavage of SARS-CoV-2-S WT (D614) and variants (G614 and FKO), and SARS-CoV-1 WT and its variant with furin site addition. Antibody detected the flag tags at the N- and C-termini of S proteins. β-Actin served as loading controls. Black arrow heads indicate bands corresponding to the S1 and S2 subunits from cleavage of S proteins. Shown are representative blots from two experiments. (D)The effect of TMPRSS2 on the antiviral efficiency of hydroxychloroquine (HCQ) and camostat was compared. Retrovirus pseudotyped against S proteins described in (C) after drug or DMSO treatment. Luciferase activity was measured at 48 hours post inoculation. Relative infection (%) was calculated from infection of DMSO-treated cells. Statistical significance between wildtypes and mutants was tested by two-way ANOVA with Dunnett’s posttest. (***: P < 0.001. *: P < 0.05).