Extended Data Fig. 4: PLSCR1 engages both the endosomal pathway and the cell-surface pathway of SARS-CoV-2 entry.
From: PLSCR1 is a cell-autonomous defence factor against SARS-CoV-2 infection
a,b, Quantification of viral entry efficiency in Huh7.5 cells inoculated with pseudoviruses bearing fusion proteins from HCoV-229E (n = 5), HCoV-OC43 (n = 3), hCoV-NL63 (n = 3), hCoV-HKU1 (n = 3), EBoV (n = 3) and HCV (n = 5). EBoV: Ebola virus, HCV: Hepatitis C virus. c, Left, relative amount of intracellular viral RNA in Huh7.5 cells infected with DENV (Dengue virus type I) at an MOI = 0.5 for 24 h. The amount of viral RNA in NC cells was normalized to 1. (n = 4) Right: Quantification of % infected cells in HeLa cells infected with HSV-1 VP26-GFP at an MOI of 0.1 for 48 h. d, Quantification of the relative entry efficiency of the indicated pseudovirus in Huh7.5 cells overexpressing (OE) PLSCR1 or IFITM3. The luminescence intensity in vector group was normalized to 1. n = 4. e, Schematic showing the dissection of the cell entry route of SARS-CoV-2. f,g, Effect of the indicated compounds on SARS-CoV-2 entry in Huh7.5 cells (MOI = 1, 48 hpi, n = 3) (f) and A549-ACE2 cells (MOI = 0.2, 24 hpi, n = 3) (g). E-64d: 20 μM, Camostat: 30 μM, Bfa (Brefeldin a): 10 μM, HCQ: 10 μM. Cells were treated with indicated compounds 2 h before infection. h, Quantification of SARS-CoV-2 infection in E-64d (20 μM) treated or untreated Huh7.5 cells overexpressing vector or PLSCR1 with or without ectopic expression of TMPRSS2 (MOI = 1, 48 hpi). (n = 4) i, Left, dose response of indicated compounds on SARS-CoV-2 infection in Calu-3 (MOI = 1, 24 hpi, n = 4). The amount of viral RNA in DMSO group was normalized to 1. E64-d and Camostat groups share the same DMSO control group. Right, quantification of SARS-CoV-2 infection in Control or PLSCR1-KO Calu-3 (MOI = 1, 24 hpi, n = 4) treated with indicated compounds (E-64d: 20 μM, Camostat: 20 μM). Cells were treated with the indicated compounds 2 h before infection. The amount of viral RNA in NC-DMSO group was normalized to 1. Data are mean ± s.d. P values were calculated using one-way ANOVA followed by Tukey’s multiple comparison test in a–c,d (HCoV-NL63 group), f,g, Brown–Forsythe and Welch ANOVA with Dunnett’s post-hoc test in d (SARS-CoV-2 and EBoV group), two-sided Student’s t-test in h, two-way ANOVA followed by Tukey’s multiple comparison test in i (left) or two-way ANOVA followed by Šídák’s multiple comparisons test in i (right). Experiments in this figure were performed three times.
