Fig. 4: The lipid scramblase and antiviral activities of PLSCR1 are uncoupled.

a, Confocal images showing the localization of endogenous (endo.) PLSCR1 and SARS-CoV-2 nucleocapsid in NC or PLSCR1-KO A549-ACE2 cells infected with SARS-CoV-2 (2 hpi, MOI = 25). b, W-4Pi-SMS nanoscopy of endogenous PLSCR1 and SARS-CoV-2 spike detected at single-molecule resolution in A549-ACE2 cells infected with SARS-CoV-2 (2 hpi, MOI = 25). c, Dynamic formation of PLSCR1-coated SARS-CoV-2-containing vesicles. Time-lapse images were obtained at 1-min intervals for around 2 h and snapshots at the indicated time points are presented. A549-ACE2 cells were infected with SARS-CoV-2-PsV-Cherry. PM, plasma membrane. d, Localization of wild-type (WT) PLSCR1 or mutant PLSCR1(5CA) (C¹⁸⁴CCPCC¹⁸⁹ to AAAPAA) on SARS-CoV-2-containing vesicles. PLSCR1-KO A549-ACE2 cells stably expressing GFP–PLSCR1(WT) or GFP–PLSCR1(5CA) were infected with SARS-CoV-2 for 2 h (MOI = 25) (WT, n = 24 cells; 5CA, n = 28 cells). e, Quantification of SARS-CoV-2 infection in Huh7.5 cells expressing the indicated mutants (MOI = 1, 48 hpi, n = 5). f, Top, AlphaFold2 prediction of surrounding amino acid residues of the Phe²⁸¹ and His²⁶² sites. Rainbow-coloured from N terminus (blue) to C terminus (red). Bottom, comparative SARS-CoV-2 infection in control or PLSCR1-KO Huh7.5 cells expressing the indicated mutants (MOI = 1, 48 hpi, n = 6). g, FACS plots showing PS externalization in NC, PLSCR1-KO or TMEM16F-KO A549-ACE2 cells in the absence or presence of 10 μM ionomycin. Ionomycin is a membrane-permeable Ca²⁺ carrier that increases intracellular Ca²⁺ levels, which triggers PS externalization in a percentage of cells (threshold, dotted line). h, FACS plots showing PS externalization in NC or TMEM16F-KO A549-ACE2 cells stably overexpressing the indicated PLSCR1 mutants in the absence or presence of 10 μM ionomycin. i, Relative PS externalization activity in NC A549-ACE2 cells treated with ionomycin, normalized to 1 (n = 3). j, Cell–cell fusion assay in Huh7.5 cells stably expressing the indicated PLSCR1 mutants co-cultured with 293T cells expressing SARS-CoV-2 spike (n = 6). k, Model of SARS-CoV-2 restriction by PLSCR1. All data are mean ± s.d. P values from two-sided Mann–Whitney test in d and one-way ANOVA followed by Tukey’s multiple comparison test in e,f,i,j. Scale bars 10 μm (a,c,d, main), 5 μm (b, main and a,c,d, inlays) and 500 nm (b, inlays). All experiments performed three times, except a (five times).

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