Figure 2. LY6E inhibits viral membrane fusion and syncytia formation.
a-b, VSV pseudoparticles (pp) harboring spike proteins from (a) HCoV-229E, MERS-CoV and G protein from VSV or (b) SARS-CoV, SARS-CoV-2 and G protein from VSV were inoculated on LY6E- or empty vector-expressing cells. Virus entry efficiency was quantified by measuring virus-encoded luciferase reporter activity. c, VSV pp expressing VSV G protein on their surface and encoding CoV S protein and GFP (VSV*ΔG(CoV S)) were inoculated with LY6E- or empty vector-expressing Huh7. Syncytia formation was analyzed by immunofluorescence microscopy. Blue: DAPI, green: GFP, red: TagRFP inserted in SCRPSY vector. d, Quantification of VSV*ΔG(CoV S)-induced syncytia depicted as percentage syncytia area. Three independent areas were analyzed per biological replicate (circle, square, triangle). e-h, Cell-cell fusion was analyzed by co-incubating cells transfected with plasmids encoding mCherry, VSV-G protein, or CoV S proteins and a split-luciferase/split-GFP construct together with CoV permissive LY6E-expressing or control cells transfected with the complementary fragment of split-luciferase/split-GFP. In e, g, GFP complementation was analyzed via immunofluorescence microscopy of GFP-positive syncytia. In f,h, four images per condition were acquired and processed. Data were normalized to empty control and are depicted as fold change mean GFP. Data represent mean of independent biological replicates, n=8 (HCoV-229E S, MERS-CoV-S) n=9 (VSV-G) (a), n=6 (b), n=3 (d), n=3 (f,h). Statistical significance was determined by two-way ANOVA followed by Sidak’s multiple comparison test (a-h). In c, scale bar is 20 μM, in e, g, scale bar is 50 μM. Error bars: SD (a-b, d, f, h). P values: a, **** p=3.3 × 10−16, **** p=3.6 × 10−15; b, **** p=1.363 × 10−6; **** p=4.200 × 10−14; d, **** p=6.0 × 10−9, p=<1.1 × 10−15; f, *** p=0.0008; h, *** p=0.0008.