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. 2021 Feb 10;29(6):1984–2000. doi: 10.1016/j.ymthe.2021.02.007

Figure 1.

Figure 1

Establishment of a NanoLuc complementation-based bioreporter for measuring ACE2 interaction with the SARS-CoV-2 spike (S) RBD

(A) SARS-CoV-2 uses ACE2 as a viral entry receptor. (B) Schematic depicting mechanism of action for the bioreporter. (C and D) 293T cell were transfected with codon-optimized ACE2 and RBD constructs. 48 h post-transfection, cell lysates and supernatants were harvested. 10 μg of protein or 10 μL of supernatant was resolved by SDS-PAGE. (C) Immunoblot analysis of HA-tagged SmBiT-ACE2 and FLAG-tagged RBD-LgBiT expression. (D) Luminescence was quantified by a luciferase assay using FMZ as substrate (n = 3 biological replicates, mean ± SD; one-way ANOVA, ∗∗∗p < 0.005 relative to RBD-LgBiT alone, Dunnett’s correction for multiple comparisons). Lysates and supernatants were analyzed independently. (E) LgBiT-YAP-15 and SmBiT-14-3-3 were co-transfected with SmBiT-ACE2 and RBD-LgBiT, respectively, to demonstrate the specificity of the bioreporter (n = 3 biological replicates, mean ± SD; one-way ANOVA, ∗∗∗p < 0.005 relative to mock, Dunnett’s correction for multiple comparisons). (F) Recombinant ACE2 purified from 293T cells was incubated for 15 min with cell lysate containing RBD-LgBiT at room temperature. Equal amounts of lysates containing SmBiT-ACE2 were added (total 20 μg) and incubated for 5 minutes. Luciferase assay was performed using CTZ as substrate. (n = 3 biological replicates, mean ± SD; one-way ANOVA, ∗∗∗p < 0.005, Tukey’s correction for multiple comparisons). (G) Recombinant RBD purified from 293T cells was incubated for 15 min with cell lysate or supernatant containing SmBiT-ACE2 at room temperature. Equal amounts of lysates or supernatants containing RBD-LgBiT were added and incubated for 5 min. Luciferase assay was performed using CTZ as substrate (n = 3 biological replicates, mean ± SD; one-way ANOVA, ∗∗∗p < 0.005, Tukey’s correction for multiple comparisons). (H) Bioreporter assay was performed on lysates of 293T cells co-transfected with SmBiT-ACE2 and either SARS-CoV or SARS-CoV-2 RBD-LgBiT constructs, respectively. IVIS imaging of bioluminescence is shown below.