Fig. 1

Analysis of SARS-CoV-2 membrane fusion by BiMuC. A Schematic representation of the BiMuC assay. The Jun and Fos proteins were fused to the Nt and Ct ends of the VFP, respectively, to create the VN-Jun and VC-Fos chimeras, from now on presented simply as Jun and Fos. Reconstitution of the structure of the VFP (represented in green), and thus its fluorescence properties, occurs exclusively after Jun and Fos interaction. When the VN-Jun and VC-Fos chimeras are expressed in separated cell pools, no reconstitution is possible (left). SARS-CoV-2 membrane fusion relies on the successful interaction between SARS-CoV-2 S and human ACE2. Expressing these two proteins on the surface of two adjacent cells leads to the fusion of the cellular membranes and creating a syncytium (middle). When the cells being fused carry the VN-Jun and VC-Fos chimeras can reconstitute the VFP native structure (depicted in green, left). On the contrary, inhibition of any of the cellular or viral processes that lead to membrane fusion, from synthesis and maturation of the S protein to its interaction with ACE2, will result in the absence of a fluorescent signal. B For the cell-cell fusion assay, Hek 293T cells were transfected with the indicated plasmid combinations. That is: Jun + SARS-CoV-2 S, Fos + hACE2, Jun + hACE2, Fos + SARS-CoV-2 S, Jun + Fos, Jun, Fos, SARS-CoV-2 S, hACE2, Fos + hACE2 + TMPRSS2. The next day cells in each cell pool were counted and mixed as indicated. After 48 h, fluorescence was measured. The mean and standard deviation of at least three independent experiments are represented (n values 7, 6, 6, 6, 3, 3, respectively). A solid dot represents the individual value of each experiment. Green bars denote those samples with fluorescence levels comparable to the positive control (Jun-Fos). Statistical differences are based on a two-tailed homoscedastic t-test (p-values are indicated above the corresponding bar, ns non-significant)