Fig. 5

Cell–cell fusion luciferase assay. a Scheme of the pMCAS(3Flag-Sync1-MS)dsRed construct used for luciferase assay. The signal peptide of Env(A) (sigPE(A)) was followed in-frame by the three-Flag epitope (F), Syncytin-1 SU and TM subunits. The first six amino acids before the splicing donor (SD) are shared by Gag and sigPE(A). The construct contained the full-length cytoplasmic tail of Syncytin-1 (C). The mutated cryptic splice acceptor is depicted by an asterisk, dashed lines indicate the alternative splicing generating Syncytin-1 and dsRed mRNAs. Numbering corresponds to original protein sequences. b Scheme of the luciferase assay using the NanoBiT technology. Two subunits of NanoLuc luciferase, LgBiT and HiBiT, were transfected into a DF-1 cell line separately and stable transfectants were selected. Cells expressing the LgBiT part of NanoLuc luciferase were transduced with FuTraP variants and sorted (green). Cells expressing HiBiT were transiently transfected by pMCAS(3Flag-Sync1-MS)dsRed (red). A mixture of LgBiT- and HiBiT-expressing cells was seeded. The interaction of human ASCT2 with Syncytin-1 triggered cell–cell fusion followed by complementation of LgBiT by HiBiT. After substrate addition, the luminescence emission signal was quantified. c Efficiency of cell–cell fusion induced by interaction of Syncytin-1 with cells expressing variants of FuTraP measured by luciferase assay. Luminescence units of individual FuTraP variants were normalised to the wild-type FuTraP-hASCT2-wt (Y-axis). The experiment was repeated three times in biological triplicates. d Inhibition of fusion by preincubation of selected FuTraP cells with sS1-containing supernatant (dark green). Supernatant containing the RCASBP(B)GFP was used as a mock control (light green). Fusions were detected by the luciferase assay and luminescence units were normalised to non-treated wild-type FuTraP-hASCT2 (Y-axis). The experiment was repeated three times in biological triplicates. e Efficiency of cell–cell fusion induced by interaction of Syncytin-1 with cells expressing variants of FuTraP measured as a fusion index. The fusion index was defined as S/N, where S is the total number of nuclei in syncytia (≥ 3 nuclei within iRFP and dsRed double-positive cells) and N is the total number of nuclei in the field. The fusion index was normalised to FuTraP-hASCT2-wt (Y-axis). The experiment was repeated twice in biological triplicates. DF-1/LgBiT cells expressing the S1 allele of chicken Tvb (ggTvb^S1) or human EAAT1 (hEAAT1) represent the negative controls (NC). Data is shown as means ± standard errors, *p < 0.05, **p < 0.01, ****p < 0.0001 Mann–Whitney two-tailed test