Fig. 4

S-M1237I mutation increases viral assembly/secretion but decreases infectivity in vitro. (A) Schematic illustration of the protocols for generation of S-pseudotyped lentiviruses (upper panel) and SC2-VLPs (lower panel), and for subsequent viral titer and viral infectivity determination. (B) Analysis of the viral titer of the lentiviruses pseudotyped with S-B117-M1237- Δ18aa or S-B117-I1237- Δ18aa produced by 293T cells (left panel) and the viral infectivity (right panel, with S-B117-M1237- Δ18aa set as 1). The results were derived from six independent experiments and are shown as the mean ± SD (P < 0.05^*). (C) Analysis of viral titers of the SC2-VLPs containing S-B117-M1237 or S-B117-I1237 produced by 293T cells (left panel) and viral infectivity (right panel, with S-B117-M1237 set as 1). The results were derived from three independent experiments and are shown as the mean ± SD (P < 0.05^*). (D) Schematic illustration of the one-hybrid reporter assay for evaluating fusion activity induced by the SARS-CoV-2 S protein. Effector 293T cells were co-transfected with the expression plasmid for S-B117-M1237 or S-B117-I1237 and the pGAL4DBD-hAR-NTD plasmid. The target 293T-hACE2 cells were transfected with pGAL4/UAS-TK-Luc. At 24 h post transfection, the effector and target cells were co-cultured for 24 h and harvested to assay luciferase activity. (E) Representative results of the cell-cell fusion activity mediated by S-B117-M1237, S-B117-I1237, S-B117-M1237-Δ18aa or S-B117-I1237-Δ18aa, which were detected by the luciferase reporter assay of the lysates harvested from co-cultured cells (P < 0.05^*) (upper panel). The results were derived from three independent experiments and are shown as the mean ± SD (P < 0.01^**). The expression of the spike protein from 293T cells transfected with the indicated plasmids was analyzed by immunoblotting and GAPDH was included as a loading control (lower panel)