Figure 2. Development of a fusion assay and identification of amino residues within the putative fusion peptide whose mutation abolishes membrane fusion.

(A) Effect of Gc cytoplasmic tail deletion on subcellular localization of Gn/Gc. BSR-T7/5 cells were transfected with plasmid encoding wt Gn/Gc or that encoding Gn/GcΔ5. At 24 h post-transfection, the cells were fixed, treated with Triton X-100 (permeabilized) or left without treatment (non-permeabilized) and stained with anti-Gn (Gn) or anti-Gc (Gc) monoclonal antibodies. (B) Syncytium formation by expressed Gn/GcΔ5. BSR-T7/5 cells were co-transfected with either plasmid encoding Gn/Gc or Gn/GcΔ5 in addition to that encoding the Venus protein. At 24 h post-transfection, cells were treated with either low-pH buffer (pH 5.2) or neutral buffer (pH 7.4) for 1 min, further incubated for 1 h, and observed under a fluorescence microscope. (C) Alignment of the putative fusion peptide of Phleboviruses. Asterisks and dots represent the conserved residues and relatively conserved residues, respectively. The numbers represent the location of the putative fusion peptide in the ORF encoded in the M RNA. SFTSV: severe fever with thrombocytopenia syndrome virus (D) Fusion activity of Gn/GcΔ5-based mutants, each carrying a single amino acid mutation within the putative fusion peptide, and their reactivity to Gn- or Gc-specific monoclonal antibodies. BSR-T7/5 cells were co-transfected with plasmids encoding the L, N, and Venus proteins and M RNA encoding Gn/GcΔ5 (WT) or a M RNA mutant encoding Gn/GcΔ5 and a single amino-acid substitution within the putative fusion peptide. Cells were treated with a low pH buffer and observed under a fluorescence microscope for the fusion assay (top row). The cells were also fixed and stained with anti-Gn monoclonal antibody (middle row) or anti-Gc monoclonal antibody (bottom row).