Membrane-fusion activity of Haals-αTfR in recipient cells. (A) Detection of surface expression of target receptors in recipient cells. Surface expression of TfR in HT1080 and HFL-1 cells was analyzed with flow cytometry by staining with PE-conjugated anti-TfR antibody (black peak) or an isotype control (white peak). (B) Schematic representation of recombinant H protein. scFv is displayed as a C-terminal extension of H glycoprotein. N; Amino-terminal cytoplasmic tail, TM; Transmembrane domain, *; Y481A, R533A, S548L and F549S mutations in H protein. (C) Syncytium formation ability differed among scFv clones. HT1080 cells were co-transfected with F and indicated H expression plasmid, and were photographed 30 hr later. (D) Fusion test by co-culture assay of donor and recipient cells. CHO cells stably expressing the F and H proteins were co-cultured with HFL-1, and were photographed 24 hr later. Yellow arrows indicate syncytia. (E) The number of resistant/GFP(+) colonies from HFL-1 cells by MV-MMCT using H or Haals-TfR. Data are the means of four independent experiments (±SD), **; p < 0.01 (unpaired t-test).