Fig 1.

Engineering of ectodomain FLAG-tagged morbillivirus F proteins. (A) Homology model of the prefusion CDV F trimer (52). Residues flanking the FLAG epitope insertion are highlighted in red. (B) Scheme of the morbillivirus F gene. Conserved regions among class I fusion proteins are shown. FP, fusion peptide; HRA and HRB, heptad repeat regions A and B, respectively; TM, transmembrane domain. The red boxes represent the positions along the gene selected for FLAG epitope insertions. (C and F) Expression and processing abilities of the different F variants. Total cell protein extraction from the various F mutants expressed in Vero cells was performed. Immunoblots were decorated with a polyclonal anti-HA (MeV F constructs) or polyclonal anti-F (CDV F constructs) antibody. (D) Quantitative fusion assay. Vero-SLAM cells (target cells) were infected with MVA-T7 (MOI of 1). In parallel, a population of Vero cells (effector cells) was transfected with the different F proteins, a plasmid encoding H, and a plasmid containing the luciferase reporter gene under the control of the T7 promoter. Twelve hours after transfection, effector cells were mixed with target cells and seeded into fresh plates. After 2.5 h at 37°C, fusion was indirectly quantified by using a commercial luciferase-measuring kit. For each experiment, the value obtained for the standard F/H combination was set to 100%. Means of data from three independent experiments in duplicate are shown. wt, wild type. (E and G) Syncytium formation assay. Shown are data for cell-cell fusion induction after the cotransfection of Vero-SLAM cells with plasmid DNA encoding various CDV F proteins and H. Representative fields of view were captured at 24 h posttransfection with a fluorescence confocal microscope (Fluoroview FV1000; Olympus).