FIG 1.

A microdomain in the F head domain controls fusion without influencing interactions with H. (A) Scheme of the morbillivirus F gene. Conserved regions among class I fusion proteins are shown for the fusion peptide (FP), heptad repeat regions A and B (HRA and HRB), and transmembrane domain (TM). The blue boxes represent the position along the gene selected for FLAG and HA epitope insertions. The region in the CDV F (A75/17) swapped by the corresponding region in MeV F (Edmonston) is shown in black. (B) Syncytium formation assay. Cell-cell fusion induction after cotransfection of Vero cells with plasmid DNAs encoding various CDV F proteins and MeV H. Representative fields of view were captured with a confocal microscope (Olympus FluoView FV1000) 24 h posttransfection. (C) Highlight of the amino acid differences between CDV F and Chim 4. (D) Homology model of the prefusion CDV F trimer (49); the segment from positions 446 to 455 in CDV F is shown in green. (E) Close-up view of the microdomain in the CDV F that controls fusion (referred to as the “cysteine loop”). (F) Syncytium formation assay. Cell-cell fusion was performed as described in panel B but with single CDV F mutants. (G) Quantitative fusion assay. Target Vero cells, Vero-cNectin-4 cells, or Vero-cSLAM cells (as indicated) were infected with MVA-T7 (MOI of 1). In parallel, another population of Vero or CHO cells (effector cells) was transfected with the different F proteins, the indicated H protein, and a plasmid containing the luciferase reporter gene under the control of the T7 promoter. Twenty hours after transfection, effector cells were mixed with target cells and seeded into fresh plates. After 2 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 1. Means of three independent experiments in duplicate are shown. (H) Surface expression and F conformational state probing. Vero cells were transfected with the F expression plasmids mentioned above. For immunofluorescence (IF) analysis, cells were stained with the different anti-F MAbs (FLAG [gray bars], 4941 [green bars], and 4068 [red bars]) 24 h posttransfection at 4°C. Alexa Fluor 488-conjugated secondary antibody was then added, and stained cells were subjected to flow cytometry to record mean fluorescence intensities (MFI). Means of three independent experiments performed in duplicate are shown. (I) Assessment of H interaction with functional F proteins. To stabilize the F-H interactions, transfected Vero cells were treated with DTSSP. MeV H- and CDV F-coexpressing Vero cells were then lysed with RIPA buffer, and complexes were immunoprecipitated (IP) with three anti-H MAbs (I41, I44, and 16DE6) and protein G-Sepharose bead treatment. Proteins were boiled and subjected to immunoblotting using a polyclonal anti-HA antibody to detect the F antigenic materials. Co-IP F proteins were detected in comparison with F present in the lysates prior to IP by immunoblotting using a polyclonal anti-F antibody (TL, total lysate; F₀, uncleaved F protein; F₁, cleaved membrane-anchored F subunit). For a control, total H proteins, obtained by direct immunoprecipitation with the above-mentioned MAbs, were revealed by immunoblotting using a polyclonal anti-H antibody (IP). (J) Semiquantitative assessment of F-H avidity of interaction. To quantify the avidities of F₁-H interactions, the signals in each of the F₁ and H bands were quantified using the AIDA software package. The avidity of F₁-H interactions is represented by the ratio of the amount of coimmunoprecipitated F₁ over the product of F₁ in the cell lysates divided by the ratio of the amount of immunoprecipitated H over the product of H in the cell lysate. Subsequently, all ratios were normalized to the ratio of the wild-type F-H interactions set to 100%. Averages represent at least two independent experiments.