FIG 4.

A single charged amino acid in the HN stalk domain determines F activation promiscuity of two rubulaviruses. (A) Sequence alignment of the PIV5 HN F activation domain and the corresponding region of MuV HN. PIV5 HN residues critical for F activation are shown in red. Arrowheads mark the MuV HN residues that were mutagenized. (B) Protein expression levels of MuV HN single point mutants, as observed after radioimmunoprecipitation of Tran³⁵S-labeled proteins from transfected 293T cell lysates using whole MuV polyclonal antisera. Samples were analyzed on a SDS–10% PAGE gel. Numbers on the left indicate molecular masses markers in kilodaltons. (C) Protein expression and transport to the surface of 293T cells were quantified in cells transfected with MuV HN point mutants. MuV HN proteins were detected on the surfaces of cells using the MuV antisera and fluorescently labeled with a goat α-rabbit FITC-conjugated secondary antibody for detection by flow cytometry. MFI values were expressed as a percentage of wt MuV HN surface expression, and the results are representative of three independent experiments (n = 3). (D) Hemadsorption assay of MuV HN stalk point mutants. Absorbance of hemoglobin from lysed RBCs that were specifically bound to HN proteins expressed on 293T cells was measured at 410 nm and expressed as a percentage of wt MuV HN hemadsorption. (n = 3). (E) Neuraminidase assay of MuV HN stalk point mutants. Emission at 450 nm on cleavage of MU-NANA was expressed as a percentage of the wt MuV HN neuraminidase activity (n = 3). (F) Luciferase reporter assay for fusion showing quantitative cell-cell fusion for MuV HN point mutant proteins coexpressed in Vero cells with MuV F (gray bars) or PIV5 F (white bars). The results are expressed in RLU as a percentage of the wt MuV F and MuV HN fusion activity (n = 3). (G) Representative micrographs showing cell-cell fusion observed when MuV HN point mutants are coexpressed with either MuV F or PIV5 F. Cells were fixed, stained, and photographed at 18 h posttransfection.