FIG 3.
The PIV5 HN F activation region is able to substitute for the NDV HN F activation domain. (A) Gapless protein sequence alignment of portions of HN, H, or G stalk domains from various paramyxoviruses. Predicted transmembrane domains (TM), identified as a stretch of hydrophobic residues flanked by charged residues, are underlined in red. The sequence of seven residues of the putative F-activating domain of PIV5 HN and those replaced in the NDV HN stalk domain to create the NDV-PIV5-Fact-HN chimera, are shown enclosed within black boxes. Color coding of boxes highlighting individual residues correspond to that described in Fig. 2B. Negatively charged aspartic acid or glutamic acid residues of rubulaviruses, including D88 of PIV5 HN and E105 of MuV HN, are colored red. (B) Schematic representation of the NDV HN wt and the NDV-PIV5-Fact-HN chimera proteins. The PIV5 HN region (residues 81 to 87) that replaced the corresponding NDV HN region (residues 90 to 96) is highlighted in black on the NDV-PIV5-Fact-HN stalk. CT, cytoplasmic tail. (C) NDV HN wt and NDV-PIV5-Fact-HN expression in 293T cells labeled with Tran35S-label. The proteins were immunoprecipitated from transfected cell lysates using a NDV HN polyclonal antibody (R4722). The proteins were analyzed on a 10% reducing gel. Numbers on the right are molecular masses in kilodaltons. (D) Surface expression of the NDV-PIV5-Fact-HN chimera protein compared to wt NDV HN surface expression determined by flow cytometry. Proteins were detected on the surfaces of transfected cells using the NDV HN R4722 antibody and labeled with a goat α-rabbit FITC-conjugated secondary antibody. The results from three independent experiments. (E) Cell-cell fusion mediated by the NDV-PIV5-Fact-HN chimeric protein when cotransfected with PIV5 F or NDV F, as measured using a luciferase reporter assay for fusion. Fusion activity was measured as RLU and is expressed as a percentage of wt NDV F and wt NDV HN fusion activity. Results from three independent experiments are shown. (F) Representative micrographs showing cell-cell fusion in BHK-21 cells transfected with NDV F or PIV5 F alone or in combination with wt NDV HN, PIV5 HN, or the NDV-PIV5-Fact-HN chimera. Cells were fixed, stained, and photographed at 18 h posttransfection. (G) Receptor-binding ability of the NDV-PIV5-Fact-HN chimera protein as measured using a hemadsorption assay. Absorbance of hemoglobin from RBCs specifically bound by the expressed proteins on 293T cells is measured at 410 nm and shown as a percentage of wt NDV HN hemadsorption activity. Asterisk indicates a P value of <0.05 from three independent experiments. (H) Receptor-destroying activity of the NDV-PIV5-Fact-HN chimera protein as measured by a neuraminidase assay using a fluorogenic substrate. The fluorescent emission is measured at 450 nm and expressed as a percentage of wt NDV HN neuraminidase activity (n = 3).