mAb αH-1347 epitope mapping.

<p>(A) Schematic representation of the full length CDV-H-protein (H-wt) and a derivative soluble form (sH-ecto). The main functional regions of the stalk are color-coded. CT: cytosolic tail; TM: transmembrane domain. (B) Schematic representation of soluble Hstalk-RFP chimeric proteins and derivative deleted mutants (1–5). Del: deletion; RFP: red fluorescent protein. Drawings are not to scale. (C, D and F) Immunoprecipitation experiments performed with the indicated mAb of soluble H forms derived from the supernatant of transfected 293T cells. Antigenic materials were detected using polyclonal anti-GCN4 (upper panel) or anti-His antibody (lower panel). IP: immunoprecipitation; IB: immunoblotting. (E) Upper part: Schematic representation of the deletion 5 soluble Hstalk-RFP. Lower part: alignment of the primary amino acid sequence of the CDV H-stalk “spacer” section and the derivative triple alanine-scan mutants. Alanines are highlighted in red. (G) Homology structural model of CDV-F and positions (in red) where the mAb αH-1347 epitope has been inserted. Lower part: primary amino acid sequence of the CDV H-stalk “spacer” section with the encompassed F-transferred mAb-1347 peptide (highlighted in red). (H) Reactivities of F-wt-tagFLAG or F-wt-tag1347 with the conformation-insensitive anti-FLAG, the F prefusion state-specific anti-F-Pre (4941) [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004880#ppat.1004880.ref061" target="_blank">61</a>, <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004880#ppat.1004880.ref063" target="_blank">63</a>] or the anti-CDV-H 1347 mAbs, probed 24 hours post-transfection in receptor-negative Vero cells. After addition of the secondary antibody, MFI values were recorded by flow cytometry analyses. Means ± S.D. of data from three independent experiments in triplicate are shown.