Functional and biochemical characterization of stalk-elongated and shortened engineered H variants.

<p>(A) Upper part: schematic representation of the main functional domains of the full length CDV H-wt protein. Middle part: schematic representation of the H-elongated construct with the precise position of the insertion indicated. The 11 residues inserted are derived from the N-terminal stalk “contact” section 112–122 that putatively assume a helical fold with an 11-mer repeat. Lower part: schematic representation of the H-shortened version with the precise position of the deletion indicated. (B) Immunoblotting of standard and size-modulated H proteins. Antigenic materials ran in an 8% SDS-Page gel under reducing conditions were detected using a polyclonal anti-H antibody. (C) Syncytium formation assay. Fusion activity in Vero-cSLAM cells triggered by co-expression of CDV H (or size-modulated mutants) and CDV F in the presence (+) of absence (-) of mAb αH-1347. Representative fields of view of cell-cell fusion induced 24h post-transfection are shown. (D) Reactivity of H-wt and size-modulated H variants to mAbs αFLAG and αH-1347. Vero cells were transfected with the different H plasmids and, after addition of the secondary antibody, MFI values were recorded 24h post-transfection. (E) SLAM-binding activity of H mutants. Standard and H mutants were expressed in Vero cells. One day post-transfection, cell surface expression (CSE; recorded by anti-FLAG staining followed by flow cytometry analyses) and SLAM binding efficiency (calculated as described in the legend of <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004880#ppat.1004880.g001" target="_blank">Fig 1D</a>, but without addition of mAbs before SLAM treatments) were determined. All values were normalized to H-wt. (F) Co-IPs, performed as described in the legend of <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004880#ppat.1004880.g004" target="_blank">Fig 4B</a>, were obtained 24 hours post-transfection of cell extracts of Vero cells transfected with standard H or derivative mutants and F-wt-expressing vectors. (G) Semiquantitative assessment of F/H avidity of interactions. To quantify the avidities of F₁-H interactions, the signals in each 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 (coIP) 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 ((coIP F₁/TL F₁)/(IP H/TL H)). 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. (H) Effect of SLAM treatment on mAb αH-1347’s H-binding activity. The assay and values were determined as described in the legend of <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004880#ppat.1004880.g004" target="_blank">Fig 4D</a> (37°C). Means ± S.D. of data from two independent experiments performed in triplicates are shown.