Fig. 5. MV-H protein expression. (A) Constructs for expression of MV-H in human HEK293-derivative cells. The gray pentagons indicate N-linked sugar sites at residues 168, 187, 200, and 215. The location of a disulfide bond (position 154) is also indicated. B, SDS-PAGE analysis of MV-H under reducing and non-reducing conditions. C, Size-exclusion chromatography of MV-H on Superdex 200 GL 10/300.

Fig. 6. Structure-based sequence alignment of MV-H and three paramyxovirus HNs (NDV, SV5, and hPIV3). The amino acid numbers above the alignment are derived from MV-H. The gray characters show the invisible loop positions at residues 237-249. Residues that are highly conserved (red) and less well conserved (orange) between the MV and three paramyxoviruses are indicated. Those residues that are highly conserved (blue) and less well conserved (green) between only the three paramyxoviruses are also indicated. Strands and helices are indicated by the arrows and rectangles, respectively. The color scheme corresponds to that shown in Fig. 1. The pentagons indicate N-linked glycosylation sites at residues 187, 200, and 215. C1-C5 above the Cys residues indicate residues involved in disulfide bond formation. The asterisk (*) denotes residues important for SLAM-binding (**: major importance and *: minor importance). The hash symbol (#) denotes residues important for CD46-binding (##: major importance and #: minor importance). The back slash symbol (//) indicates the invisible loop position at residues 165-183. The blue squares indicate conserved residues at the active site in HNs.

Fig. 7. Secondary structure of the MV-H. Strands and helices are indicated by the arrows and rectangles, respectively. The color scheme corresponds to that shown in Fig. 1. Purple: stem region, blue: b-sheet 1, forest green: b-sheet 2, chartreuse: b -sheet 3, yellow: b-sheet 4, orange: b-sheet 5, red: b-sheet 6. The putative SLAM-binding sites are located on the loops of b-sheet 5. The dotted lines indicate the invisible loop positions at 165-183 and 237-249. The pink arrowed line (b6s4) derives from the feature located between a0 and a1.

Fig. 8. N215-linked sugar position of MV-H. A, 2Fo-Fc map (1.0s) around the GlcNAc2 (green-based stick model) attached to N215. B, C, The MV-H monomer top site is fully solvent exposed in the crystals. B: top view, C: side view. The color representation is the same as in Fig. 1. The black circle represents the assumed location of the N215-linked sugar.

Fig. 9. The N-linked complex-sugar model of MV-H. The light and dark cyan surfaces indicate the four N-linked sugars derived from each monomer. The N-linked sugars were manually built using the complex-type penta-antennary sugars whose coordinates were obtained from the web site http://www.glycosciences.de. N168-and N187-linked N sugars are not visible in the crystal. Red and blue arrows indicate the SLAM and CD46 binding sites, respectively.