FIG. 1.

Insertion of additional N-glycans into the MeV H stalk domain at membrane-proximal and membrane-distal positions. glyc., glycosylated; deglyc., deglycosylated. (A) Ribbon representation of hypothetical envelope glycoprotein interaction modes and the positions of additional N-glycans engineered to probe the proximity of the F trimer to H. Staggered alignment (left panel) predicts that only N-glycans added at a membrane-proximal position (shown as dark spheres for H residue 72) but not a membrane-distal position (highlighted residue 111; engineered N-glycan not shown for spatial constraints) are compatible with the formation of functional fusion complexes. Parallel alignment of the head domains (right panel) permits H and F hetero-oligomerization with added N-glycans at either position in the H stalk (shown for residues 72 and 111 as dark spheres). Prefusion F and H head and stalk domains, F residue A121 (previously identified to contribute to MeV H and F interaction) (36), and cysteine residues in the H stalk (C139 and C154) engaged in disulfide bonds for covalent H dimerization are shown. H is represented as a dimer in accordance with available X-ray information, although a tetramer (dimer-of-dimers) may in fact be the physiological oligomer. Either order is predicted to tolerate additional N-glycans at position 72. (B) Of three N-glycosylation sites engineered at positions 70, 71, and 72, only that at position 72 is efficiently glycosylated and surface expressed. Surface-exposed proteins were biotinylated, precipitated with immobilized streptavidin, and subjected to PNGase F treatment for deglycosylation or mock treated in equal aliquots. Electrophoretic mobility of MeV H species was determined by SDS-PAGE and decoration of immunoblots with a specific antiserum directed against the cytosolic tail of MeV H. (C) Engineered N-glycans at positions 110, 111, and 112 are efficiently recognized. Treatment of samples was as described for panel B.