Abstract
Paramyxoviruses initiate entry through the concerted action of the tetrameric attachment glycoprotein (HN, H, or G) and the trimeric fusion glycoprotein (F). The ectodomains of HN/H/G contain a stalk region important for oligomeric stability and for the F triggering resulting in membrane fusion. Paramyxovirus HN, H, and G form a dimer-of-dimers consisting of disulfide-linked dimers through their stalk domain cysteines. The G attachment protein stalk domain of the highly pathogenic Nipah virus (NiV) contains a distinct but uncharacterized cluster of three cysteine residues (C146, C158, C162). On the basis of a panoply of assays, we report that C158 and C162 of NiV-G likely mediate covalent subunit dimerization, while C146 mediates the stability of higher-order oligomers. For HN or H, mutation of stalk cysteines attenuates but does not abrogate the ability to trigger fusion. In contrast, the NiV-G stalk cysteine mutants were completely deficient in triggering fusion, even though they could still bind the ephrinB2 receptor and associate with F. Interestingly, all cysteine stalk mutants exhibited constitutive exposure of the Mab45 receptor binding-enhanced epitope, previously implicated in F triggering. The enhanced binding of Mab45 to the cysteine mutants relative to wild-type NiV-G, without the addition of the receptor, implicates the stalk cysteines in the stabilization of a pre-receptor-bound conformation and the regulation of F triggering. Sequence alignments revealed that the stalk cysteines were adjacent to a proline-rich microdomain unique to the Henipavirus genus. Our data propose that the cysteine cluster in the NiV-G stalk functions to maintain oligomeric stability but is more importantly involved in stabilizing a unique microdomain critical for triggering fusion.