Cysteines in the Stalk of the Nipah Virus G Glycoprotein Are Located in a Distinct Subdomain Critical for Fusion Activation

Abstract

c 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 disulfidelinked 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.

Identification of a Region in the Stalk Domain of the Nipah Virus Receptor Binding Protein That Is Critical for Fusion Activation

“…To investigate whether the chimeric pro- teins G 1-180 -HN 124-571 and G 1-(2A)186 -HN 124-571 are defective at initial F triggering or at this later stage in the fusion process, we determined whether the defect is before or after insertion of the fusion protein into the target cell. Insertion of F's fusion peptide into the target cell indicates that the prehairpin intermediate has formed (18,28,(41)(42)(43); at this stage, F mediates attachment to the target cells, and disengagement of the receptor binding protein does not lead to release from the target cell (13,18,28,40,41,44). To determine whether the G 1-180 -HN 124-571 and G 1-(2A)186 -HN 124-571 chimeric proteins activate NiV F up to the stage of the prehairpin intermediate with fusion peptide inserted but then fail to complete the fusion process, we modified the assay described in Fig.…”

“…The oligomeric state of paramyxovirus receptor binding proteins has been shown to be essential for fusion promotion for NiV (44) and has been suggested to be critical for fusion promotion by HPIV3 (38), measles virus (65,66), and other paramyxoviruses (24). It was unknown whether, within a tetramer, the receptorengaged signal can be transmitted from the head of one monomer via the stalk of a different monomer, or whether each monomer acts individually in terms of signal transmission; the chimeric receptor binding proteins provided the answer.…”

Fusion Activation through Attachment Protein Stalk Domains Indicates a Conserved Core Mechanism of Paramyxovirus Entry into Cells

“…3A) shows some variability in the putative distances at which F-interacting/activating residues are found within these different attachment protein stalks. From biochemical studies, F-activating regions in henipavirus G proteins are considered to be located higher up in the stalk (30,40,66). It was also shown using a series of insertion and deletion mutants of MeV H (35) that altering the distances of F-activating residues from the membrane has major effects on functionality.…”

Stimulation of Nipah Fusion: Small Intradomain Changes Trigger Extensive Interdomain Rearrangements

“…Nipah's G protein assembles as a homo-tetramer and its ectodomain contains both the ephrin binding and F activation sites (4,5,(11)(12)(13)(14)(15)(16)(17)(18)(19)(20)(21)(22)(23)(24). The F activation site is located in the N-terminal portion of the ectodomain (Fig.…”

“…The F activation site is located in the N-terminal portion of the ectodomain (Fig. 1), which is generally referred to as the stalk domain, or the F activation domain (FAD) (13)(14)(15)(16)(17)(18)(19)(20)(21)(22)(23)(24). The receptor binding sites are located in the C-terminal portion of the ectodomain, and are >2 nm away from the F activation site.…”