Fusion activation by a headless parainfluenza virus 5 hemagglutinin-neuraminidase stalk suggests a modular mechanism for triggering

Abstract

The Paramyxoviridae family of enveloped viruses enters cells through the concerted action of two viral glycoproteins. The receptor-binding protein, hemagglutinin-neuraminidase (HN), H, or G, binds its cellular receptor and activates the fusion protein, F, which, through an extensive refolding event, brings viral and cellular membranes together, mediating virus-cell fusion. However, the underlying mechanism of F activation on receptor engagement remains unclear. Current hypotheses propose conformational changes in HN, H, or G propagating from the receptor-binding site in the HN, H, or G globular head to the F-interacting stalk region. We provide evidence that the receptor-binding globular head domain of the paramyxovirus parainfluenza virus 5 HN protein is entirely dispensable for F activation. Considering together the crystal structures of HN from different paramyxoviruses, varying energy requirements for fusion activation, F activation involving the parainfluenza virus 5 HN stalk domain, and properties of a chimeric paramyxovirus HN protein, we propose a simple model for the activation of paramyxovirus fusion. Paramyxovirus-mediated fusion depends on the concerted actions of two glycoproteins, an attachment protein [hemagglutinin-neuraminidase (HN), H, or G] and its cognate fusion (F) protein, the latter initially folding into a metastable form. The attachment protein is thought to trigger the fusion protein in a receptor-dependent manner (1-5). This triggering of the metastable F (6) by the receptor-binding protein couples receptor binding of HN, H, or G to lowering the activation energy barrier of F so that F refolds into a highly stable postfusion form (7). In the process, F undergoes a series of structural rearrangements involving several intermediates and brings about membrane merger (8).HN proteins bind sialic acid as their receptor and also have neuraminidase (receptor-destroying) activity. The PIV5 HN protein comprises 565 residues and has a short N-terminal cytoplasmic tail followed by a single transmembrane domain and a large ectodomain (residues 37-565). The protein consists of a stalk region (residues 1-117) that supports a large globular head domain (residues 118-565) containing the receptor-binding and neuraminidase-active site. X-ray crystal structures of the globular head domain of PIV5, NDV, Nipah virus, Hendra virus, measles virus, and human parainfluenza virus 3 (hPIV3) attachment proteins have been obtained (9-16). The PIV5 HN globular head structure (16) reveals a neuraminidase-like fold with a six-bladed β-propeller structure that is a common feature of the other paramyxovirus HN/H/G head domains, regardless of receptor specificity. The sialic acid-binding site is placed centrally within the β-propeller. PIV5 HN exists as a pair of disulfide-linked dimers with the disulfide bond at cysteine 111 (16,17). These dimers are associated noncovalently to form a dimer-of-dimer oligomer (16,18). The PIV5 HN dimer-of-dimer structure showed the dimers arranged at an approximately 90°angle to each oth...

Morbillivirus and henipavirus attachment protein cytoplasmic domains differently affect protein expression, fusion support and particle assembly

“…Since the fusion-triggering function of paramyxovirus attachment glycoproteins resides in the extracellular domain, fusion support is dependent on correct expression of functional protein on the cell surface (Bose et al, 2012;Liu et al, 2013;Navaratnarajah et al, 2011). We therefore assessed the fusion support function of the parental and cytoplasmic domain exchange mutants in a quantitative fusion assay with the homologous or heterologous F proteins.…”

“…The attachment protein-mediated initiation of the F protein rearrangement and subsequent membrane fusion seem to be highly conserved, despite widely diverse receptors (Chang & Dutch, 2012). Interaction with the respective cellular receptors induces conformational changes in the attachment protein globular head (Bose et al, 2012;Liu et al, 2013;Navaratnarajah et al, 2011), which are in turn transferred to the stalk region (Bose et al, 2011;Liu et al, 2013Liu et al, , 2015Navaratnarajah et al, 2012;Yuan et al, 2011). This F-triggering function of the attachment protein can be dissociated from its other functions, such as intracellular transport, association with F and receptor binding, which indicates that it resides in a distinct region.…”

“…This F-triggering function of the attachment protein can be dissociated from its other functions, such as intracellular transport, association with F and receptor binding, which indicates that it resides in a distinct region. Indeed, the attachment protein stalk domain is still able to trigger the F protein even when the globular head is removed (Bose et al, 2012(Bose et al, , 2014Brindley et al, 2013;Liu et al, 2013).…”

Immobilization of the N-terminal helix stabilizes prefusion paramyxovirus fusion proteins

“…4). Together with biochemical and structural data of paramyxovirus attachment proteins (17)(18)(19)(20)(21)(22)(23)(24)(25)(26)(27)(28), a general model of paramyxovirus fusion has been proposed: upon activation by the attachment protein, metastable prefusion F undergoes a series of large-scale, ATP-independent conformational changes, going down an energy gradient from a metastable prefusion state to a highly stable postfusion state. The energy released during F refolding is believed to facilitate membrane fusion to create a pore between the virus and host cell through which the viral ribonucleoprotein complex can enter the target cell.…”

Probing the Functions of the Paramyxovirus Glycoproteins F and HN with a Panel of Synthetic Antibodies

“…Upon binding receptor, the attachment protein globular heads are thought to initiate a rearrangement, and this movement is believed to be responsible for F activation during the F-HN/H/G interaction (1,(41)(42)(43)(44).…”