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...
Paramyxoviruses enter host cells by fusing the viral envelope with a host cell membrane. Fusion is mediated by the viral fusion (F) protein, and it undergoes large irreversible conformational changes to cause membrane merger. The C terminus of PIV5 F contains a membrane-proximal 7-residue external region (MPER), followed by the transmembrane (TM) domain and a 20-residue cytoplasmic tail. To study the sequence requirements of the F protein C terminus for fusion, we constructed chimeras containing the ectodomain of parainfluenza virus 5 F (PIV5 F) and either the MPER, the TM domain, or the cytoplasmic tail of the F proteins of the paramyxoviruses measles virus, mumps virus, Newcastle disease virus, human parainfluenza virus 3, and Nipah virus. The chimeras were expressed, and their ability to cause cell fusion was analyzed. The chimeric proteins were variably expressed at the cell surface. We found that chimeras containing the ectodomain of PIV5 F with the C terminus of other paramyxoviruses were unable to cause cell fusion. Fusion could be restored by decreasing the activation energy of refolding through introduction of a destabilizing mutation (S443P). Replacing individual regions, singly or doubly, in the chimeras with native PIV5 F sequences restored fusion to various degrees, but it did not have an additive effect in restoring activity. Thus, the F protein C terminus may be a specific structure that only functions with its cognate ectodomain. Alanine scanning mutagenesis of MPER indicates that it has a regulatory role in fusion since both hyperfusogenic and hypofusogenic mutations were found.
cCysteines were introduced into the membrane-proximal external region (MPER) of the paramyxovirus F protein. A disulfide bond formed, and the mutant protein was expressed at the cell surface but was fusion inactive. Reduction of the disulfide bond restored fusion activity. The data indicate that in addition to dissociation of the three-helix bundle stalk domain of prefusion F, the MPER region also needs to separate for F to be able to refold and cause fusion.
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