The paramyxovirus parainfluenza virus 5 (PIV5) enters cells by fusion of

The paramyxovirus parainfluenza virus 5 (PIV5) enters cells by fusion of the viral envelope with the plasma membrane through the concerted action of the fusion (F) protein and the receptor binding protein hemagglutinin-neuraminidase. on F1 that contains a hydrophobic region, known as the FP, which intercalates target membranes during F protein refolding. The crystal structure of the soluble ectodomain of the uncleaved form of PIV5 F is known; here we report the crystal structure of the cleavage-activated prefusion form of PIV5 F. The structure shows minimal movement of the residues adjacent to the protease cleavage site. Most of the hydrophobic FP residues are buried in the uncleaved F protein, and only F103 at the Ercalcidiol newly created N terminus becomes more solvent-accessible after cleavage. The conformational freedom of the charged arginine residues that compose the protease recognition site increases on cleavage of F protein. are enveloped, negative-strand RNA viruses that are significant pathogens in humans and animals (1). The family includes parainfluenza viruses 1C5 (PIV1C5), mumps virus, measles virus, Newcastle disease virus, Sendai virus, Hendra virus, Nipah virus, respiratory syncytial virus, and metapneumovirus. To enter cells, paramyxoviruses, like all enveloped viruses, must fuse the viral envelope with a membrane of a host cell. For paramyxoviruses, this process involves two viral spike glycoproteins: a receptor binding protein, variously called HN, H, or G, and the fusion protein, F (2, 3). The paramyxovirus F protein is a class I viral fusion protein that initially folds in the endoplasmic reticulum into a trimeric metastable prefusion form and on triggering undergoes major irreversible conformational changes (refolding) to form the trimeric postfusion conformation. F protein refolding couples the energy released with membrane fusion (4). The F protein is synthesized as a precursor (F0) that must be Ercalcidiol cleaved either by a host protease (furin or furin-like protease) in the Golgi apparatus or by an extracellular trypsin-like enzyme to form the biologically active molecule F1,F2. Cleavage creates a new N terminus on F1 that contains a highly conserved hydrophobic region known as the fusion peptide (FP) (5). Cleavage of F is a prerequisite for fusion and virus infectivity (6, 7), and intracellular cleavage of F correlates with virus pathogenicity (8). For PIV5, the receptor-binding protein hemagglutinin-neuraminidase (HN) binds to sialic acid moieties on the cell surface and is required for the activation of F occurring at the plasma membrane and at neutral pH. It is thought that F interacts with the stalk domain of HN (9C17). On fusion activation, F undergoes refolding, resulting in formation of a trimeric coiled coil composed of a heptad repeat A region that extends away from the viral membrane (18C20). Peptide inhibitor studies and available atomic structures indicate that many of the key elements of this entry mechanism are common to other class I Tmem34 viral fusion proteins, such as the hemagglutinin (HA) of influenza virus, gp120/41 of HIV, S protein of severe acute respiratory syndrome coronavirus, and glycoprotein (GP) of Ebola virus (reviewed in ref. 4). Although X-ray structures of the six-helix bundle of many type Ercalcidiol I fusion proteins have been determined, more complete postfusion ectodomain structures are known only for PIV3 F, NDV F, and RSV F (21C25). Furthermore, structures of the prefusion conformation of type I fusion proteins have been solved only for influenza virus HA, PIV5 F, and Ebola virus GP (20, 26C28). The atomic structures of both uncleaved and protease-cleaved prefusion forms are available only for influenza virus HA (26, 28). The before and after cleavage HA structures are largely superimposable, except for residues near the protease cleavage site that compose a surface loop. The structures yield valuable information that helps explain observations regarding the protease recognition site and provides insight into the acid lability of HA after cleavage activation (28). Earlier work using antisera to peptides derived from the F sequence suggested considerable change in antibody reactivity occurring on cleavage of F0 to F1,F2 (29). Here we present the crystal structure of the cleaved, prefusion form of the soluble ectodomain of PIV5 F. Similar to influenza virus HA, the paramyxovirus F uncleaved and cleaved structures are largely superimposable, except for the residues composing and surrounding the protease.