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Structure of the Cleavage-Activated Prefusion Form of the Parainfluenza Virus 5 Fusion Protein

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Structure of the Cleavage-Activated Prefusion Form of the Parainfluenza Virus 5 Fusion Protein Structure of the cleavage-activated prefusion form of the parainfluenza virus 5 fusion protein Brett D. Welcha,b,1, Yuanyuan Liua,b,1, Christopher A. Korsa,b, George P. Lesera,b, Theodore S. Jardetzkyc,2, and Robert A. Lamba,b,2 aHoward Hughes Medical Institute and bDepartment of Molecular Biosciences, Northwestern University, Evanston, IL 60208; and cDepartment of Structural Biology, Stanford University School of Medicine, Stanford, CA 94305 Contributed by Robert A. Lamb, August 9, 2012 (sent for review June 28, 2012) The paramyxovirus parainfluenza virus 5 (PIV5) enters cells by refolding, resulting in formation of a trimeric coiled coil com- fusion of the viral envelope with the plasma membrane through posed of a heptad repeat A region that extends away from the the concerted action of the fusion (F) protein and the receptor viral membrane (18–20). binding protein hemagglutinin-neuraminidase. The F protein folds Peptide inhibitor studies and available atomic structures in- initially to form a trimeric metastable prefusion form that is trig- dicate that many of the key elements of this entry mechanism are gered to undergo large-scale irreversible conformational changes common to other class I viral fusion proteins, such as the hem- to form the trimeric postfusion conformation. It is thought that agglutinin (HA) of influenza virus, gp120/41 of HIV, S protein of F refolding couples the energy released with membrane fusion. severe acute respiratory syndrome coronavirus, and glycoprotein The F protein is synthesized as a precursor (F0) that must be (GP) of Ebola virus (reviewed in ref. 4). Although X-ray struc- cleaved by a host protease to form a biologically active molecule, tures of the six-helix bundle of many type I fusion proteins have F1,F2. Cleavage of F protein is a prerequisite for fusion and virus been determined, more complete postfusion ectodomain struc- infectivity. Cleavage creates a new N terminus on F1 that contains tures are known only for PIV3 F, NDV F, and RSV F (21–25). a hydrophobic region, known as the FP, which intercalates target Furthermore, structures of the prefusion conformation of type I membranes during F protein refolding. The crystal structure of fusion proteins have been solved only for influenza virus HA, the soluble ectodomain of the uncleaved form of PIV5 F is known; PIV5 F, and Ebola virus GP (20, 26–28). The atomic structures of here we report the crystal structure of the cleavage-activated both uncleaved and protease-cleaved prefusion forms are avail- prefusion form of PIV5 F. The structure shows minimal movement able only for influenza virus HA (26, 28). The before and after of the residues adjacent to the protease cleavage site. Most of the cleavage HA structures are largely superimposable, except for hydrophobic FP residues are buried in the uncleaved F protein, and residues near the protease cleavage site that compose a surface only F103 at the newly created N terminus becomes more solvent- loop. The structures yield valuable information that helps explain accessible after cleavage. The conformational freedom of the observations regarding the protease recognition site and provides charged arginine residues that compose the protease recognition insight into the acid lability of HA after cleavage activation (28). site increases on cleavage of F protein. Earlier work using antisera to peptides derived from the F sequence suggested considerable change in antibody reactivity cleavage activation | F protein structure | paramyxoviruses occurring on cleavage of F0 to F1,F2 (29). Here we present the crystal structure of the cleaved, prefusion form of the soluble he Paramyxoviridae are enveloped, negative-strand RNA ectodomain of PIV5 F. Similar to influenza virus HA, the Tviruses that are significant pathogens in humans and animals paramyxovirus F uncleaved and cleaved structures are largely (1). The family includes parainfluenza viruses 1–5 (PIV1–5), superimposable, except for the residues composing and sur- mumps virus, measles virus, Newcastle disease virus, Sendai vi- rounding the protease recognition site. Unlike HA, there is no rus, Hendra virus, Nipah virus, respiratory syncytial virus, and concerted movement or burying of the N-terminal residues of metapneumovirus. To enter cells, paramyxoviruses, like all the FP. However, because PIV5 F is triggered for fusion by its enveloped viruses, must fuse the viral envelope with a membrane receptor binding protein HN at neutral pH, there is no need for of a host cell. For paramyxoviruses, this process involves two an HA-like mechanism for priming sensitivity to low pH. viral spike glycoproteins: a receptor binding protein, variously called HN, H, or G, and the fusion protein, F (2, 3). Results and Discussion The paramyxovirus F protein is a class I viral fusion protein Expression and Crystallization of the Prefusion PIV5 F-GCNt Protein in that initially folds in the endoplasmic reticulum into a trimeric its Cleaved Form. PIV5 F is a type I transmembrane GP with a metastable prefusion form and on triggering undergoes major 19-residue signal sequence, a large (465-residue) ectodomain, irreversible conformational changes (refolding) to form the tri- a C-terminal transmembrane anchor, and a short cytoplasmic meric postfusion conformation. F protein refolding couples the tail. F is synthesized as a precursor (F0) that must be cleaved for energy released with membrane fusion (4). The F protein is synthesized as a precursor (F0) that must be cleaved either by a host protease (furin or furin-like protease) in the trans Golgi Author contributions: B.D.W., Y.L., T.S.J., and R.A.L. designed research; B.D.W., Y.L., C.A.K., and G.P.L. performed research; Y.L. and C.A.K. contributed new reagents/analytic tools; apparatus or by an extracellular trypsin-like enzyme to form the B.D.W., G.P.L., T.S.J., and R.A.L. analyzed data; and B.D.W., T.S.J., and R.A.L. wrote biologically active molecule F1,F2. Cleavage creates a new N the paper. terminus on F1 that contains a highly conserved hydrophobic The authors declare no conflict of interest. region known as the fusion peptide (FP) (5). Cleavage of F is Database deposition: The atomic coordinates and structure factors have been deposited a prerequisite for fusion and virus infectivity (6, 7), and in- in the Protein Data Bank, www.pdb.org (PDB ID code: 4GIP). tracellular cleavage of F correlates with virus pathogenicity (8). See Commentary on page 16404. For PIV5, the receptor-binding protein hemagglutinin-neur- 1B.D.W. and Y.L. contributed equally to this work. aminidase (HN) binds to sialic acid moieties on the cell surface 2To whom correspondence may be addressed. E-mail: [email protected] or ralamb@ and is required for the activation of F occurring at the plasma northwestern.edu. membrane and at neutral pH. It is thought that F interacts with This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. the stalk domain of HN (9–17). On fusion activation, F undergoes 1073/pnas.1213802109/-/DCSupplemental. 16672–16677 | PNAS | October 9, 2012 | vol. 109 | no. 41 www.pnas.org/cgi/doi/10.1073/pnas.1213802109 Downloaded by guest on September 26, 2021 F to be biologically active. Proteolytic cleavage of F0 to form the the cleavage protocol, thereby facilitating a homogenous pop- disulfide-linked chains F1 and F2 generates an N terminus on F1 ulation of cleaved prefusion F-GCNt. After inactivation of SEE COMMENTARY that contains the hydrophobic FP. WT PIV5 (strain W3A) F trypsin with protease inhibitors and repurification of F-CGNt by contains five arginine (Arg) residues at the cleavage site, and nickel chromatography (Ni-NTA), protein gel analysis (SDS/ cleavage occurs intracellularly in the trans Golgi network by furin PAGE) indicated the protein was cleaved into F1-GCNt and F2 or a furin-like protease during transport of F to the cell surface and was >95% pure (Fig. 1B). Electron microscopy revealed that (1). The atomic structure of uncleaved prefusion F, a mutant F the cleaved F-GCNt was stable and remained in the prefusion containing three Arg residues at the cleavage site (residues Arg- form when stored at 4 °C for days, as demonstrated by the “ ” C 98, -99, and -100, with Arg-101 and -102 deleted), indicates that characteristic tree-like morphology (Fig. 1 ). However, heat- the cleavage site residues form a small surface-exposed loop (20). ing the sample to 60 °C for 10 min (a surrogate for HN activa- tion) converted cleaved F to the postfusion conformation, with To express a soluble ectodomain of F0 protein suitable for “ ” crystallization, we used an F protein (F-GCNt) that contains its characteristic golf tee morphology with rosette formation (30) (Fig. 1D). three Arg residues at the cleavage site and a coiled-coil trime- rization domain fused in a helical frame with the C-terminal Structure of Cleaved Prefusion PIV5 F-GCNt. The cleaved prefusion heptad repeat B (HRB) region in place of the transmembrane PIV5 F-GCNt crystallized in the space group C2 and diffracted anchor and cytoplasmic tail (20). This trimerization domain is X-rays anisotropically up to 2.0–3.0 Å resolution (Table S1). The necessary for stabilizing the PIV5 F-soluble protein in its pre- structure was solved by molecular replacement using the fusion form (20, 22) (Fig. 1A). uncleaved prefusion PIV5 F-GCNt structure [Protein Data Bank F-GCNt was expressed from insect cells using a recombinant (PDB) ID code: 2B9B], and a single trimer was found in the baculovirus expression system, secreted into serum-free medium, asymmetric unit. and affinity-purified via a 6× His tag that was appended to the C Each F-GCNt monomer consists of four domains—DI, DII, terminus of the GCNt domain.
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