Structure and stabilization of the Hendra virus Fglycoprotein in its prefusion formJoyce J. W. Wonga, Reay G. Patersonb, Robert A. Lambb,c,1, and Theodore S. Jardetzkya,1

aDepartment of Structural Biology, Stanford University School of Medicine, Stanford, CA 94305; bDepartment of Molecular Biosciences, NorthwesternUniversity, Evanston, IL 60208-3500; and cHoward Hughes Medical Institute, Northwestern University, Evanston, IL 60208-3500

Contributed by Robert A. Lamb, November 30, 2015 (sent for review October 30, 2015; reviewed by Rebecca Ellis Dutch, Felix A. Rey, and Charles Russell)

Hendra virus (HeV) is one of the two prototypical members of theHenipavirus genus of paramyxoviruses, which are designated bio-safety level 4 (BSL-4) organisms due to the high mortality rate ofNipah virus (NiV) and HeV in humans. Paramyxovirus cell entry ismediated by the fusion protein, F, in response to binding of a hostreceptor by the attachment protein. During posttranslational process-ing, the fusion peptide of F is released and, upon receptor-inducedtriggering, inserts into the host cell membrane. As F undergoes adramatic refolding from its prefusion to postfusion conformation,the fusion peptide brings the host and viral membranes together,allowing entry of the viral RNA. Here, we present the crystal struc-ture of the prefusion form of the HeV F ectodomain. The structureshows very high similarity to the structure of prefusion parainfluenzavirus 5 (PIV5) F, with the main structural differences in the membranedistal apical loops and the fusion peptide cleavage loop. Functionalassays of mutants show that the apical loop can tolerate perturba-tion in length and surface residues without loss of function, exceptfor residues involved in the stability and conservation of the F proteinfold. Structure-based disulfide mutants were designed to anchor thefusion peptide to conformationally invariant residues of the F head.Two mutants were identified that inhibit F-mediated fusion by sta-bilizing F in its prefusion conformation.

paramyxovirus F protein | F-protein atomic structure |metastable F-protein stabilization | Hendra virus | membrane fusion

The Paramyxoviridae family of viruses includes many speciesknown to cause human and animal disease, including Nipahvirus (NiV) and Hendra virus (HeV) of the genus Henipavirus(1). This emergent genus was first described in 1994 with a dis-ease outbreak of HeV, followed by a disease outbreak of NiV in1999 (2), and was recently expanded by the discovery of the Cedarvirus (3) and evidence for 19 new species of African henipaviruses(4). Both NiV and HeV have caused outbreaks of encephalitic andrespiratory illness in humans in Malaysia, Bangladesh, Australia,and several neighboring countries, with high morbidity and mor-tality, and are designated biosafety level 4 (BSL-4) organisms (5,6). The animal reservoir for NiV and HeV is Pteropus spp. fruitbats. These viruses are transmitted to humans from an intermediateanimal vector, pigs in the case of NiV and horses in the case ofHeV. Serological and genetic evidence for henipaviruses has beendiscovered in Pteropus far from known locations of disease in-cidence (6).Like other paramyxoviruses, the henipaviruses are enveloped

viruses that are densely studded on their outer surfaces with thetwo transmembrane-anchored glycoproteins involved in entry ofthe virion into host cells via membrane fusion (7). These glyco-proteins are the fusion glycoprotein, F, which mediates fusion ofthe viral lipid envelope with the host cell plasma membrane, andthe attachment glycoprotein, G, in henipaviruses, which acts as atrigger for fusion upon specific recognition of the host cell re-ceptors ephrinB2 and ephrinB3 (8, 9). Triggering is thought tooccur via sequential conformational changes in G that arecommunicated to F while they associate in F–G complexes (10,11). Exposure of stalk residues in G, which are thought to be

occluded by its receptor-binding head domains before triggering,appear key to initiating F refolding and membrane fusion.The trimeric F protein in its prefusion form has a globular

conformation consisting of three domains (DI, DII, and DIII),followed by a C-terminal stalk, transmembrane domain, and cy-toplasmic tail (12). DI and the Ig-like fold DII are implicated ininteractions with the attachment protein (13, 14). F also containstwo heptad repeats, HRA in DIII and HRB in the stalk. Duringposttranslational processing, the F0 precursor is cleaved by thecellular protease cathepsin L at a defined site following a basicresidue, K109 in HeV F, resulting in release of the fusion peptidesegment located C-terminal to the cleavage site. Cleavage results intwo disulfide-linked fragments, F1 and F2 (15, 16). Upon activation,the F protein undergoes large-scale refolding, mostly in DIII. HRAis extended into a long α-helix, which forms a six-helix bundle withthe HRB region of the stalk. This refolded F forms a golf tee-shaped postfusion conformation (17), which is modeled to bringthe host and virus membranes together as the fusion peptidesinserted into the host-membrane oligomerize with the virion-embedded portion of the HRB stalk (12).The atomic resolution structures of the prefusion forms of the

F protein have been solved for two paramyxoviruses, para-influenza virus 5 (PIV5) and respiratory syncytial virus (RSV)(12, 18). These two proteins exhibit fairly low sequence identityand significant differences in their structures, although severalkey domain features are conserved (18). HeV F shares low se-quence similarity with PIV5 and lower sequence similarity withRSV (27.9% and 21.3% identity, respectively, calculated with

Significance

Hendra virus (HeV) is a deadly member of the Henipavirusgenus of paramyxoviruses, which causes high mortality in hu-mans and horses. We determined the crystal structure of theHeV fusion protein, F, in its metastable, prefusion conformation.The structure is highly conserved compared with parainfluenzavirus 5 (PIV5) F, but divergent from respiratory syncytial virus(RSV) F. The structural similarities suggest a common mode ofactivation for PIV5 and HeV F despite low sequence homology.Structural differences in the HeV F cleavage/activation loop areobserved that may be explained by a requirement for cleavageby cathepsins. The HeV F structure was used to predict disulfidebonds that stabilize its prefusion conformation, providing aconstruct for vaccine and functional studies.

Author contributions: J.J.W.W., R.G.P., R.A.L., and T.S.J. designed research; J.J.W.W. andR.G.P. performed research; J.J.W.W., R.G.P., R.A.L., and T.S.J. analyzed data; and J.J.W.W.,R.G.P., R.A.L., and T.S.J. wrote the paper.

Reviewers: R.E.D., University of Kentucky; F.A.R., Institut Pasteur; and C.R., St. Jude Childrens’Research Hospital.

The authors declare no conflict of interest.

Data deposition: The atomic coordinates and structure factors have been deposited in theProtein Data Bank, www.pdb.org (PDB ID code 5EJB).1To whom correspondence may be addressed. Email: ralamb@northwestern.edu ortjardetz@stanford.edu.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1523303113/-/DCSupplemental.

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ClustalX). The fusion (F)-attachment (HN) protein pair of PIV5,along with the fusion-attachment protein pair of Newcastle diseasevirus and human parainfluenza virus type 3, behave accordingto the “association” model of fusion activation. Although in-teraction is required for fusion, the F-HN pairs of these proteinshave relatively low affinity for each other and host receptorbinding is thought to bring the proteins into greater association atinitiation of fusion. The F protein of RSV does not require itscognate attachment protein for virus fusion and replication. Incontrast, the fusion-attachment protein pairs of the Morbillivirusgenus (measles and canine distemper virus) and henipaviruses,NiV and HeV, behave more consistently with the “dissociation”model of fusion activation. Biochemical evidence suggests a rel-atively higher affinity for the fusion-attachment proteins and

preassociation in complexes where dissociation only occursupon receptor binding (8, 9, 19). To date, no high-resolutionstructural information has been available for fusion proteins inthe dissociation class.Here, we present the crystal structure of the HeV F ectodo-

main. Its structure has a high degree of structural similarity tothe structure of PIV5 F despite low sequence similarity. The areasof greatest structural deviation are at the pair of loops at the apicalregion of the trimer and at the fusion peptide cleavage site. Site-directed mutagenesis within the apical loops showed that residuesthat have a role in maintaining tertiary structural integrity of theregion have effects on fusion and cell surface expression. Based onthe crystal structure, amino acid pairs with a likelihood of formingdisulfide bonds were predicted computationally. Double-Cys

Fig. 1. Crystal structure of prefusion HeV F ectodomain and comparison with prefusion F structures from other paramyxoviruses. (A) HeV F fusion glyco-protein ectodomain in prefusion conformation, orthogonal views. (B) HeV F prefusion monomer. (C) Superposition of prefusion HeV F and PIV5 F (PDB IDcode 2B9B), orthogonal views. HeV F is shown in green, and PIV5 F is shown in orange. Crystallographically refined N-linked carbohydrates are circled andindicated by Asn residue number. (D) Superposition of prefusion HeV F and RSV F (PDB ID code 4JHW), orthogonal views. HeV F is shown in green, and RSV F isshown in purple. (E) Rotation between domains of HeV F and RSV F. The axes of rotation are indicated by yellow rods.

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mutants of HeV F were generated with the intent of preventingrefolding of the HRA region that is crucial for fusion. Two ofthe mutants show loss of fusion activity, while being expressed atthe cell surface and recognized by a mAb (5B3) specific for theprefusion conformational state.

Results and DiscussionOverall Structure of HeV F Is Similar to PIV5 F. The HeV F ecto-domain consisting of residues 26–482 fused to the GCN4 trime-rization domain (12) was expressed in 293F cells and crystallized.X-ray diffraction data were collected to 3.2 Å, and a molecularreplacement solution was found with the PIV5 prefusion F ecto-domain (12) as a search model (Fig. S1 A–C and Table S1). Thecrystal structure of the HeV F ectodomain (Fig. 1 A–C) shows ahigh degree of structural conservation with the crystal structure ofPIV5 F. In addition to high similarity between the folds of DI, DII,and DIII (Fig. 1B and Fig. S1), relative domain orientations aresimilar, resulting in an rmsd by secondary structure matching(SSM) of 2.27 Å between the ectodomains of HeV F and PIV5 F(Fig. 1C). Although conserved fold elements exist between theindividual domains of RSV F (18) and HeV F (Fig. 1D), the overallcorrespondence of the structures is much poorer. In contrast toPIV5 F, significant differences in relative domain orientations existbetween all three domains of the globular head between HeVF and RSV F (Fig. S1C). These differences are expressed asrotation of a domain about an axis following SSM superposition ofa fixed domain, calculated with DynDom (20). The positions of DIIin HeV F and RSV F relative to DI differ by 43°, and the positionsof DIII relative to DI differ by 64° (Fig. 1E), resulting in an rmsd of5.11 Å between the RSV and HeV F structures.N-linked carbohydrate residues were observed for all of the

previously reported glycosylation sites of HeV F, N67, N99,N414, and N464 (21) (Fig. 1C). N67 is on the N-terminal apicalloop, and N99 is at the base of the fusion peptide cleavage siteloop. The only N-glycan site location conserved between HeV Fand PIV5 is HeV N464, which coincides with PIV5 457 on theHRB stalk. Notably, there is no functional conservation betweenthe sites, because HeV F N464 is dispensable for fusion and

processing (21), whereas the stalk glycan of PIV5 is essential forstability, cell surface expression, and proteolytic processing (22).The only HeV F glycan necessary for F expression and function,N414 (21), is in DII on the lower extremity of the globular head.

Investigation of Apical Loop Differences in HeV F. A notable dif-ference between the HeV F and PIV5 F structures occurs in thepair of loops at the topmost surface of the trimer. Compared withthe PIV5 loops, the HeV F loops are more oriented toward thecentral axis and have more of an α-helical than β-sheet secondarystructure (Fig. 2 A and B) and the N-terminal loop is shorter byone amino acid (Fig. 2C). These differences result in a slightoverall compaction in structure in the apical region. V68 has a rolein structural stabilization of the double loops due to insertion of itsside chain in a hydrophobic pocket that includes I179, L183, V184,and I187 from the second apical loop (Fig. 3A). The disulfidebond between C71 and C185 joining the first and secondapical loops is conserved with PIV 5. Both HeV and PIV5 F haveN-linked glycans on the N-terminal apical loop, but the N-linkedglycan of HeV at N67 is N-terminal, whereas the N-linked glycanof PIV5 at N65 is C-terminal to the loop (Fig. 3A).Site-directed mutants were generated in the apical loop region of

HeV F to assess the contribution of individual residues to fusionfunction (Fig. 3A), because these residues could represent structuraldifferences associated with G interaction and/or activation. SingleAla insertions were made following residues 65, 68, and 70 (mutants65B, 68B, and 70B) to mimic the longer PIV5 loop and to test ifloop length has an impact on HeV F function. Ala mutants weremade of the polar surface residues K70 and D185 and the aliphaticburied residue V68. Residue T164 was mutated to Leu because it is

Fig. 2. Apical loops of HeV F in comparison to PIV5 F. (A) Apical portion of theHeV F trimer with PIV5 F superposed. HeV F is shown in green, and PIV5 F isshown in orange, with the apical loops lightened to pale green in HeV F and toorange in PIV5 F. The threefold axis of rotational symmetry is indicated. Thedisulfide bond between the two apical loops is indicated with sticks, and thesulfur atoms are colored olive-yellow. Carbohydrates of HeV F are shown asgreen sticks, and carbohydrates of PIV5 F are shown as orange sticks. (B) Viewdown the rotational axis of the HeV F and PIV5 F trimers. (C) Sequence align-ment of the apical loops of HeV F, PIV5 F, and NiV F generated with ClustalW.Loop structures confirmed by available crystal structures are lightened. Degreeof conservation is indicated as follows: *, perfect; :, strong; ., weak.

Fig. 3. Fusion activity of HeV F apical loopmutants. (A) Site-directedmutants ofthe apical region of HeV F. Residues mutated to Ala are indicated in purple, andAla insertion mutants are indicated by B. (B) Fusion levels of site-directed mu-tants measured by luciferase reporter gene assay. R.L.U., relative light units.

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highly exposed at the end of a protruding β-strand hairpin, where itcould potentially interact with G.Of the site-directed mutants, only V68A, T164L, and 65B (an

insertion mutant), listed in order of increasing defect, show sig-nificant effects on fusion (Fig. 3B and Fig. S2). Mutant V68Ashows reduced cell surface protein expression, which wouldcontribute, at least partially, to its decreased fusogenicity (Fig. S3and Table S2), but the mutation may also affect the stabilityof the prefusion conformation. Mutant T164L has robust totalprotein expression but undergoes abnormal proteolysis, resultingin a smaller F1 fragment, which is a likely contributor to its de-creased cell surface expression and fusogenicity (Fig. S3). Mutant65B has normal proteolytic processing, a mild decrease in totalexpression level, and a more significant decrease in cell surfaceexpression to ∼25% of WT, but it shows negligible fusion activity(Fig. S3 and Table S2). Therefore, Ala insertion after residue 65affects fusion activity more severely than cell surface expression.Based on the fusion and protein expression assays, we con-

clude that the protein surface residues K70 and D188 alone areunlikely to be crucial residues involved in HeV F interaction withpotential binding partners. The one-residue-length decrease ofthe first apical loop in HeV F is not crucial for function, becauseonly 65B of the three loop insertion mutants resulted in a fusionlevel or protein processing defect (Fig. 3B and Fig. S3). The 68Binsertion has the additional effect of disrupting the N-X-S glyco-sylation motif beginning at residue N67, resulting in an F0 frag-ment with decreased molecular weight relative to WT (Fig. S3),showing that this region is tolerant of various types of perturba-tions. The common feature of V68A and 65B, the two mutantsthat disrupt total fusion activity, and the amount of cell surfaceexpression is that they could disrupt the secondary structure of theapical loop region. V65 of HeV F is in a strand that is structurallyconserved with PIV5 just before it begins to diverge in the apicalloop region, and 65B may disrupt a conserved feature that is im-portant for the function of F. The hydrophobic packing of the twoapical loops by V68 may substitute for the β-sheet interactionsbetween the loops in PIV5. Maintaining these contacts may benecessary to avoid trafficking defects or the early protein degra-dation that may lead to the decreased amount of V68 and 65Bmutant proteins on the cell surface.

HeV F Fusion Peptide Cleavage Site Region. Residues 100–116 ofHeV F, which surround the F1/F2 cleavage site following residueK109, form an elongated, antiparallel β-sheet–like structure withthe cleavage site at the turn. This structure is in contrast to residues94–109 of PIV5, which form a circular, open loop (Fig. 4). Theresidues C-terminal to the cleavage site in HeV F form a moredefined β-strand structure. This strand makes β-sheet backboneinteractions with a neighboring β-strand consisting of residues 425–428 fromDII that is conserved between HeV F and PIV5 F (Fig. 4).These differences in fusion peptide conformation may reflect

their recognition by different proteases during posttranslationalprocessing. Unlike HeV F, which is cleaved by cathepsin L, PIV5is cleaved into its fusion-active form by furin (23). Crystalstructures exist for furin in complex with peptide-derived syn-thetic inhibitors (24) and for cathepsin L in complex with anatural peptide substrate (25), peptide-derived inhibitors (26),and full-length protein inhibitors (27, 28). The furin substrate-binding pocket is well-suited to accommodate an open-loopconformation (Fig. S4A). Its wide and rounded shape wouldforce a linear peptide substrate to curve around to exit. Theentirety of the binding pocket surface is electronegative, which issuitable for complementary electrostatic interactions with thepoly-Arg preceding the cleavage site. In comparison to furin, thesubstrate-binding pocket of cathepsin L forms a longer andnarrower groove (Fig. S4B). The crystal structures of cathepsin Lin complex with the MHC class II invariant chain p41 fragmentand chagasin show that the turn of an elongated loop binds in the

positions of the substrate-binding groove immediately precedingthe cleavage site, whereas additional binding from the rest of theprotein inhibitor is provided by insertion of two additional turnsat separate points in the binding groove (Fig. S4C). The elon-gated loop structure surrounding the cleavage site in HeV Fsuggests that it may insert into the cathepsin L active site ina similar fashion.

Prefusion HeV F Disulfide Bond Mutants. The crystal structure of theHeV F protein provides the opportunity to predict F variantsthat could be stabilized in the prefusion conformation, whichwould be useful for functional studies, antibody (Ab) epitopemapping, and vaccine antigen design. To stabilize prefusion F,double-Cys mutants were generated throughout the HRA regionof HeV F with the intent of locking it in its prefusion confor-mation via disulfide bonds. Potential pairs of disulfide bond-forming residues within the HeV F crystal structure were foundwith Disulfide by Design (29), and six were chosen for furtheranalysis (Fig. 5A). All six mutants expressed to appreciable lev-els, but only two, Y97C-G131C and N100C-A119C, had normalproteolytic processing and were expressed at WT levels on thecell surface (Fig. S3 and Table S2). The other four mutants hadsome combination of decreased or abnormal proteolytic pro-cessing and greatly decreased cell surface expression. All sixmutants exhibited negligible fusion (Fig. 5B). This result suggeststhat the Y97C-G131C and N100C-A119C mutants are locked inthe prefusion conformation while on the cell surface and areunable to undergo the prefusion-to-postfusion conformationaltransition triggered by G. Although both residues are adjacent inthe prefusion conformation, they end up on essentially oppositesides of the protein after fusion due to Y97 and N100 being onone side of the fusion peptide cleavage site and F131 and A119on the other (Fig. 5A). Residues N-terminal to the cleavage siteare adjacent to the head in the postfusion conformation, whereasresidues C-terminal to the cleavage site are embedded in themembrane at the base of the stalk (Fig. 5A).To confirm if the HeV F protein on the cell surface is in the

prefusion conformation, HEK293T cells transfected with HeVF-expressing vector were analyzed by flow cytometry with ananti-HeV F mAb (5B3) specific for its prefusion conformation

Fig. 4. Fusion peptide region of HeV F in comparison to PIV5 F. (A) HeV andPIV5 F fusion peptides and preceding regions superimposed. HeV F is shown inlight green, and PIV5 F is shown in light orange, with the fusion peptidesdarkened in HeV F and PIV5 F. Basic protease cleavage site residues are in-dicated by spheres, and the N- to C-terminal fusion peptide direction is in-dicated by arrows. (B) Sequence alignment of the fusion peptide regions ofHeV, PIV5, and NiV F generated with ClustalW. The basic residue upstream ofthe N terminus of the fusion peptide is boxed in red, and the remainder of thefusion peptide is darkened. The degree of conservation is indicated as in Fig. 2.

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(30). The median fluorescence intensities (MFIs) (Fig. 5C, Fig. S5,and Table S3) show that the Y97C-G131C and N100C-A119Cmutants are present at the surface in the prefusion conformationat ∼60% of the WT level, consistent with the predicted disulfidebond stabilization. The radioimmunoprecipitation assay indicatesthat the total amount of HeV F on the cell surface may be similar

to the total amount of HeV F of WT (Table S2), potentiallysuggesting that a lower percentage of prefusion F accumulates forthe disulfide bond mutants. However, variability in the radio-immunoprecipitation assay and in transfection efficiency, whichcould have an impact on the MFI, is too significant to determinethe efficiency of folding of the disulfide bond mutants to theprefusion state. Nonetheless, the observed fusion defects in Y97C-G131C and N100C-A119C are much greater than the reduction inthe amount of prefusion F at the cell surface detected by mAb 5B3compared with WT. Therefore, we conclude that the majority ofthe decrease in fusion is most consistent with the F protein beinglocked in the prefusion conformation by disulfide bonds.Engineering of vaccine antigens with introduced disulfide

bonds has been explored as a means of generating more stableand/or more immunogenic variants (31, 32). The metastability ofviral fusion glycoproteins can be an obstacle for their expression,purification, and antigenicity due to their tendency to transitionspontaneously from the prefusion to postfusion conformation (18,33). The successful engineering of disulfide bonds for maintainingfusion proteins in the prefusion conformation has been reportedfor measles, RSV, PIV5 F, influenza HA, and HIV gp120-gp41trimers (32, 34–37). An additional benefit of prefusion-stabilizingdisulfide bonds may be increased thermal stability during storage,because the WT prefusion-to-postfusion transition can be trig-gered by heat (38). Disulfide bond-enhanced thermal stability andshelf life have been demonstrated in subunit vaccines for Lymeborreliosis (39) and ricin A toxin (40). Our results with the HeV FY97C-G131C and N100C-A119C mutants provide tools for fur-ther studies of HeV F fusion and antigenicity, as well as an ad-ditional example of how the engineering of disulfide bonds intometastable fusion proteins can be used to control their confor-mational and functional properties.

ConclusionsThe HeV F ectodomain has a structure very similar to thestructure of PIV5 F, suggesting that the mechanism for receptor-dependent triggering of fusion is conserved. However, HeV Fpreassociates with its attachment protein, G, before receptorbinding, whereas PIV5 F does not. Significant structural differ-ences are observed in their fusion peptide regions and the doubleapical loops. The more narrow and elongated loop of HeV Fsurrounding the K109 cleavage site likely reflects differences inthe recognition site of its specific activating protease, cathepsin L,as opposed to furin, which is used by PIV5. The functional role ofthe double apical loops appears limited to its structural integritybased on our functional studies. Residues in these loops involvedin structure stabilization and conservation are important formaintaining protein expression and function. Other residueswithin the F trimer must promote the preferential assembly ofHeV into prefusion complexes with G, in contrast to PIV5 F, andthese residues have yet to be identified. However, the overallstructural conservation is consistent with a similar mode of Factivation for Henipavirus and parainfluenza viruses. We havegenerated two HeV F disulfide mutants that fold to the prefusionform, while blocking F fusion activity. Both disulfide mutantsgenerate covalent links between residues immediately precedingand following the fusion peptide cleavage site, so that they areunable to move apart in the conformational transition to thepostfusion state. Fusion peptide immobilization via the structure-based introduction of such disulfide bonds provides a promisingroute for design of other stabilized viral fusion protein variants.

Materials and MethodsProtein Expression and Purification. The HeV F ectodomain (Fecto) was expressedin HEK293F cells transiently transfected with the HeV Fecto-pCAGGS3 plasmidaccording to the high-density method of Backliwal et al. (41). Cell culture me-dium was collected 5 d after transfection and dialyzed against 25 mM sodiumphosphate (pH 7.6), 200 mM NaCl, and 10 mM imidazole. HeV F was purified

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Fig. 5. Fusion activity of predicted disulfide bond-forming mutants in HeVF. (A) Disulfide bond double mutants of HeV F HRA. Prefusion conformationof HeV F with residue pairs chosen for Cys mutations (Left) and a model ofpostfusion HeV F based on postfusion hPIV3 F (PDB ID code 1ZTM) (Right) areshown. Y97, the only ordered aligned residue in the crystal structure, iscolored purple. The distance between the last visible residue preceding thefusion peptide and first visible residue of HRB is shown as a yellow dashedline. (B) Fusion levels of disulfide mutants measured by luciferase reportergene assay. (C) Binding of prefusion-specific mAb 5B3 to HeV F disulfidemutants measured by flow cytometry. PE, phycoerythrin.

1060 | www.pnas.org/cgi/doi/10.1073/pnas.1523303113 Wong et al.

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http://www.pnas.org/lookup/suppl/doi:10.1073/pnas.1523303113/-/DCSupplemental/pnas.201523303SI.pdf?targetid=nameddest=SF5http://www.pnas.org/lookup/suppl/doi:10.1073/pnas.1523303113/-/DCSupplemental/pnas.201523303SI.pdf?targetid=nameddest=ST3http://www.pnas.org/lookup/suppl/doi:10.1073/pnas.1523303113/-/DCSupplemental/pnas.201523303SI.pdf?targetid=nameddest=ST2www.pnas.org/cgi/doi/10.1073/pnas.1523303113

from the medium by nickel-nitrilotriacetic acid (Ni-NTA) chromatography andeluted with a stepwise imidazole gradient. The peak fractions were furtherpurified by size exclusion on a Superdex S200 (GE Healthcare Life Sciences) col-umn in 25 mM Tris (pH 8.0), 200 mM NaCl, and 100 mM imidazole buffer.

Crystallization and Structure Solution of HeV Fecto. The His 8-tag was removedfrom HeV Fecto before crystallization by digestion with Factor XA (New EnglandBiolabs) in 20 mM Tris (pH 7.4), 100 mM NaCl, and 2 mM CaCl2 at a ratio of 1 μgof protease to 20 μg of F overnight at 4 °C. Cleaved His-tags and uncleavedprotein were removed by incubation with Ni-NTA beads, followed by removal ofprotease by size exclusion chromatography. HeV Fecto peak fractions werebuffer-exchanged into 20 mM Tris (pH 8.0), 100 mM NaCl, and 1 mM EDTA, andthen concentrated to 5 mg/mL for crystallization setup. Crystals were grown byvapor diffusion in a 1:1 protein/reservoir solution ratio at room temperature. Thereservoir composition was 100 mM sodium acetate (pH 5.0), 1.75 M lithiumsulfate, 100 mM magnesium sulfate, and 3.4% (vol/vol) isopropanol. Crystalswere washed in reservoir solution before flash-freezing in liquid nitrogen. Dif-fraction data were collected at Advanced Light Source Beamline 8.2.2, and datawere scaled and indexed with XDS (42). A molecular replacement solution wasfound using MOLREP (43) and the PIV5 prefusion F structure [Protein Data Bank(PDB) ID code 2B9B] as a search model. The model was refined with Crystal-lography & NMR System (CNS) (44)-simulated annealing and REFMAC5 (45).Model building was done with Coot (46) and the lego_ca function of O (47).Coordinates have been deposited in the PDB with ID code 5EJB.

Flow Cytometry Assay of Prefusion HeV F Conformation. HEK293T cells in six-well plates were transfected with 1 μg of full-length HeV F pCAGGS, 5 μL of

lipofectamine, and 4 μL of Plus Reagent (Life Technologies). After overnightincubation, cells were detached with 500 μL of enzyme-free cell dissociationbuffer, PBS (Life Technologies). A total of 25,000 cells were washed by dilution in1 mL of FACS buffer (1× PBS, Corning Cellgro; 1% BSA) and pelleting at 400 × gfor 5 min. Cells were resuspended in 100 μL of FACS buffer for staining with 1 μgof mAb 5B3 for 1 h, and then were washed by dilution and pelleting with 2 mLof FACS buffer. Cells were resuspended in 100 μL of FACS buffer for stainingwith 1 μg of goat anti-mouse F(ab′)2 fragment-phycoerythrin (eBioscience) and0.5 μL of LIVE/DEAD Fixable Aqua stain (Life Technologies) for 1 h. Cells werewashed by dilution and pelleting with 2 mL of FACS buffer twice and were thenresuspended in 100 μL of FACS buffer and fixed in 1% paraformaldehyde. Datawere collected on ∼7,000 cells per sample on a Becton Dickinson FACScan flowcytometer (Becton Dickinson) with Cytek DxP multicolor upgrades. MFIs werecalculated on live gated cells with FlowJo (TreeStar). Variation in transfectionefficiency was not taken into account in calculations of MFI.

ACKNOWLEDGMENTS. We thank the staff at Advanced Light Source Beam-line 8.2.2 and Stanford Synchrotron Radiation Lightsource for their assis-tance in crystallographic data collection; Rebecca Dutch and members ofthe Dutch laboratory for providing the HeV F pCAGGS, HeV G pCAGGS,polyclonal anti-HeV F 527-540 Ab, and HeV F radioimmunoprecipitation as-say protocols; Gabriele Fuchs and the Peter Sarnow laboratory for adviceand facilities for performing radioimmunoprecipitation assays; ChristopherBroder for providing the mAb 5B3; and Luke Pennington and the KariNadeau laboratory for assistance and instrumentation for performing flowcytometry. This work was supported, in part, by NIH Research Grants AI-23173 (to R.A.L.) and GM-61050 (to T.S.J.). R.A.L. is an Investigator of theHoward Hughes Medical Institute.

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