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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:
[email protected] [email protected].
This article contains supporting information online at
www.pnas.org/lookup/suppl/doi:10.1073/pnas.1523303113/-/DCSupplemental.
1056–1061 | PNAS | January 26, 2016 | vol. 113 | no. 4
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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
K60C-T173C
P63C-V175C
A137C-V267C
Y97C-G131C
N100-A119CN100-A116C
A
B
CK60C-T173C
P63C-V175C
Y97C-G131C
N100C-A116C
N100C-A119C
A137C-V267C
Wt Vector
R.L
.U. (
norm
aliz
ed to
Wt)
VectorWtY97C-G131CN100C-A119C
0
0.2
0.6
0.4
0.8
1.0
1.2
1.4
102
142
97
109-141
Cou
nts
PE0 10^3 10^4 10^5
100
0
50
150
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
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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
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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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