Structure of the infectious salmon anemia virusreceptor complex illustrates a unique bindingstrategy for attachmentJonathan D. Cooka, Azmiri Sultanaa, and Jeffrey E. Leea,1

aDepartment of Laboratory Medicine and Pathobiology, Faculty of Medicine, University of Toronto, Toronto, ON M5S 1A8, Canada

Edited by Peter Palese, Icahn School of Medicine at Mount Sinai, New York, New York, and approved February 22, 2017 (received for review November1, 2016)

Orthomyxoviruses are an important family of RNA viruses, whichinclude the various influenza viruses. Despite global efforts toeradicate orthomyxoviral pathogens, these infections remainpervasive. One such orthomyxovirus, infectious salmon anemiavirus (ISAV), spreads easily throughout farmed and wild salmo-nids, constituting a significant economic burden. ISAV entry requiresthe interplay of the virion-attached hemagglutinin-esterase andfusion glycoproteins. Preventing infections will rely on improvedunderstanding of ISAV entry. Here, we present the crystal structuresof ISAV hemagglutinin-esterase unbound and complexed withreceptor. Several distinctive features observed in ISAV HE are notseen in any other viral glycoprotein. The structures reveal a uniquemode of receptor binding that is dependent on the oligomericassembly of hemagglutinin-esterase. Importantly, ISAV hemagglutinin-esterase receptor engagement does not initiate conformational re-arrangements, suggesting a distinct viral entry mechanism. This workimproves our understanding of ISAV pathogenesis and expands ourknowledge on the overall diversity of viral glycoprotein-mediatedentry mechanisms. Finally, it provides an atomic-resolution model ofthe primary neutralizing antigen critical for vaccine development.

orthomyxovirus | viral fusion | viral attachment | hemagglutinin |infectious salmon anemia virus

Infection caused by infectious salmon anemia virus (ISAV)remains a serious threat to the global aquaculture industry.

ISAV is the causative agent of one of the most economicallydestructive viral aquaculture pandemics in recent history, causingbillions of dollars in economic losses in an increasing number ofcountries in the last 30 y. The disease appears as a systemiccondition characterized by severe anemia, impaired circulation,viral-induced red blood cell destruction, and hemorrhaging inseveral organs, with mortality rates of 30–90% (1). No treatmentis currently available, and vaccination efforts have only resultedin partial protection.ISAV belongs to the Isavirus genus in the family Orthomyx-

oviridae and consists of eight single-stranded RNA segments(14.3 kb) that encode for at least 10 proteins. Although ISAV isclosely related to the various influenza viruses, its mechanism ofviral entry is unique. In influenza viruses, attachment to the hostis achieved through a sialic acid binding domain found directlyon the viral fusion protein. ISAV is the sole example of anorthomyxovirus that encodes a fusion (F) glycoprotein thatlacks either receptor binding or destroying activities. Theroles have been redistributed to a distinct hemagglutinin-esterase (HE) protein (2). Initial attachment of ISAV toterminal 4-O-acetylsialic acid (4-OAS) glycans on host cells ismediated through HE, whereas F mediates virus–host fusion.The ISAV HE and F glycoproteins colocalize on virions andform a heteromeric preentry complex of unknown stoichi-ometry (3). Binding of erythrocyte ghosts to cells expressingthe ISAV preentry complex induces dissociation of ISAV Ffrom HE, presumably acting as a trigger for membrane fusion.Interestingly, HE proteins from low-virulence strains of ISAV

show a higher affinity to the F protein on erythrocyte ghostbinding and correlate with a lower fusion activity and a longerhighly polymorphic region (HPR) (3).Little is known about ISAV entry and two-component viral

glycoprotein entry complexes. In the current study, we de-termined the high-resolution crystallographic structures of ISAVHE unbound and in complex with 4-OAS. Our structures provideinsight into the mechanism of ISAV viral entry and contribute toour understanding of the diversity of viral entry mechanisms.

Results and DiscussionExpression, Purification, and Biochemical Analysis.Recombinant ISAVHE17–353 was secreted from Drosophila melanogaster S2 cells andpurified from culture media in milligram quantities (SI Appendix,Fig. S1A). Size exclusion chromatography revealed that ISAVHE17–353 elutes at a volume consistent with an elongated trimer (SIAppendix, Fig. S1B). The resulting ISAV HE17–353 exhibits properesterase activity, as measured by a 4-nitrophenol acetate hydrolysisassay (SI Appendix, Fig. S1C and Table S1).

Overall Structure of ISAV HE Is Trimeric. Crystals of apo and holoISAV HE17–353 were obtained and diffracted at 1.8 and 2.0 Åresolution, respectively. The apo ISAV HE17–353 structure wasdetermined by a multicrystal native sulfur single-wavelength anom-alous dispersion (S-SAD) method and subsequently used to phasethe holo structure by molecular replacement. Three noncovalentlyattached monomers (A, B, and C) of ISAV HE17–353 adopt a

Significance

The infectious salmon anemia virus (ISAV), an aquatic patho-gen with lethal hemorrhagic potential, decimates farmed andfreshwater fish populations globally. Here, we determined thecrystallographic structures of the hemagglutinin-esterase (HE)viral glycoprotein responsible for the dynamic attachment ofthe virus to its receptor in Atlantic salmon. We identified sur-face features of ISAV HE that are conserved across isolatesknown to cause significant economic burden to fisheriesworldwide. This provides a molecular blueprint for the designof a broadly protective vaccine. Furthermore, we showed thatISAV HE has a distinct receptor recognition strategy from thoseof other influenza-like viruses and coronaviral HE proteins,contributing to our understanding of the diversity of viralentry mechanisms.

Author contributions: J.D.C. and J.E.L. designed research; J.D.C. performed research; J.D.C.and A.S. analyzed data; J.E.L. supervised research; and J.D.C. and J.E.L. wrote the paper.

The authors declare no conflict of interest.

This article is a PNAS Direct Submission.

Data deposition: The atomic coordinates have been deposited in the Protein Data Bank,www.pdb.org (PDB ID codes 5T9Y and 5T96).1To whom correspondence should be addressed. Email: jeff.lee@utoronto.ca.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1617993114/-/DCSupplemental.

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“broccoli floret” shape with overall dimensions of ∼130 × 70 × 70 Å(Fig. 1). The structure observed in the crystal is consistent withpreviously reported electron micrographs of intact ISAV particles,which revealed a viral particle with clear spike protrusions extending∼130 Å from the viral membrane (4). Each protomer is divided intothree functional domains: the receptor binding domain (RBD), theesterase domain, and the stalk domain. Small-angle X-ray scattering(SAXS) reconstructions of apo ISAV HE17–353 indicate that thecrystal structure is representative of the molecular conformationobserved in solution (Fig. 2).ISAV HE17–353 trimerization is stabilized by an extensive in-

terface involving 58 residues from all three domains with aburied surface area of ∼3,870 Å2 (SI Appendix, Fig. S2). Thisinterface is mainly hydrophobic in nature, with no salt bridgesand only 12 hydrogen bonds identified between adjacent proto-

mers. ISAV is a cold-adapted pathogen and a facultative halo-phile; thus, the fusion of host and cellular membranes occurs at12–15 °C. Maintenance of the trimeric state of ISAV HE17–353 ina highly saline ocean environment is likely a result of the hy-drophobic nature of the ISAV HE oligomerization interface. Incontrast, terrestrial orthomyxoviruses use extensive intermolecularsalt bridges to stabilize interactions between the RBD and stalkdomain before fusion and promote the rearrangement of thehemagglutinin (HA) and hemagglutinin-esterase-fusion (HEF)proteins into their irreversible postfusion conformation (5).

The ISAV HE Receptor Binding Domain Engages Its Sialic AcidReceptor at Protomer Interfaces. The ISAV HE RBD forms atwo-layer β-sandwich of eight antiparallel β-strands arranged intoan Ig-like domain. It is linked to the esterase domain by a smallantiparallel two-stranded β-sheet comprised of β5 and β13 of the

viral membrane

A

Esterase

RBD

Stalk

TM

cytoplasm

monomer A

monomer B

monomer C

C

N 130 Å

B C

90o

1 YEsterase RBD Stalk

354 378

391SP17

Esterase127 212 299 342

S S

19 279

S S

91 233118 221

S S

173 190

S S

S S

292 297

TM CT

C

C19-C279

C292-C297

C118-C221

C91-C233

C173-C190

extracellular

333 HPR

Fig. 1. Overall structure of ISAV HE. A schematic representation of the domain structure of ISAV HE. (A) The predicted N-linked glycosylation site at N333 isdepicted as a red Y. CT, cytoplasmic tail; SP, signal peptide; TM: transmembrane domain. (B) Ribbon diagram of ISAV HE with domains in one monomercolored according to A. Disulfide bonds are numbered and colored red. (C) Molecular surface representations of ISAV HE.

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esterase domain (Fig. 1 and SI Appendix, Fig. S3). Our cocrystalstructure of ISAV HE17–353 in complex with 4-OAS shows abinding mode different from prototypical orthomyxoviruses andother viruses that use a functionally equivalent protein for re-ceptor binding (6–11) (Fig. 3). 4-OAS is coordinated in a water-filled, positively charged pocket that is generated at the interfaceof two adjacent ISAV HE protomers. β6, β9, β12, and theβ7 strands on the adjacent protomer form extensive interactionswith 4-OAS. Binding is accomplished through a lock-and-keybinding mechanism, as the conformations of apo and holostructures are nearly identical (0.21 Å rmsd) (SI Appendix, Fig.S4). The positively charged character of the pocket is comple-mentary to the negatively charged 4-OAS. Receptor specificity isdictated by the presence of the 4-O-acetyl group, which forms ahydrogen bond with the backbone nitrogen of R139 on protomer1 and docks its methyl group into a shallow hydrophobic cleftformed by A132, I158, and V160 of protomer 2 and L136 ofprotomer 1 (Fig. 4). The carbonyl oxygen and guanidinyl groupof R139 also make hydrogen bonds with N5 and the carboxylateat carbon C1 of 4-OAS, respectively. Further coordination of thereceptor by protomer 1 occurs through hydrogen bonding of the4-OAS hydroxyl groups at C7 and C9 with the D141 backbonenitrogen and sidechain carboxylate, respectively. Finally, R198on the adjacent protomer partially spans the bound sugar andlocks the receptor into the pocket through a hydrogen bond tothe hydroxyl group at the anomeric carbon of 4-OAS.

The ISAV HE Esterase Domain. The esterase domain is composed oftwo discontinuous regions (E1: residues 17–126; E2: 213–299)separated by the RBD (Fig. 1 and SI Appendix, Fig. S3). Theesterase domain has an SGNH (Ser/Gly/Asn/His)–hydrolase fold(12) and contains a central four-stranded parallel β-sheet thatacts as a core around which three α-helices and several small 310helices pack to generate the esterase active site. There are five

intramolecular disulfide bonds per protomer, with four of thefive located in the esterase domain. Three of the esterasedisulfide bonds (C19–C279, C91–C233, and C118–C221) pin thedisparate halves of the domain together.The ISAV HE esterase active site is located ∼29 Å away from

the RBD sugar-binding site and contains two pockets (P1 andP2) for 4-OAS coordination. Analysis of the primary sequence ofISAV HE indicates that residues S32, D261, and H264 generatea canonical Ser-His-Asp catalytic triad with esterase activity (2).S32 is the nucleophilic residue in the ISAV HE triad, as muta-tion of this residue to an alanine ablates esterase activity (2). Aformate ion, observed in both apo and holo structures in theactive site, mimics the acetate product of the ISAV HE esterasereaction (Fig. 4D).Our structures of ISAV HE illustrate a formate ion bound in

an expanded P1 pocket at the esterase active site, whereas theP2 pocket is shallow and poorly defined. The distance betweenthe P1 and P2 pockets in the ISAV HE active site is ∼6 Å, withV39 generating a hydrophobic platform at the base of P2 for theterminal methyl of the 5-N-acetyl moiety and N58 generating acap for the coordination of the polar 5-N-acetyl moiety (Fig. 4E).This formate ion is coordinated through hydrogen bonding viathe side chain of S32, which recapitulates a bond to the labileoxygen in the enzymatic reaction. The ion is further coordinatedby hydrogen bond interactions with the sidechain of N89 and thebackbone nitrogen of S32, which, along with G59, generate anoxyanion hole that would stabilize the negative charge impartedto the transition state of the terminal oxygen of the 4-O-acetylgroup of the ISAV receptor during hydrolysis. Residues directlyinvolved in coordinating the pyranose ring or the other substit-uents of 4-OAS within the esterase domain cannot be deter-mined from our structures; however, comparisons to type 2coronaviral HE proteins that use 4-OAS as a substrate mayprovide some insight. For instance, accommodation of 4-OAS by

~165 Å

~98 Å

~61 Å

Rg = 37.8 ÅDmax= 135 Å

A

C D

B E

q2 X I(

q)

q (Å-1)

3.50-

3.00-

2.50-

2.00-

1.50-

1.00-

0.50-

0.05 0.10 0.15 0.20 0.25 0.30 0.35 0.40 0.45 0.50

log

[I(q)

]

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3.00-

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2.00-

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resi

dual

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[I(q

)]

q2 (Å-2)

7.95-

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r (Å)0 20 40 60 80 100 120 140

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6.2 mg/mL5.3 mg/mL4.4 mg/mL3.5 mg/mL2.6 mg/mL

90˚180˚

6.2 mg/mL5.3 mg/mL4.4 mg/mL3.5 mg/mL2.6 mg/mL

Fig. 2. Analysis of ISAV HE17–353 by small-angle X-ray scattering. SAXS scattering experiments performed on a concentration series of PNGase F-treated ISAVHE17–353. (A) SAXS intensity curves of ISAV HE17–353 at multiple concentrations. (B) Kratky plots of ISAV HE17–353 at multiple concentrations. (C) Refinedscattering intensity curve from ISAV HE17–353 at a concentration of 5.3 mg/mL (Inset, Upper) Guinier analysis for extrapolation of I(0) and determination of theradius of gyration (Rg). (Inset, Bottom) Residuals from the data used in the Guinier analysis. (D) Paired distribution function [P(r)] for ISAV HE17–353 at 5.3 mg/mLwith a D-max of 135 Å. (E) ISAV HE17–353 crystallographic structure superimposed on the ab initio SAXS envelope reconstruction.

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a type 2 murine coronaviral HE (8) illustrates that the P1 andP2 pockets in these enzymes are also ∼6 Å apart, housing the4-O-acetyl and the 5-N-acetyl moiety in P1 and P2, respectively(SI Appendix, Fig. S5). In comparison, 9-O-acetylsialic acid bind-ing type 1 HE esterases bind a 9-O-acetylsialic acid via P1 and P2pockets that allow the coordination of the 9-O-acetyl moiety inP1 and the 5-N-acetyl moiety in P2. This can be clearly observedin structures of porcine and bovine torovirus HE (13), bovinecoronavirus HE (14), and the structures of influenza C virus andinfluenza D virus HEF proteins (7, 9). In these structures, thedistance between the P1 and P2 pockets is ∼8 Å and explains thedifferences in sialic acid substrate observed between type 1 andtype 2 HE proteins.

The ISAV HE Stalk Distinctively Coordinates Divalent Cations, Ratherthan a Typical Cl− Ion, at Its Core. An extended α-helical stalk re-gion (residues 283–320), ∼68 Å in length, links the esterasedomain to the transmembrane anchor and viral membrane sur-face (Fig. 1 and SI Appendix, Fig. S3). The stalk domains fromthe monomers in the trimer complex form a parallel coiled-coil.It exhibits a modified heptad repeat motif, wherein two chargedresidues (D313 and H323) pack into the core. Accommodationof H323 in the hydrophobic center of the coiled-coil occurs viaan internal coordinated water molecule, whereas accommoda-tion of the negatively charged D313 occurs via coordination ofMg2+ along the central threefold axis (SI Appendix, Fig. S6). TheMg2+ ion forms octahedral geometry with three water moleculesand three Oδ atoms from D313. The average distance betweenthe ion and the interacting oxygen atoms is 2.2 Å, which ischaracteristic of reported values for Mg2+ coordination by pro-teins (15). The presence of the Mg2+ ion in the ISAV HE crystalstructure is likely a result of the presence of the magnesium for-

mate used as a precipitant in protein crystallization. In contrast,results from inductively coupled plasma atomic emission spectros-copy indicate that recombinant ISAV HE17–353 copurifies withnear-stoichiometric amounts of Ca2+ and detectable amounts ofZn2+ and Cu2+ (Table 1 and SI Appendix, Fig. S7A). Copurificationof Cu2+ is a possible artifact of the CuSO4 induction used to expressthe recombinant protein. Chelation of ISAV HE17–353 by incuba-tion with either 5 or 12.5 mM EDTA results in negligible differ-ences in thermal stability (Tm = 49 °C) (SI Appendix, Fig. S7B).ISAV is transmitted through ocean water, which on average con-tains 10 mM Ca2+ and 50 mM Mg2+ (16). We found that incuba-tion of ISAV HE17–353 with CaCl2 at concentrations equivalent tothose found within the ecological niche of ISAV does not alter thethermal stability of the recombinant protein (SI Appendix, Fig.S7C). However, incubation of ISAVHE17–353 with 50 mMMgCl2 ismildly destabilizing (ΔTm= −2 °C) and shows dose-dependency (SIAppendix, Fig. S7D). This suggests that divalent cation binding inthe stalk domain does not play a role in stabilizing the ISAV HEtrimer. Coordination of ions within a central coiled-coil is charac-teristic of numerous viral glycoproteins. At this time, most class1 viral glycoproteins (including ISAV F) coordinate a negativelycharged Cl− ion at the trimer interface of its fusion subunit, whereasISAV HE is an example of a viral hemagglutinin that binds divalentcations within the central coiled-coil.

ISAV HE Is Unique Compared with Other Hemagglutinins and ViralGlycoproteins. Pairwise sequence alignments between ISAV HEand various viral glycoproteins reveal limited sequence identity(<10%) to the orthomyxoviral HA or HEF proteins (SI Appen-dix, Fig. S8). Moreover, ISAV HE shares no sequence similarityto influenza A and B virus neuraminidases or the paramyxoviralhemagglutinin-neuraminidase, which, similar to ISAV, constrain

IAV HA Murine CoV HE

A B C D4-OAS

4-OAS9-OAS

Neu5Chain A

Chain B

ISAV HE

Chain A

Chain AChain A

IDV HEF

Receptor Binding SiteAxis of Symmetry

Fig. 3. ISAV receptor-binding is distinct from other viral hemagglutinin-esterase proteins. An illustration of the ISAV HE RBD and esterase active site withcomparisons to other sialic acid binding viral glycoproteins. (A) Backbone and ribbon diagrams of receptor-binding domains in ISAV HE, (B) influenza A virusHA, (C) influenza D virus HEF, and (D) murine coronavirus (CoV) HE.

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their receptor-binding and destroying properties to the sameprotein. Primary sequence alignments of ISAV HE indicatesome similarity to torovirus and coronavirus HE proteins (<25%)(SI Appendix, Fig. S9A). However, the primary sequence of theISAV HE protein appear to be most similar (∼32%) to ORF 95 ofthe anguillid herpesvirus 1 (AngHV-1), an aquaculture pathogenthat causes hemorrhagic lesions in farmed Japanese eel (17). Al-though the percentage identity of ISAV HE compared withAngHV-1 ORF 95 is less than 20%, conservation of the locationsof cysteine residues within the primary sequence and stretches ofamino acids within the esterase domain and the RBD are worthnoting (SI Appendix, Fig. S9B). It has been suggested previouslythat AngHV-1 ORF 95 is homologous to the ISAV HE protein,originating from a possible gene capture event in the evolutionary

history of AngHV-1 (18). However, it is clear from the primarysequence alignment that AngHV-1 ORF-95 lacks most of theC-terminal coiled-coil stalk domain observed in ISAV HE and haslost the catalytic triad conserved in other viral hydrolases.Structural conservation of the ISAV HE protomer was assessed

using the DALI pairwise search (19). These results paint a verydifferent picture of ISAV HE than what was indicated by pri-mary sequence alignment. The global organization of the RBDand esterase domain of ISAV HE protomer is comparable tothat of coronaviral HE proteins, toroviral HE proteins, and in-fluenza C and D virus HEF1 protomers. The results from theDALI search also identified several putative acylhydrolases, li-pases, and esterases from the bacteria Aspergillus and Bacteroidesthat contain the SGNH–hydrolase fold.

4-OAS

A131

A132

I158

V160R198

G138

R139D141

Q205

T140

C

-5.0 kT e-1 +5.0 kT e-1

A

B

Val160

Arg198

NHN

H3

HN

+

Ile158

Ala131

Ala132

Arg139

Asp141

OO

_

OH

Thr140

NH

2

OGln205

Gly138

H2O

H2O

4-OAS

N 2.5 Å

2.9 Å3.3 Å

3.0 Å

NH

NH

3

HN

2.8 Å

2.8 Å

2.8 ÅO

OH

O

HN

OH

O

O

CH3O

HO

OH

O

CH3

Arg139N

O 2.7 Å

3.1 Å

3.0 Å

_

2.8 Å

H2O 3.4 Å

+

D261

N89

G59 S32

H264

FMT

D E

G59

S32

H264FMT

P1

P2

C1

C2

C3C5 C4

C6

C7

C8

C9

-5.0 kT e-1 +5.0 kT e-1

V39N58

Fig. 4. ISAV receptor-binding and esterase active site. Coordination of 4-O-acetylsialic acid (4-OAS) by ISAV HE is mediated by extensive intermolecularinteractions within both the RBD and the esterase domain. (A) Ribbon diagram of the 4-OAS receptor (yellow) bound to the interface of two ISAV HEmonomers (lilac and green). (B) Schematic representation of the van der Waals interactions and hydrogen bonding network between ISAV HE and 4-OAS.(C) Coulombic surface of ISAV HE RBD bound to 4-OAS. (D) Ribbon diagram of the ISAV HE esterase active site illustrating the Ser-His-Asp catalytic triad. Aformate ion (FMT) bound into the oxyanion hole is visualized as a stick model. (E) Coulombic surface of the ISAV HE esterase active site. The P1 and P2 esterasepockets are outlined by a dashed line.

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Despite the similarities mentioned here, ISAV HE has uniqueadaptations to allow for engagement of the viral receptor.Corona- and toroviral HE proteins are dimeric proteins thatlack the extended stalk domain found in ISAV HE, and unlikeISAV HE, each protomer of the toroviral and coronaviral HEproteins bind to a single viral receptor independently. Indeed,orthomyxoviral HA and HEF protomers also bind to a singleviral receptor independently. Orthomyxoviral HEF proteins andthe toro- and coronaviral HE proteins classically recognize 9-O-acetylsialic acid as their viral receptor. Coronaviruses that rec-ognize 9-O-acetylsialic acid carry the designation of type 1 HEproteins, whereas type 2 HE proteins evolved from their type 1counterparts to recognize 4-OAS (20). This shift in recognitionwas achieved through a modest but coordinated evolutionaryswitch in RBD and esterase architecture that lead to a rotationof the 4-OAS binding mode by 90° to that of the 9-O-acetylsialicacid while keeping the global fold of the RBD intact (21). Ac-commodation of sialic acids in the RBD of influenza A, in-fluenza B, influenza C, and influenza D virus followed a similarevolutionary path wherein amino acid substitutions within aconserved RBD-fold lead to the recognition of several differentsialic acids (7, 10, 22, 23). Conversely, recognition of 4-OAS byISAV HE occurs through the generation of a distinct bindingsite found at the trimerization interface between ISAV HEprotomers, as described earlier (Fig. 3).

Insights into the Entry Mechanism of ISAV. ISAV viral entry isthought to occur via a multistep mechanism unique from those ofinfluenza A virus and other viruses with class I viral glycopro-teins. ISAV HE forms a complex with the F fusion protein on thesurface of the virus before the initiation of viral attachment andentry (3, 24). On receptor binding, ISAV HE and F dissociate,and the virus is subsequently endocytosed by the host cell. In thewild, freshwater lakes can experience diurnal oscillations in pH(0.2–0.4 pH units) as a result of photosynthetic respiration byaquatic organisms (25–27), and acidic lakes (pH < 6.5) can reachthe optimal pH for ISAV fusion when this cycle reaches its nadir.Acidification of Salmo salar ecosystems via acid deposition fromanthropogenic sources (28) could also generate a fusogenic mi-lieu, which in the absence of the ISAV HE protein could resultin premature ISAV F protein rearrangement. Studies of avianinfluenza, another aquatic orthomyxovirus, show that pH < 6.6in freshwater habitats can negatively affect viral infectivity (29).Formation of the ISAV HE:F complex may act to protect andsequester the ISAV F protein from environmental pH fluctuations.

Traversing the endosomal pathway introduces the dissociatedISAV F protein to a steady decrease in pH, which is the final,irreversible signal for ISAV F protein rearrangement, consistentwith previous studies that ISAV F is stabilized in its postfusionconformation at low pH (30). Interestingly, the expression ofISAV F on the surface of cultured cells is sufficient for syncytiaformation when these cells are exposed to low-pH media (31).Our structures clearly show that ISAV HE is not analogous tothe paramyxoviral HN protein, which like ISAV HE has receptor-binding and receptor-destroying activities. Receptor binding ofparamyxoviral HN causes a conformational rearrangement of HNthat transmits the bound-state signal to the paramyxoviral F pro-tein, triggering a rearrangement that culminates in the mergerof host and viral membranes (32). In contrast, 4-OAS receptorbinding does not induce a conformational change in ISAV HE,and thus is not likely the trigger to activate ISAV F for host–virusmembrane fusion.Fusion of the viral and endosomal membranes allows the re-

lease of the segmented viral genome into the cytoplasm. Theviral genomic RNA is subsequently translocated into the nucleus.ISAV proteins are synthesized in the cytoplasm and ER, andthen subsequently directed to the plasma membrane for assem-bly. In the final step of the cycle, the esterase activity of ISAVHE then destroys the 4-OAS-containing receptors on themembrane to release the virus from the host cell (33).

Insights into ISAV Vaccine Design. ISAV remains a worldwidethreat to wild and farmed salmon because of orthomyxovirusesexhibiting high mutation rates leading to new pathogenicstrains. Vaccination of salmon against ISAV is an importantstrategy for controlling outbreaks by directly protecting stocksor preventing spread. ISAV HE is the main target for thedesign of vaccines (34). Immunization of mice with infectedISAV culture supernatant has allowed the development of ananti-HE monoclonal antibody that is neutralizing and can interferewith the hemagglutination activity of HE, demonstrating the po-tential of anti-HE vaccination strategies (35). Although no specificneutralizing epitopes have yet been identified within the ISAV HEglycoprotein, a vaccine based on a salmonid alphavirus (SAV)replicon designed to induce the expression of HE provides pro-tective immunity (36). Furthermore, the inclusion of SAV repli-cons that express ISAV F protein or the ISAV matrix protein didnot improve the efficacy of the vaccine, indicating that presenceof the HE protein is sufficient to generate a protective immuneresponse. More recently, an oral subunit vaccine that contains anequimolar mixture of recombinant ISAV HE and ISAV F proteinsexpressed in yeast has also been shown to generate sustained andprotective humoral immunity (37), but the exact contribution ofISAV HE vs. the ISAV F protein was not addressed in this study.The innate immune response to ISAV infection includes increasedexpression of Mx and interferon-stimulated gene 15 (ISG15) viaan IFN-independent mechanism (38), as well as retinoic acid-inducible gene I (RIG-I) and major histocompatibility complex classI (MHC-I) expression, which correlate with viral load. Interestingly,this characteristic innate immune response is not observed in salmonwith protective immunity to ISAV induced by vaccination resultingin humoral immunity (39). Inducing humoral immunity is veryimportant in generating a protective immune response againstISAV, as innate responses to ISAV infection are vigorous, butunable to control the virus.Comparative sequence and structural analysis reveals a high

degree of overall surface conservation between ISAV HE iso-lates (sequence identity >95%), especially in the esterase do-main (Fig. 5). In the head of the RBD, only three residues (P156,K185, and I187) are poorly conserved. The greatest variationexists in the HPR of the stalk domain. Moreover, ISAV HEcontains only a single N-linked glycan at the base of the stalkregion; thus, ISAV HE does not have the glycan shield, as observed

Table 1. Copurification of divalent cations with ISAV HE17–353

Cation (spectral line, nm) ΔConcentration (ppb)Molar ratio (TrimericISAV HE17–353:ion)

Mg2+ (279.553) −0.40 ± 0.70 —

Mg2+ (280.271) −0.76 ± 0.84 —

Mg2+ (285.213) −0.90 ± 1.1 —

Ca2+ (317.933) 46.9 ± 7.2 1.7: 1.2Ca2+ (393.366) 45.1 ± 1.1 1.7: 1.1Mn2+ (257.610) DL* —

Mn2+ (259.372) DL* —

Fe2+ (238.204) DL* —

Co2+ (228.616) DL* —

Ni2+ (221.648) DL* —

Cu2+ (324.752) 15.1 ± 3.4 1.7: 0.2Zn2+ (206.200) 19.4 ± 3.6 1.7: 0.3Cd2+ (228.802) DL* —

ppb, parts per billion.*DL, Results were below the detectable limits.

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Fig. 5. ISAV HE surface conservation across ISAV HE isolates. A comparison of ISAV surface residues indicates that there is extensive conservation throughoutthe global reservoir of ISAV isolates. (A) Representation of ISAV HE surface residue conservation calculated using the alignment in B. Conserved residues aredepicted in purple, whereas residues that vary across isolates are colored from pink through green. (B) Alignments of ISAV HE isolates from Norway, Chile,and Canada illustrate several polymorphic sites in the ISAV HE protein. The HPR begins at residue 338 and continues to the transmembrane domain. Theesterase catalytic triad is marked with an asterisk and is highlighted in purple. Alignments were generated with ClustalΩ and illustrated with ESPript 3.0.

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in other viral glycoproteins. Our results suggest that ISAV HEcould be exploited for the development of an ISAV subunit vaccinesuch that immune responses are exclusively directed againstbiologically important and conserved epitopes.

Material and MethodsISAV HE17–353 was stably expressed in Drosophila S2 cells at 20 °C with aC-terminal deca-histidine tag for purification by Ni-affinity chromatography.ISAV HE17–353 was deglycosylated using PNGaseF, thrombin digested, andfurther purified by anion exchange and size exclusion chromatography.Purified ISAV HE17–353 was characterized for stability, using a fluorescence-based thermal shift assay and circular dichroism (CD) spectroscopy. A4-nitrophenol acetate hydrolysis esterase assay was used to ensure therecombinant protein was functional. Small-angle X-ray scattering experimentswere performed at the Advanced Light Source SIBYLS beamline 12.3.1, viatheir mail-in SAXS data collection service (40–42). Crystals were grown by sit-ting drop vapor diffusion in 0.2 M magnesium formate and 20% (wt/vol) PEG3350. Phases were obtained by multicrystal native S-SAD phasing, with the finalmodel refined to 1.8 Å resolution and final Rwork/Rfree values of 13.4%/17.1%.Receptor-bound ISAV HE17–353 was obtained by soaking 4-OAS into the ISAVHE17–353 apo crystal, and its structure was determined by molecular replace-ment to 2.0 Å resolution, using the refined apo ISAV HE17–353 model. The holoISAV HE structure was refined to final Rwork/Rfree values of 13.9%/18.3%.Crystals were diffracted at the Canadian Light Source and Advanced PhotonSource. Small-angle X-ray scattering was performed at the SIBYLS beamline,Advanced Light Source to determine the conformation of ISAV HE17–353 in

solution. Details of all experimental procedures can be found in the SI Ap-pendix, SI Methods and Materials.

ACKNOWLEDGMENTS. We thank Kathryn Burnett and Greg Hura at SIBYLSBeamline 12.3.1 (Advanced Light Source); Shaun Labiuk, Michel Fodje,and Pawel Grochulski on Beamline 08ID-1 (Canadian Light Source); andK. Rajashankar on Beamline 24-ID-E (Advanced Photon Source) for synchro-tron support. Funding from a Natural Sciences and Engineering ResearchCouncil of Canada (NSERC) Discovery Grant (RGPIN 435607-13), an OntarioEarly Researcher Award (ER-13-09-116), Canada Foundation for Innovation,and a Canada Research Chair (to J.E.L.) is gratefully acknowledged. J.D.C.received stipend support from a Vanier-Canada Graduate Scholarship andOntario Graduate Scholarships. The Canadian Light Source is supportedby Canada Foundation for Innovation, NSERC, National Research Council(NRC) Canada, Canadian Institutes of Health Research (CIHR), Province ofSaskatchewan, Western Economic Diversification Canada, and University ofSaskatchewan. Advanced Light Source is a national user facility operated byLawrence Berkeley National Laboratory on behalf of the US Department ofEnergy, Office of Basic Energy Sciences, through the Integrated DiffractionAnalysis Technologies (IDAT) program, supported by Department of EnergyOffice of Biological and Environmental Research. Additional support onSIBYLS comes from the NIH project MINOS (R01GM105404). Beamline24-ID-E at Advanced Photon Source is a part of the Northeastern Collabo-rative Access Team beamlines, which are funded by the National Instituteof General Medical Sciences from the NIH (P41 GM103403). AdvancedPhoton Source is a US Department of Energy Office of Science User Facilityoperated for the Department of Energy Office of Science by Argonne NationalLaboratory under Contract DE-AC02-06CH11357.

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