Recent evolution of equine and the origin of

Patrick J. Collinsa,b,1, Sebastien G. Vachieria,b,1, Lesley F. Haireb, Roksana W. Ogrodowiczb, Stephen R. Martinc, Philip A. Walkerb, Xiaoli Xionga,b, Steven J. Gamblinb, and John J. Skehela,2

Divisions of aVirology, bMolecular Structure, and cPhysical Biochemistry, Medical Research Council, National Institute for Medical Research, London NW7 1AA, United Kingdom

Edited by Robert A. Lamb, Northwestern University, Evanston, IL, and approved June 24, 2014 (received for review April 10, 2014) In 2004 an hemagglutinin 3 neuraminidase 8 (H3N8) equine human and avian viruses indicates that in general they are closely influenza virus was transmitted from horses to dogs in Florida similar. However, the structures of α-helix A, in the fusion sub- and subsequently spread throughout the United States and to domain (13), of the HAs of Ef and C are distinctly different from Europe. To understand the molecular basis of changes in the those of the HAs of all other known equine, avian, or human antigenicity of H3 hemagglutinins (HAs) that have occurred during influenza viruses. We have defined the genetic and structural virus evolution in horses, and to investigate the role of HA in the basis of the novel fusion subdomain structure by X-ray crystal- equine to canine cross-species transfer, we used X-ray crystallog- lography of site-specific mutant HAs and consider its possible raphy to determine the structures of the HAs from two antigen- consequences for HA stability and function in membrane fusion. ically distinct equine viruses and from a canine virus. Structurally Because of the importance of receptor binding by HA in virus all three are very similar with the majority of amino acid sequence transmission and cross-species transfer, we have used biolayer differences between the two equine HAs located on the virus interferometry to compare the avidity and specificity of equine membrane-distal molecular surface. HAs of canine viruses are and canine virus binding to a range of sialoside receptor analogs. distinct in containing a Trp-222→Leu substitution in the receptor binding site that influences specificity for receptor analogs. In the We have also used X-ray crystallography to determine the struc- fusion subdomain of canine and recent equine virus HAs a unique tures of equine and canine virus HAs in complex with some of difference is observed by comparison with all other HAs exam- these receptor analogs. From these studies we deduce the mo- MICROBIOLOGY ined to date. Analyses of site-specific mutant HAs indicate that a sin- lecular basis of the observed differences in specificity and avidity gle amino acid substitution, Thr-30→Ser, influences interactions and we consider their possible role in virus transmission. between N-terminal and C-terminal regions of the subdomain that are important in the structural changes required for membrane fu- Results and Discussion sion activity. Both structural modifications may have facilitated the Equine and Canine HA Structures. All three structures can be seen transmission of H3N8 influenza from horses to dogs. in Fig. 1 to be very similar to each other and to other HAs of the H3 subtype described before (14). This similarity was expected quine influenza viruses of the hemagglutinin 3 neuraminidase from their sequence identities: Ee vs. Ef, 95%; Ef vs. C, 96%; E8 (H3N8) subtype were first isolated in 1963 from race horses in Miami (1). Since then they have caused numerous outbreaks Significance of infection in horses around the world with serious disease and economic consequences (2). In 2004, again in Florida, an H3N8 Equine influenza viruses of the H3N8 subtype have caused virus was isolated from an outbreak of canine influenza (3) and outbreaks of respiratory disease in horses throughout the similar viruses have since been isolated from dogs in the United world since their discovery in 1963 in Florida. In 2004 an equine States and in Europe (4, 5). Genetic comparisons indicate that virus in circulation was transmitted to dogs and subsequently the canine viruses are closely related to equine viruses that were spread throughout the United States and to Europe. Compar- in circulation in horses around 2000 (3, 5). In studies of differ- ative analyses of the structures of hemagglutinin glycoproteins ences in equine viruses isolated since 1963 (6–8) and between of equine and canine viruses by X-ray crystallography locate equine and canine viruses (3, 5), the sequences of genes for the the sites of variation on the molecules, indicate a role in de- hemagglutinin membrane glycoprotein (HA) have been com- termining binding specificity for an amino acid sequence dif- pared. Sequence data for equine virus HAs indicate the evolu- ference in the receptor binding site, and describe a unique tion of four distinct lineages. The first was associated with structural difference in the membrane fusion region in recent antigenic drift, between 1963 and 1980 (6, 7, 9), and following equine and canine virus HAs by comparison with all other this three separate branches formed a “Eurasian” lineage, an known HAs. These differences are proposed to have facilitated “American” lineage, and a divided lineage containing two clades, cross-species transfer. “Florida” clade 1 and Florida clade 2 (10, 11). The HAs of the canine viruses are most similar to those of Florida clade 1 Author contributions: P.J.C., S.G.V., L.F.H., R.W.O., S.R.M., S.J.G., and J.J.S. designed research; P.J.C., S.G.V., L.F.H., R.W.O., S.R.M., P.A.W., X.X., S.J.G., and J.J.S. performed equines. The majority of amino acid sequence changes revealed research; P.J.C., S.G.V., L.F.H., R.W.O., S.R.M., P.A.W., X.X., S.J.G., and J.J.S. contributed from the analyses are in the HA1 component of HA, some in new reagents/analytic tools; P.J.C., S.G.V., S.R.M., S.J.G., and J.J.S. analyzed data; and regions known to be antigenically important in H3 HAs, and P.J.C., S.G.V., S.J.G., and J.J.S. wrote the paper. several near the receptor binding site (12) (Fig. 1). The authors declare no conflict of interest. To understand the structural consequences of these changes, This article is a PNAS Direct Submission. in particular those that distinguish equine from canine virus Freely available online through the PNAS open access option. HAs, we have used X-ray crystallography to determine their Data deposition: The atomic coordinates and structure factors have been deposited in the structures. We have examined the HAs from two equine viruses Protein Data Bank, www.pdb.org (PDB ID codes 4UNW–4UNZ, 4UO0–4UO9, and 4UOA). and one canine virus: A/Equine/Newmarket/2/93, from the Eurasian 1P.J.C. and S.G.V. contributed equally to this work. lineage, “Ee”; A/Equine/Richmond/07, from Florida clade 2, 2To whom correspondence should be addressed. Email: [email protected]. “ ” “ ” Ef ; and A/Canine/Colorado/06, C . Comparison of the overall This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. structures of the three HAs with those of other H3 HAs from 1073/pnas.1406606111/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1406606111 PNAS | July 29, 2014 | vol. 111 | no. 30 | 11175–11180 Downloaded by guest on October 6, 2021 Fig. 1. The structure of monomers of Ee, Ef, and C HAs compared with A/Duck/Ukraine/63 avian H3 HA. (A) Superposition of the A/Duck/Ukraine/63 avian H3 HA (14) (colored in green) with the Ee structure (colored in blue for the HA1 chain and in red for the HA2 chain). The position of Thr-30 of chain HA1 is also indicated. (B) Structure of the Ef HA (light shades of blue and red for the HA1 and HA2 chains, respectively). The side-chain atoms of amino acids differing from those of the Ee HA are shown as spheres. The position of Ser-30 of chain HA1 and the location of the modified HA2 α-helix are indicated. (C) Structure of the C HA (darker shades of blue and red for the HA1 and HA2 chains, respectively). The positions of the five amino acids specific to canine HAs are indicated and colored as corresponding to the chain they belong to. Also indicated is the position of HA1 Ser-30 and the location of the modified HA2 α-helix.

and Ef vs. the HAs of the H3 avian and H3 human viruses, Ef and C HAs, Asn-54→Lys, Asn-83→Ser, and Ile-328→Thr (3), A/duck/Ukraine/63, 86%, and A/Aichi/2/68, 85%. It is also is not clarified by comparison of the HA structures. Residues 83 reflected in the rmsd of the α-carbon atoms shown in Table S1. and 54 are on the surface of HA, about 20 Å and 35 Å from the Of the 20-aa sequence differences noted between Ee and Ef receptor binding site, respectively, toward the virus membrane. (Fig. 1B and Fig. S1) 19 are accessible on the surface of HA. Of Amino acid substitutions at either position might influence HA these, 15 by comparison with the locations of amino acid changes antigenicity (Fig. 1). Residue 328 is the C terminus of HA1 (Fig. 1). in antigenic variants of human H3 HAs might result in antigenic The substitution Ile-328→Thr, which is conserved in canine differences (12). viruses, could have been selected to ensure the required cleavage In HA1, in the receptor binding site just one change, the of precursor HA0 into HA1 and HA2. This could be achieved by amino acid substitution HA1 Trp-222→Leu distinguishes canine optimizing the Gln/Glu–X–Arg− recognition site for a canine HAs from both Ee and Ef HAs. The clearest structural differ- protease like tryptase Clara (15) or by increasing the ability of ence between Ee and Ef is in the fusion subdomain between HA0 to compete with mucus protease inhibitors known to be HA2 residues 48 and 57. This difference is shared by Ef and C. present in the respiratory tract (16). In Ee, as in all other known HA structures, residues 48–57 are part of the five-turn α-helix A (residues 38–58), which, with Receptor Binding Avidity and Specificity. To probe the impact on the longer antiparallel α-helix B, forms a prominent α-helical receptor binding of the sequence differences between the three hairpin in each HA subunit. HAs, we determined, by biolayer interferometry, the avidity and In contrast, in Ef and C HAs, α-helix A is unfolded at residues specificity of the viruses for five different receptor analogs (Fig. 49 and 50 and a salt bridge between group-conserved HA2 Arg-54 2). The strong preference observed for α2,3-linked over α2,6- and conserved HA2 Glu-97 of the neighboring subunit is lost. linked sialosides is in agreement with previously reported results The significance of other sequence differences in HA1 between for equine viruses (17). Similarly, the greater avidities of equine

Fig. 2. Biolayer interferometry (BLI) data for the binding of Ee, Ef, and C viruses to different receptor analogs. (A–C)Ee(A), Ef (B), and C (C) binding curves where 30-kDa polymers containing 20% mol sugar and 5% mol biotin linked to a polyacrylamide backbone were immobilized to different extents on streptavidin-coated biosensors. Data are plotted as fractional saturation of the sensor surface as a function of relative sugar loading for a fixed virus con- centration of 100 pM.

11176 | www.pnas.org/cgi/doi/10.1073/pnas.1406606111 Collins et al. Downloaded by guest on October 6, 2021 viruses for analogs containing sulfated GlcNAc3 residues have The Different Structures of α-Helix A. As mentioned before, the been reported before (18–20). Ef virus binds 3′ sialylactosamine clearest structural difference between the HAs of the Ee and Ef (3′SLN) with about 1/3rd the avidity of Ee and the other three equine viruses is in α-helix A of HA2 (Figs. 1, 4, and 5). In Ee analogs, Sialyl Lewis X (SLex), sulfated Sialyl Lewis X (Su-SLex), HA, as in all human and avian H3 HAs, and probably in all and Su-3′SLN, with about 1/10th the avidity of Ee (Fig. 2). equine H3 HAs of viruses isolated before 2000 (3, 5), α-helix A is In contrast, the properties of the canine virus are very similar five turns long (Fig. 4A). In Ef HA, in contrast, the helix is to those of Ee, binding 3′SLN and SLex with closely similar interrupted at HA2 residues 49 and 50. It is continued for one avidity and Su-SLex and Su-3′SLN with about one-third to one- turn between residues 51 and 54 and then it terminates (Fig. 4B). half the avidity of Ee. The difference in sequence between Ef HA2 residue 55 becomes the N terminus of the loop that links and C HA in the receptor binding site, HA1 Trp-222→Leu, is the C terminus of α-helix A to the N terminus of α-helix B, likely to be involved in these receptor binding differences. In (residues 55–75). This structural feature of Ef HA is shared by C other respects the receptor binding sites are identical. The HAs HA (Fig. 4C). In both cases, HA2 residues 49 and 50 are adja- of canine H3N2 viruses isolated in South Korea and southern cent to a prominent loop of the neighboring subunit of the tri- China in 2005 also differed from their proposed avian HA pre- mer, the 30 loop formed by HA1 residues 25–35 (Fig. 4). In this cursor in containing the Trp-222→Leu substitution (21, 22). region the sequence of Ee HA differs from that of Ef and C at H3N2 viruses generated by reverse genetics that contained HA1 residue 30: threonine in Ee rather than serine in Ef and C. either Trp-222 or Leu-222 were observed to have similarly small The HAs of all equine H3N8 viruses isolated since 2000 con- differences in receptor binding properties to those reported here, tained the HA1 Thr-30→Ser substitution and the HAs of all which led to the suggestion that this amino acid substitution canine H3N8 viruses contained HA1 Ser-30. could facilitate infection of dogs (23). To determine whether this sequence change is responsible for the structural difference between Ee vs. Ef and C, we analyzed Structures of Equine and Canine HAs in Complex with Receptor the structure of a site-specific mutant C HA containing the Analogs. The differences detected in the structures of the com- substitution Ser-30→Thr. The results show (Fig. 5B) that in the plexes formed between the HAs and the receptor analogs are CSer-30→Thr mutant the α-helical structure between HA2 consistent with the results of the binding assays (Fig. 3 and Figs. residues 47 and 57 is restored. In addition, HA2 Arg-54, which S2–S4). All three HAs bind the sialosides with the Sia1–Gal2 is conserved in all H3 HAs, is repositioned to form a salt bridge α2,3-glycosidic linkage in trans conformation (24). Characteris- with conserved HA2 Glu-103 of the same subunit. The structural

tically, HA1 Gln-226 interacts with α2,3-linked receptors by effect of the Thr-30→Ser substitution appears to be a conse- MICROBIOLOGY forming hydrogen bonds with the 4-OH group of Gal2 and the quence of the ability of serine to occupy the polar region formed glycosidic oxygen between Sia1 and Gal2 (25). Gln-226 also by HA2 His-106 of α-helix B and HA2 Gln-105 of the neigh- assumes a slightly higher position in the complexes [up to 1.5 Å boring α-helix B (Fig. 5A). Ser-30 forms hydrogen bonds with toward the 190-helix (Fig. S2)] than in unliganded HA. In the both residues. As a result, the 30 loop is lower by about 2 Å and structures of C HA complexed with Su-3′SLN and Su-SLeX, a cavity is created that is lined by residues of α-helix A and of ionic bonds are formed between the SO4 and the terminal amino the 30 loop (Fig. 5A). HA2 Arg-54 occupies this cavity and in- group of Lys-193. These interactions appear to be the dominant teracts, through hydrogen bonds, with the main chain carbonyls determinants of the avidity increases observed for these analogs, of HA1 residues 29 and 30 of the neighboring 30 loop and the as noted before for H5 HAs (26). In the complex structure carbonyls of HA2 residues 46, 47, 49, and 50 of α-helix A. In formed by C HA with SLeX, the carbonyl oxygen of residue 225 contrast, in the vast majority of HAs, like Ee, that have threonine forms an additional hydrogen bond with the 4-OH of GlcNAc3- rather than serine at HA1 residue 30, the 30 loop adopts a po- linked fucose (Fig. 3). This interaction is also observed in the sition about 2 Å higher and the size of the cavity is reduced. In complex formed by Ef HA but the hydrogen bond appears this conformation Arg-54 adopts a different rotamer that enables longer in this case (3.1 Å instead of 2.7 Å in C HA; Fig. S4). This it to interact with Glu-97 of the neighboring subunit (Fig. 4A). is probably a result of the Trp-222→LeusubstitutioninCvs. We introduced the Ser-30→Thr mutation into Ef HA and ob- Ef/Ee, a probability that would be consistent with the observed served the same recovery of α-helix A structure (Fig. S5). How- importance of the size of the 222 side chain in influencing re- ever, because Ef HA, like the HAs of about 10% of equine viruses ceptor binding affinity of avian HAs (19, 20). Conversely, in isolated since 2000, contains the substitution HA2 Gly-50→Glu, complexes formed by C and Ee HAs with Su-SLeX,thelength the 30 loop in the Ef Ser-30→Thr mutant is restricted to a slightly of this hydrogen bond appeared very similar (2.7 Å and 2.6 Å, lower position and HA2 Arg-54 forms a hydrogen bond with the respectively), possibly because of the interaction of Lys-193 with carbonyl of HA1 29 and the side chain of Glu-50, rather than a the SO4 group (Fig. 3 and Fig. S4). salt bridge with HA2 Glu-97 (Fig. S5). In other respects the effects

Fig. 3. Receptor binding of Sialyl Lewis X (SLeX) and sulfated Sialyl Lewis X (Su-SLeX) by C HA. Su-SLeX bound to C HA (A) and SLeX bound to C HA (B). C HA HA1 main chain carbon residues are colored pink, and SLeX and Su-SLeX carbon atoms are colored yellow. Nitrogen and oxygen atoms are colored blue and red, respectively, in both balls and sticks and sphere representations. Hydrogen bonds are represented by black dashes; spheres are main chain atoms, with the exception of main chain 225 carbonyl.

Collins et al. PNAS | July 29, 2014 | vol. 111 | no. 30 | 11177 Downloaded by guest on October 6, 2021 Fig. 4. The local structure formed by HA2 residues 38–55 and HA1 residues 25–35 in the Ee, Ef, and C structures. (A)EeHAisshownwithoneHA2chain colored blue and the HA1 and HA2 chains of the closest monomer colored teal and white, respectively. (B) Ef HA is shown with one HA2 chain colored green and the HA1 and HA2 chains of the closest monomer colored blue and white, respectively. (C) C HA is shown with one HA2 chain colored pink and the HA1 and HA2 chains of the closest monomer colored dark blue and white, respectively. Hydrogen bonds are represented by black dashes, and spheres represent main chain atoms. The turns of HA2 helix A are numbered and referenced.

of the Ser-30→Thr substitution were similar in Ef and C site- of the 30 loop (29). Group 2-conserved HA2 His-106 becomes specific mutant HAs. To complete this comparison, C HA also the N terminus of a 180° turn in α-helix B, formed at fusion pH differs from Ef HA at HA1 residue 29, containing methionine by the refolding of HA2 residues 106–111 (30). HA2 His-106 rather than isoleucine. Our results with a Met-29→Ile site-specific makes a hydrogen bond with conserved HA2 Lys-51 at neutral mutant HA indicate that the substitution has no effect on the pH (31), and HA1 residues 29 and 30 around which the 30 loop structure of C HA (Fig. 5C). This result was expected because Ef turns (Fig. 4) are adjacent to HA2 residues 50 and 51 of the contains HA1 Ile-29 together with the modified α-helix A. neighboring subunit. In addition, group 2-conserved HA2 Gln- 105 makes a hydrogen bond with Ser-30 and also, via water, with Potential Consequences of the Ef/C-Specific α-Helix A Structure. conserved HA2 Asp-109, which also makes a hydrogen bond via Sequences of HAs from equine H3N8 viruses isolated since 1963 a water molecule with the main chain amide of HA2 Leu-2, the indicate that the Thr-30→Ser mutation was first detected in 1999 second residue of the “fusion peptide.” Because the Thr-30→Ser (3, 5). The change in structure of α-helix A presumably occurs substitution appears to enable these interactions, it is possible coincidentally. A biological consequence of this change may be that some feature of HAs containing Ser-30 related to HA- to enable transfer of the novel equine virus to dogs and between mediated membrane fusion or its activation may be favorable for dogs. The location of HA2 residues 47–54 in the Ef and C HA infection and transmission in dogs as well as horses. structures is in a region of HA that is prominent in the structural Our analysis of the pH at which the conformational change in changes in HA required for HA-mediated membrane fusion and C and other HAs containing Ser-30 occurs (Table S2)indicates has been highlighted in studies of mutant HAs that change that the characteristic change in protease susceptibility of HA structure at higher pH than wild-type HA (27, 28). Conserved at fusion pH occurs 0.2 pH higher for these HAs than for Ee HA. HA1 Lys-27 in the 30 loop becomes susceptible to trypsin after Because fusion by Ee HA occurs at pH 5.0, which is lower than incubation of HA at fusion pH, indicating structural rearrangement the pH at which other H3 HAs have generally been observed to

Fig. 5. The local structure formed by HA2 residues 38–55 and HA1 residues 25–35 in the C wt, S30T, and M29I structures. (A–C)C(A), S30T mutant (B), and M29I mutant (C) HA structures for HA2 residues 38–55 and HA1 residues 25–35. C wt, M29I, and S30T mutants are shown with one HA2 chain colored pink and the HA1 and HA2 chains of the closest monomer colored dark blue and white, respectively. Hydrogen bonds are represented by black dashes, spheres represent main chain atoms. The turns of HA2 helix A are numbered and referenced.

11178 | www.pnas.org/cgi/doi/10.1073/pnas.1406606111 Collins et al. Downloaded by guest on October 6, 2021 fuse membranes (28), the slightly higher pH of fusion of viruses to the biosensor at different relative sugar loadings was calculated from the with HA1 Ser-30 is consistent with an effect of the Thr-30→Ser amplitude of the response at the end of the association step. substitution to decrease HA stability and facilitate HA-mediated membrane fusion. Expression and Purification of HAs. HAs for wild-type A/Equine/Newmarket/2/ 93, A/Equine/Richmond/1/07, and A/Canine/Colorado/17864/06 along with Conclusion A/Equine/Richmond/1/07 mutant (S30T) and A/Canine/Colorado/17864/06 mutant (S30T) were subcloned, as previously described, into a modified For the five mutations that distinguish the HAs of canine from pAcGP67A vector that carries a TEV protease site, a trimerization foldon HAs of equine H3N8 viruses (Fig. 1C) our structural and re- domain, and a 6× His tag (38). Large-scale protein expression was performed ceptor binding analyses primarily provide support for a role of with 2–3 L of SF9 cells. Seventy-two hours postinfection cells were removed the Trp-222→Leu mutation in virus transmission in canines. This by centrifugation, supernatant concentrated, and loaded onto a talon cobalt conclusion gains support from the established importance of column (Clontech). HA fractions were pooled and buffer was exchanged residues in the 220 loop in determining receptor specificity, in by concentrating into 20 mM Tris, pH 8.0, 150 mM NaCl. Concentrated particular HA1 residues 225 and 226 (32, 33). The difference in protein was digested with trypsin [10:1 HA:trypsin (wt:wt ratio); 4 °C, 16 h] to remove the foldon and the His tag. The digestion was stopped using receptor binding specificity with which the substitution is asso- soybean trypsin inhibitor. HAs were further purified by gel filtration chro- ciated may be a reflection of the relative abundance of sialosides matography, using a superdex-200 16/60 column (GE) in 20 mM Tris·HCl, pH on the surfaces of canine cells that are targets for influenza in- 8.0, 150 mM NaCl. HA fractions were pooled and buffer was exchanged into fection but as yet such studies have not been reported. The other 10 mM Tris·HCl, pH 8.0, 50 mM NaCl and concentrated to 12 mg/mL for mutation, for which a role in transmission can be suggested, is at crystallization. the C terminus of HA1, which may be involved in recognition by a protease used to cleave the HA0 precursor in infected dogs. Crystallization of HAs. Canine HA crystals were obtained from 29% (vol/vol) There is precedence for the probable involvement of such a pro- pentaerythritol propoxylate (PEP) 17/8, 0.1 M Tris, pH 8.07, 0.2 M ammonium – tease in human influenza, from studies of the specificity of tryptase sulfate. Canine HA receptor complexes were prepared by soaking HA crys- Clara (15). For two of the three remaining mutations in HA1, we tals in crystallization solution [35% (vol/vol) PEP 5/4, 0.1 M Hepes, 0.2 M ammonium sulfate] supplemented with 40 mM receptor analog 3′SLN and can deduce no structural consequences. This is also the case for ′ X X → 12 mM of analogs Su-3 SLN, Su-SLe , and SLe . Canine S30T mutant crystals the single substitution in HA2, Asn-154 Thr, which results in were obtained from 45% (vol/vol) PEP 17/8, 0.1 M Hepes, pH 7.5, 0.3 M the loss of an otherwise strictly conserved carbohydrate side potassium chloride. Canine M29I mutant crystals were obtained from 35% chain. A role for this side chain in maintenance of HA structure (vol/vol) PEP 629, 0.1 M Hepes, pH 7.5, 0.2 M ammonium sulfate.

or chaperone recognition is therefore not necessary for canine A/Equine/Richmond/1/07 HA crystals were obtained from 12% PEG 2KMME, MICROBIOLOGY influenza replication. However, a role for HA1 Ser-30 in mod- 0.1 M Mes, pH 5.7. Crystals were cryoprotected with crystallization solution ulating HA stability or in membrane fusion activity or activation plus 25% ethylene glycol and HA–receptor analog complexes were prepared is a distinct possibility. This also leads to the suggestion that a by soaking HA crystals in cryoprotected crystallization solution supplemented with 40 mM 3′SLN and 12 mM SLeX. A/Equine/Richmond/1/07 S30T mutant combination of mutations that involve changes in receptor bind- was crystallized from 18% PEG 2KMME, 0.1 M Cacodylate, pH 6.5. ing, HA1 Leu-222, and membrane fusion properties, HA1 Ser-30, A/Equine/Newmarket/2/93 crystals were obtained from 30% PEG400, 0.1 M may be necessary for equine to canine cross-species infection. In Tris·HCl, pH 8.0. HA–receptor complexes were obtained from soaking HA this regard, it is noteworthy that the HA of an H5N1 aerosol- crystals in crystallization solution supplemented with 24 mM Su-3′SLN transmissible mutant also had mutations that modified its receptor and Su-SLeX. binding specificity and affinity, its stability, and probably its membrane fusion activity (34, 35). Our studies of the structure Structure Determination. All crystals were frozen by direct immersion in liquid of the aerosol-transmissible H5 mutant HA (36) indicate that nitrogen and diffraction datasets were collected at 100 K at the IO2 and IO4 the HA1 Thr-318→Ile substitution gains increased stability for beamlines at the Diamond light source (Harwell, UK). Datasets were indexed the aerosol-transmissible mutant HA through hydrophobic inter- and integrated with XDS (39) and imported in the CCP4 suite of programs α (40), and structures were solved and refined using CCP4 or Phenix (41). actions between HA1 Ile-318 and -helix A residues HA2 Val-48 Crystallographic statistics are summarized in Tables S3 and S4. and Val-52. The results of both studies indicate the region in which HA1 Ser-30 of canine and recent equine HAs interacts Tryptic Digestion After Low-pH Incubation. HA solutions (0.15 mg/mL) were with HA2 residues of α-helix A and α-helix B is an important incubated at various pH values (obtained by adding 0.15 M citrate buffer, pH contributor to the structure and stability of H3 hemagglutinins. 3.5), adjusted to neutral pH by using 1 M Tris·HCl, pH 8.0, and digested with trypsin at a HA/trypsin ratio of 20:1 (wt:wt) for 30 min at room temperature. Materials and Methods The digestion was stopped using an equal amount, to trypsin, of soybean Influenza Viruses. Wild-type influenza viruses A/Equine/Newmarket/2/93, trypsin inhibitor. Tryptic digestion products were analyzed by SDS/PAGE. Ee; A/Equine/Richmond/1/07, Ef; and A/Canine/Colorado/17864/06, C were grown in hens’ eggs and purified by sucrose density gradient centrifuga- ACKNOWLEDGMENTS. We thank Debra Elton and Jenny Mumford of the tion, according to standard protocols (37). Animal Health Trust for supplying viruses A/Equine/Newmarket/2/93, A/Equine/Richmond/1/07, and A/Equine/South Africa/4/03. We thank Dr. Takashi Yamanaka of the Equine Research Institute, Japan Racing Association, Biolayer Interferometry. Virus binding to receptor analogs was measured on for supplying A/Equine/Ibaraki/1/07. We thank Dr. Edward J. Dubovi and an Octet RED instrument (ForteBio) as previously described (38). Biotinylated Dr. Colin Parrish (Cornell University) for providing A/Canine/Colorado/ 3′SLN (Neu5Acα2–3Galβ1–4GlcNAcβ) and 6′SLN (Neu5Acα2–6Galβ1–4GlcNAc) 17864/06. We thank Nicolai Boivin at the Shemyakin Institute of Bioorganic were purchased from Lectinity Holdings. Su-3′SLN [Neu5Acα2–3Galβ1–4(6- Chemistry for supplying sulphated sialosides. We are grateful to Dr. Stephen HSO3)GlcNAcβ], Su-SLeX [Neu5Acα2–3Galβ1–4(Fucα1–3)-(6-HSO3)GlcNAcβ] Wharton and Dr. John McCauley, Division of Virology, Medical Research and SLeX [Neu5Acα2–3Galβ1–4(Fucα1–3)GlcNAcβ] were obtained from N. Boivin Council (MRC) National institute for Medical Research, for assistance during the project. We thank members of the National Institute for Medical Re- at the Shemyakin Institute of Bioorganic Chemistry, Russian Academy search World Health Organization Collaborating Centre for Reference and of Sciences, Moscow. Binding of viruses (100 pM) was measured at 25 °C in a Research on Influenza for their assistance throughout the studies. We are μ 30- to 50-min association step. All solutions also included 10 M grateful to staff at the Diamond Light Source Synchrotron for assistance and carboxylate (Roche) and 10 μM (GSK) to prevent cleavage of the beamline access under Proposal 7707. This work was funded by the MRC sugar analogs by viral neuraminidase. The (relative) amount of virus bound through Programmes U117584222 and U117570592.

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