Insight into structural diversity of influenza virus haemagglutinin


Journal of General Virology (2013), 94, 1712–1722

DOI 10.1099/vir.0.051136-0

Insight into structural diversity of influenza virus haemagglutinin Ki Joon Cho,1 Ji-Hye Lee,1 Kwang W. Hong,2 Se-Ho Kim,2 Yiho Park,1 Jun Young Lee,1 Seokha Kang,1 Sella Kim,1 Ji Hoon Yang,1 Eui-Ki Kim,1 Jong Hyeon Seok,1 Satoru Unzai,3 Sam Yong Park,3 Xavier Saelens,4,5 Chul-Joong Kim,6 Joo-Yeon Lee,7 Chun Kang,7 Hee-Bok Oh,7 Mi Sook Chung8 and Kyung Hyun Kim1 Correspondence Kyung Hyun Kim [email protected]


Department of Biotechnology & Bioinformatics, College of Science & Technology, Korea University, Sejong 339-700, Korea


Antibody Engineering Laboratory, Central Research Center, Green Cross Corp., Yongin Kyunggi 446-799, Korea


Protein Design Laboratory, Yokohama City University, Yokohama 230-0045, Japan


Department for Molecular Biomedical Research, VIB, 9052 Ghent, Belgium


Department of Biomedical Molecular Biology, Ghent University, 9052 Ghent, Belgium


College of Veterinary Medicine, Chungnam National University, DaeJeon 305-764, Korea


Influenza Virus Team, Center for Infectious Diseases, Korea Centers for Disease Control and Prevention, Osong Chungbuk 363-951, Korea


Department of Food and Nutrition, Duksung Women’s University, Seoul 132-714, Korea

Received 6 January 2013 Accepted 24 April 2013

Influenza virus infects host cells through membrane fusion, a process mediated by the low pH-induced conformational change of the viral surface glycoprotein haemagglutinin (HA). We determined the structures and biochemical properties of the HA proteins from A/Korea/01/2009 (KR01), a 2009 pandemic strain, and A/Thailand/CU44/2006 (CU44), a seasonal strain. The crystal structure of KR01 HA revealed a V-shaped head-to-head arrangement, which is not seen in other HA proteins including CU44 HA. We isolated a broadly neutralizing H1-specific monoclonal antibody GC0757. The KR01 HA-Fab0757 complex structure also exhibited a headto-head arrangement of HA. Both native and Fab complex structures reveal a different spatial orientation of HA1 relative to HA2, indicating that HA is flexible and dynamic at neutral pH. Further, the KR01 HA exhibited significantly lower protein stability and increased susceptibility to proteolytic cleavage compared with other HAs. Our structures provide important insights into the conformational flexibility of HA.

INTRODUCTION The past three pandemic influenza viruses, viz. 1957 H2N2, 1968 H3N2 and 2009 H1N1 originated from reassortants between the genes of animal influenza viruses and the virus that had been circulating in humans since 1918. The haemagglutinin (HA) genes specifically originated from avian influenza viruses (Garten et al., 2009; Salomon & Webster, 2009; Zimmer & Burke, 2009). The recent H1N1 2009 pandemic (2009pdm) influenza virus that had been in worldwide circulation was genetically homogeneous (99.5 % genome sequence identity) (Garten et al., 2009). Although the current genetic variation has now accumulated in the Three supplementary tables and supplementary methods are available with the online version of this paper.


circulating viruses, all 2009pdm isolates sequenced to date encode a glutamate at position 627 in viral PB2 instead of lysine, carry truncations of both PB1-F2 and NS1, and lack a multibasic proteolytic activation site in HA. They therefore lack previously identified molecular determinants for assessing pandemic potentials implicated in the pathogenicity and transmission of the 1918 H1N1 and H5N1 viruses (Conenello et al., 2007; Garten et al., 2009; Lazarowitz & Choppin, 1975; Steel et al., 2009). However, they replicate more extensively in the respiratory tract of infected ferrets, mice and non-human primates than seasonal influenza viruses (Huang et al., 2011; Itoh et al., 2009; Maines et al., 2009; Munster et al., 2009). Clinical severity associated with the 2009pdm virus is akin to the 1957 H2N2 pandemic outbreak (Fraser et al., 2009).

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Structural diversity of influenza virus haemagglutinin

The influenza virus surface protein, HA, is a prime target of the host immune response. HA is synthesized as precursor HA0 that is folded into an oxidized form in the endoplasmic reticulum, and is transported to the cell surface via the Golgi apparatus. HA0 is cleaved by cellular proteases into HA1 and HA2, a fusion-competent form (Skehel & Wiley, 2000). When the HA1:HA2 molecule encounters low pH, it undergoes conformational changes. HA1, acting as a clamp to maintain the metastable pre-fusion state of HA2, separates from HA2 which then undergoes a loop-to-helix transition and inserts the fusion peptide into the target cell membrane, leading to formation of the extended intermediate (Harrison, 2008; Skehel & Wiley, 2000). HA2 bends outward to collapse into a conformation that brings together the cell-embedded fusion peptide and the viral membrane, generating the final post-fusion conformation. The crystal structures of HA in pre- and post-fusion conformations have been known for a while (Bullough et al., 1994; Wilson et al., 1981), and those of the recombinant HA ectodomains from two 2009pdm isolates, A/California/04/ 2009 (CA04) and A/Darwin/2001/2009 (DA01), were already reported (Xu et al., 2010; Yang et al., 2010; Zhang et al., 2010). They were found to be similar to that of the 1918 H1N1 HA, providing an explanation for age-related immunity to the pandemic in the human population. When the two structures were reported, we also obtained a crystal of recombinant HA protein from a 2009pdm isolate, A/Korea/ 01/2009 (KR01). We later determined the crystal structure, and noticed a significant conformational difference between KR01 HA and other HAs that is not seen in other HA structures. Conformational diversity of KR01 HA was found to be more pronounced in the complex structure of KR01 HA, with the Fab fragment of a neutralizing monoclonal antibody GC0757 that binds to various H1 subtype strains. In this study, we present biochemical and structural features of KR01 HA and its complex with Fab0757, which were compared with those of recombinant HA proteins derived from other influenza virus isolates, A/Thailand/CU44/2006 (CU44), A/Brisbane/59/2007 (BR59) and A/Gyeongnam/ 684/2006 (Gy684), including CA04 and DA01.

kDa, which agreed with the value of 65.9 kDa determined by mass spectrometry, taking into account glycosylation. KR01 HA exhibited a concentration dependent binding to immobilized sialylated fetuin, whereas no binding was observed with asialofetuin, very similar to the binding of CU44 HA to the receptor (Fig. 1b). Infectivity of influenza viruses depends on the receptor binding as well as activation by host proteases capable of cleaving HA precursors (Zambon, 2001). KR01 HA exhibited greatly enhanced susceptibility in solution to proteolytic activation by TPCK-treated trypsin as compared to CU44 HA or Gy684 HA (Fig. 1c). KR01 HA was readily cleaved by trypsin into smaller protein fragments, more susceptible to degradation by chymotrypsin, but resistant to thrombin. Human airway trypsin-like protease (HAT), which was reported to cleave newly synthesized HA in vivo (Bo¨ttcher et al., 2006), cleaved KR01 HA approximately twofold faster than CU44 HA (Fig. 1c). Proteolytic cleavage of HAs by trypsin was not affected by deglycosylation (data not shown). The effects of temperature on protein stability as measured by differential scanning fluorimetry revealed that the melting temperature (Tm) of KR01 HA was 54 uC, whereas it was 59 uC and 62 uC for CU44 and Gy684 HA proteins, respectively (Fig. 1d). These findings are in line with a previous report showing that bacterially-expressed 2009pdm HA behaved as a properly folded protein with a Tm of around 52 uC, although no glycosylation was present (Khurana et al., 2010). When the stability of HAs was examined at different pH values, the Tm value for CU44 H1 HA (active form) significantly decreased with gradual lowering of the pH from 59 uC to 52.5 uC, while no change was observed in the precursor form (Fig. 1d). In contrast, Tm values of the precursor and active forms of KR01 HA remained consistently low upon pH change. The precursor showed a surprisingly low Tm of approximately 48 uC, indicating that KR01 HA has an average stability that is roughly equivalent to that of CU44 HAs at low pH. Overall structure of KR01 HA

RESULTS Characterization of HA proteins To gain insight into the structural and biochemical properties of KR01 HA, we produced and purified recombinant KR01 HA in insect cells. We similarly produced and characterized HAs from CU44 and BR59 (seasonal H1N1) and Gy684 (seasonal H3N2). CU44, BR59 and Gy684 HA proteins were found to be trimeric, as typical HA proteins in their fusionactive state are (Harrison, 2008). However, the purified recombinant KR01 HA was identified as a monomer in solution, based on the results of size-exclusion chromatography, mass spectrometry (Fig. 1a) and analytical ultracentrifugation (data not shown). The molecular mass of KR01 HA deduced from the nucleotide sequence was 60.1 (±0.1)

The overall structures of KR01 HA and CU44 HA looked very similar to those of CA04 and DA01 HA, which were previously known (Fig. 2a) (Gamblin et al., 2004; Stevens et al., 2004; Xu et al., 2010; Yang et al., 2010; Zhang et al., 2010). The structure comprised a long, extended stem region and a globular head region that included the receptorbinding and vestigial esterase domains. The stem region was composed mainly of the long central helix of HA2. In the case of KR01 HA, residues Pro322–Phe336 and Asn387– Lys409, which contain the HA1/HA2 cleavage site, could not be reliably modelled, possibly due to high flexibility. Nevertheless, the overall electron density map showed unambiguous connectivity in most places. When the structure of KR01 HA was superimposed on that of either CA04 HA or CU44 HA, the head region of KR01 HA was surprisingly found to be rotated clockwise by ~20u relative to

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75 kDa 44 kDa 158 kDa 440 kDa


13.5 ml




HA1 130606.4

Gy684 HA

Absorbance (280 nm)


194513.1 0 S

13.8 ml

Elution HA1 HA2



15.0 ml

Elution HA0

130612.4 194387.0








Elution volume (ml)


0.7 KR01 HA



Abs. (450 nm)

0.5 0.4 0.3 0.2 0.1 0.0 0.0








HA (mg ml–1)

Fig. 1. (continued)

the stem region, yielding an overall RMSD of 3.2 A˚ (Fig. 2b). Given that superposition of the head or stem region individually yielded RMSD values of 0.9 A˚ and 1.1 A˚, respectively, the conformations of CA04 and KR01 HA (or CU44 and KR01 HA) overall structures differ substantially. In addition, parts of the hinge region (residues 65–75 and 1714

260–275) of KR01 HA have high B factors ranging from 50 to 90 A˚, conferring high flexibility. CU44 HA is crystallized as a typical trimer like CA04 and DA01 HAs. However, there are two molecules per asymmetrical unit in KR01 HA crystals, which reveals strikingly distinct arrangements of molecules. The mol-

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Journal of General Virology 94

Structural diversity of influenza virus haemagglutinin Trypsin

(c) Conc. (mg ml–1) 0.01

KR01 H1

Time (min) 173 kDa 117 kDa 74 kDa 49 kDa 38 kDa


Chymotrypsin 1



Thrombin 1




50 10 100 0 1 4 8 1 4 8 1 4 8 (h)

0 10 3060 10 306010 3060 10 1 30 60 10 30 60 10 30 60 10 30 60 10 30 60 10 30 60


25 kDa


18 kDa


13 kDa 173 kDa 117 kDa 74 kDa 49 kDa 38 kDa CU44 25 kDa H1 18 kDa


13 kDa 173 kDa 117 kDa 74 kDa 49 kDa Gy684 38 kDa 25 kDa H3 18 kDa



13 kDa

Fig. 1. (continued)

ecules of KR01 HA interact with each other to create a Vshaped head-to-head structure (Fig. 2c). The molecular interactions of KR01 HA in this arrangement initially appeared to be very different from those of other HA molecules, but almost the same amino acid residues involved in the head domain interactions in CA04, DA01 or CU44 trimers were found to contribute to the head-tohead interactions in KR01 HA (Table S1, available in JGV Online). Electrostatic interactions between Asp94 and Arg226, His181 and Glu213, and Glu213 and Asn228 and Tyr230 make a large contribution to the head-to-head interactions in KR01 HA. Although we cannot rule out the possibility that this arrangement of HA molecules was produced due to crystal packing, it was recently shown that the recombinant receptor-binding domain of HA from a 2009pdm virus expressed in E. coli was in a head-to-head arrangement (DuBois et al., 2011). The stem regions of KR01 HA form two parallel coiled-coil helices, resembling a leucine zipper structure, compared to triple coiled coils in other HA proteins (Fig. 2c). A long helix of HA2 of one molecule interacts with that of a symmetry-related molecule. Highly conserved residues Ile418(Ile91), Leu425(Leu98) and Leu429(Leu102) are involved in the leucine zipper motif as in triple coiled coil interactions in HA (Table S2). The stem-to-stem interface has a buried surface area of 1115 A˚, which is greater than that of a typical leucine zipper (Wang et al., 2008). Importantly, receptor-binding and antigenic sites of KR01 HA are nevertheless structurally identical to those of CU44,

CA04 and Fig. 2(d).







Structure of KR01 HA-Fab0757 complex In addition to the KR01 HA structure, we isolated an H1-specific monoclonal antibody, GC0757, that showed significant neutralizing activity against pandemic H1N1 strains (CA04, A/California/07/2009 and A/Brevig Mission/ 1/1918) and seasonal H1N1 strains (CU44 and BR59) (Table S3). We then produced microcrystals of KR01 HA in complex with the Fab fragment from monoclonal antibody GC0757, whose structure was determined at 2.8 A˚ resolution. The complex structure revealed head-to-head arrangement of HA (Fig. 3a), which was identical to that in the free KR01 HA structure. Fab0757 binds to the head region of KR01 HA on the opposite side of the head-to-head interface, resulting in a linear arrangement of the complex, consisting of one Fab, two head regions of HA and another Fab molecule. Unexpectedly, the electron density of the stem region was largely invisible, even though there is empty space available for it and its presence was confirmed in SDSPAGE (data not shown). A small part of the stem region could nevertheless be modelled from residues 50–62 and 84– 107 in a-helical conformation (Fig. 3b), which exhibited rather high B factors ranging from 57–95 A˚ and 50–143 A˚, respectively. Close examination of both free and complex structures of KR01 HA further revealed unique features that have not been

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Fluorescence (–R′(T))



–300 CU44 Gy684 KR01


–700 40

50 60 Temperature (°C)



Tm (°C)




45 7




pH CU44 HA0 seasonal precursor

CU44 HA seasonal active

(precursor) pandemic precursor

(active) active pandemic

KR01 HA0




reported previously. When viewed parallel to the leucine zipper motif in the stem region, the head region HA1 looked overly bent outward from HA2, different from that observed in other HA structures (Fig. 4a). Superposition of KR01 HA free and complex structures including CU44 or CA04 HA clearly showed that the head or stem region superposed well individually (0.8–1.2 A˚ on average). However, the head regions adopted a surprisingly wide range of conformations when the HA molecules were superimposed based on the stem regions (Fig. 4b, upper). When the orientation of the head regions was fixed, it was obvious that both the free and complex structures of KR01 HA displayed conformational diversity of the stem regions, compared to that of CA04 HA (Fig. 4b, lower). In the HA-Fab0757 complex structure of KR01, Fab0757 binds to conserved regions of the KR01 HA globular domain that are on the opposite side of the monomer–monomer interface in a trimeric form, indicating similar binding of Fab0757 to either monomeric or trimeric forms of HA. Indeed, GC0757 binds to a broad spectrum of H1 HAs, as shown in Table S3. More interestingly, when trimeric KR01 HA was constructed based on the stem region of CA04 HA structure, it was found to adopt a blown-out conformation, resembling a bouquet of HA flowers in full bloom (Fig. 4c). In contrast, a trimeric HA model constructed from the KR01 HA–Fab complex structure showed a fully closed conformation of head regions, resembling HA flowers in tight cluster, which could not possibly be due to severe steric clashes. Our results thus strongly suggest that conformational flexibility of KR01 HA may have a significant impact on diverse conformations of HA1 over HA2.

DISCUSSION Conformational nature of KR01 HA

Fig. 1. Characterization of HA proteins. (a) Size-exclusion chromatographic analysis of standard proteins with known molecular masses (upper panel), purified HA from CU44 (A/Thailand/ CU44/2006) (second panel), Gy684 (A/Gyeongnam/684/2006) (third panel) and KR01 (A/Korea/01/2009) (lower panel). CU44 and Gy684 HA proteins were isolated as a trimer, whereas KR01 HA was as a monomer based on size-exclusion chromatography with SDS-PAGE and mass spectrometry (insets). (b) Solid-phase binding assay of HA derived from KR01 (left panel) and CU44 (right panel) to fetuin (white bars) and asialofetuin (black bars). (c) Proteolytic cleavage of KR01 HA (upper), CU44 HA (middle) and Gy684 HA (lower) by trypsin, chymotrypsin, thrombin, and HAT. HA proteins were treated with trypsin at 25 6C, or HAT at 37 6C, and then analysed by SDS-PAGE. Proteases were used at concentrations of 0.01 to 1 mg ml”1 for trypsin, chymotrypsin and thrombin and 10–100 mg ml”1 for HAT. Arrows indicate the positions of HA fragments and HAT. (d) Differential scanning fluorimetry transition curves of HA proteins (upper) and at different pH conditions (lower). Each HA protein in the precursor and active forms were incubated at 25 6C for 30 s, and then the temperature was increased by 0.5 6C every 30 s for 50 min. 1716

Influenza viruses rely on host proteases in the airways for maturation of HA. Cleavage of HA0 with extracellular trypsin-like proteases occurs at a monobasic site in human influenza viruses, whereas HA0 with a multibasic cleavage site in highly pathogenic avian influenza viruses is cleaved by ubiquitously present intracellular subtilisin-like proteases (Zambon, 2001). Although less is known about specific proteases that cleave influenza virus HA0 under conditions of natural infection, HAT was reported to promote viral spread in the human airways (Bo¨ttcher et al., 2006; Chaipan et al., 2009). We showed that KR01 HA0 precursor is much more susceptible to proteolytic activation by proteases such as HAT, trypsin and chymotrypsin, compared with the CU44 and Gy684 HA0 proteins. The precursor of KR01 HA exhibited a low thermal stability (Tm of 48 uC) similar to that of CU44 HA at low pH. KR01 HA may adopt a conformation close to the putative prefusion state at low pH, which can also explain the high susceptibility to proteolytic cleavage observed in KR01 HA. During membrane fusion, the large-scale conformational

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Journal of General Virology 94

Structural diversity of influenza virus haemagglutinin






Asn87 Asn276


Asn287 Asn23



Asp187 Tyr192 His180 Trp150 Gly225 Gln223 Tyr91 Lys219 L



Ca Cb

Val132 Asp222








Fig. 2. Overview of the structures of HA. (a) Monomer structures of KR01 (orange), CA04 (blue, 3LZG), DA01 (green, 3M6S), and CU44 (lime green) HA. Glycosylation (yellow) and amino acid variation sites among KR01, CA04 (cyan) and DA01 (violet) HAs are represented in a ball-and-stick model. No glycosylation was modelled in CU44 HA. (b) Superposition of KR01 (orange and yellow) and CA04 (grey) HAs. The top view (upper) and side view (lower) after superposition based on alignment of the stem regions, and top view (right) after superimposition based on the head regions are shown. (c) Structure of KR01 HA. Headto-head arrangement of KR01 HA in the asymmetrical unit of the crystal (orange and yellow) and symmetry-related molecules (grey). The side view (upper) and top view (lower) are shown. (d) Structures of the receptor-binding site (upper) and antigenic sites (lower). Superposition of the receptor-binding sites of KR01 (orange) and CA04 (grey) HAs is shown with key conserved residues that determine receptor specificity. Antigenic Sa site, raspberry; Sb site, pink; Ca site, lime green; and Cb site, lemon. Amino acid substitutions in the Ca site of DA01 HA are marked.

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(a) Fab757








Fig. 3. Overall structure of KR01 HA-Fab0757 complex. (a) Head regions of KR01 HA are orange and light brown, heavy chains are blue and lime green, and light chains are light blue and aqua marine. (b) Superposition of free KR01 HA and HA-Fab0757 complex. The stem region of KR01 HA is represented by a transparent cartoon.

rearrangement of HA at low pH is triggered by a loop-tohelix transition of an interhelical loop (B loop), referred to as the spring-loaded mechanism (Carr & Kim, 1993; Carr et al., 1997). It was previously suggested that the conformation of HA2 that is metastable at neutral pH is temporarily stabilized by HA1 in close spatial proximity; HA1 separates from HA2 during membrane fusion (Harrison, 2008). The conformational changes of HA that alleviate the constraints imposed in the pre-fusion state provide the energy that is required to induce membrane fusion. Both the structures of KR01 HA and its complex with Fab0757 revealed the unusual conformation in head-to-head arrangements with diverse conformations of HA1 over HA2, possibly equivalent to dissociated conformations of HA1 from HA2. With regard to true conformation of KR01 HA, naturally a transmembrane protein, at the virus surface in vivo, the viral protein may behave as a loosely assembled trimer that is able 1718

Fig. 4. Structural features of KR01 HA and its complex with Fab0757. (a) Side view of KR01 HA dimer in Fig. 2c (upper) when viewed parallel to the plane passing through the stem regions. Each monomer belonging to different dimers is in orange and yellow. (b) Comparison of HA structures of CA04 HA (grey), KR01 HA (orange) and the HA molecule in the KR01 HA-Fab complex (magenta). Side view of HAs after superposition (upper) and each monomer structures (lower). (c) Constructed model of KR01 HA (left), CA04 HA (middle) and HA in the KR01 HA-Fab0757 complex (right). Shown are the top view (upper) and side view (lower).

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to readily adopt uncapped conformations of HA1 relative to HA2. Recently, it was reported that a crystal structure of an early intermediate state revealed reorganization of ionic interactions at the HA1–HA2 interface and release of the B loop was assisted by deformation of HA1 (Xu & Wilson, 2011). Cryo-electron tomography was applied to visualize influenza A virus at acidic pH and to probe intermediate states of HA (Fontana et al., 2012). Two intermediate conformations prior to fusion appeared to reflect an outwards movement of the fusion peptide and rearrangement of the HA1 subunits, and the proposed dispersal of HA1 appears to reflect the dissociation of HA1. Both CA04 and DA01 HA proteins were purified as a monomer and crystallized as a trimer (Xu et al., 2010; Yang et al., 2010). An independent report of the crystal structure of CA04 HA also revealed that CA04 HA is a monomer in solution (Zhang et al., 2010). Unlike CA04 and DA01 HAs of which the structures showed a trimer, KR01 HA demonstrated an unusual head-to-head arrangement and its sequence differs at three amino acid positions from that of CA04 and DA01, respectively. Ser83(Pro83)*, Ala187(Thr187) and Val321(Ile321) of KR01(CA04) HA are located at the tip of the head domain or at the C-terminal region of the stem domain, which are remote from the monomer–monomer interface. In case of KR01 and DA01 HAs, Ser203(Thr203) and Arg205(Lys205) are remote from the monomer–monomer interface and Val411(Ile411) is close to the monomer–monomer interface but the residues have similar chemical properties. In addition, the difference in the crystallization conditions for KR01, CA04 and DA01 HAs was merely the presence of 100 mM NaCl. The bacterial or mammalian cell expressed HA ectodomain from A/ California/07/2009 was predominantly monomeric and could adsorb neutralizing activity from H1N1 immune sera (Khurana et al., 2010). It is thus very likely that the recombinant HA ectodomains derived from 2009pdm isolates are monomeric in solution. In fact, it was reported that a full-length recombinant HA derived from A/California/ 07/2009 is a monomer, which contains both transmembrane and cytosolic domains (Feshchenko et al., 2012). The existence of monomeric intermediates during the conformational changes in fusion was previously described for vesicular stomatitis virus G protein (Danieli et al., 1996). Although the 2009pdm HAs are essentially monomeric in solution, why KR01 HA shows significantly different conformations from that of CA04 and DA01 is not clear. Monomeric KR01 HA has receptor-binding and antigenic sites identical to those of trimeric CU44, CA04 and DA01 HAs (Fig. 2d), with a concentration dependent binding to sialylated fetuin very similar to that of CU44 HA (Fig. 1b). Furthermore, despite high proteolytic sensitivity of KR01 HA0, it requires protease treatment for activation as do trimeric HAs. We postulate that KR01 HA on the virus surface adopts flexible conformations with substantially low energy barrier, as a loosely assembled trimer. It will be interesting to see whether the interactions of HA with adjacent HA molecules would have a direct effect on generating the unusual conformations that we observe in this study. We targeted several amino acid residues that are

located at the monomer–monomer interface as well as the HA1–HA2 interface, and mutation experiments are currently under way. Implication of KR01 HA Although the pathogenesis of 2009pdm viruses is still controversial, they were reported to replicate more extensively in the respiratory tract of infected ferrets than seasonal H1N1 viruses (Munster et al., 2009; Maines et al., 2009). Human infections appeared to be mild, but an alarming number of young individuals presented with symptoms atypical for seasonal influenza (Safronetz et al., 2011). The 2009pdm viruses also showed a sustained human-to-human transmissibility and higher reproduction ratio than common seasonal viruses. However, the actual pathogenic potential of the circulating virus pool remains unknown. It is surprising to observe that the overall structure of KR01 HA remains largely intact even after dissociation from the trimer, which is stabilized by head-to-head or stem-to-stem interactions. Nevertheless, the question still remains: what would be the role of this unusual HA structure? It is known that triggering of the conformational change in an individual HA trimer is affected by the proximity of other HA molecules, which initiate fusion that may involve interactions with adjacent trimers (Danieli et al., 1996; Floyd et al., 2008; Lee et al., 2006; Markovic et al., 2001). Decrease in the surface density of HA on viral particles arrests fusion. If KR01 HA were to behave as a loosely assembled trimer on the viral surface, it is possible that HA molecules would readily interact with adjacent molecules. Taken together, our study provides important insights into the structural and molecular properties of HA derived from a 2009pdm isolate, suggesting that KR01 HA may be a conformational variant.

METHODS Viruses, cells and PCR. KR01, seasonal H1N1 (CU44 and BR59) and H3N2 (Gy684) strains were isolated from nasopharyngeal swabs, and viruses were propagated in MDCK or Vero cells, as described in the supplementary material. Viral RNA was extracted from culture supernatant using an Extragen II kit (Kainos). RNA was transcribed using the influenza A virus universal primer, and the HA gene was amplified using segment-specific primers (Kendal, et al., 1982). PCRamplified fragments were purified using a MicroSpin S-300 HR column (GE Healthcare), labelled using a BigDye Terminator v3.1 cycle sequencing kit (Applied Biosystems), and then analysed on an ABI 3100 automatic DNA sequencer. Cloning and baculovirus production of HA. KR01, CU44, BR59

and Gy684 HAs were produced in insect cells using recombinant baculovirus expression vectors. HA gene sequences (1-503 of HA0 based on H3 numbering) were cloned downstream of the gp67 secretion signal sequence of the transfer vector pAcGP67A (BD Biosciences). A thrombin cleavage site, foldon region and 6xHis-tag were inserted downstream of the HA gene sequence (Bhardwaj et al., 2008), which contained additional plasmid-encoded residues (ADPG for H1 CU44 and BR59 and H3 Gy684; ADPGYLLEF for KR01 H1) at their N- and C-termini (RSLVPR for all three HA proteins). Nucleotide sequences were confirmed by sequencing (Macrogen).

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Table 1. Data collection and refinement statistics Data statistics Wavelength (A˚) Number of unique reflections Resolution range (A˚) Completeness (%) Rmerge (%)* I/sigma Space group Unit cell parameters (A˚)

Refinement statistics Resolution range (A˚) Number of reflections R/Rfree (%)D


KR01 HA-Fab0757


0.980 773 207 (44 808) 50.0–2.7 (2.8–2.7) 99.6 (95.7) 12.1 (77.5) 21.5 (1.6) P6 a5b5208.13 c565.77 a5b590u, c5120u

0.999 313 582 (66 891) 50.0–2.8 (2.9–2.8) 87.9 (81.0) 12.8 (49.8) 21.5 (1.6) P21 a573.70, b590.13 c5238.18 a5c590u, b590.1u

0.999 1 670 465 (83 089) 50–2.5 (2.6–2.5) 100 (100) 11.7 (76.8) 28.4 (4.0) R32 a5b5217.12 c5266.02 a5b590u, c5120u

50.0–2.7 44 808 23.7/28.9

49.7–2.8 66 880 23.3/29.0

50.0–2.5 78 974 23.9/27.4

0.007 1.21 63 86.5

0.009 1.67 115 32.7

0.006 1.136 16 48.0

91.4 8.5 0.1

83.4 15.6 1.1

81.4 14.3 4.3


Bonds (A˚) Angles (u) Number of water molecules Average B (A˚2) Ramachandran statistics (%)d Favoured Allowed Disallowed

*Rmerge5S|I2,I.|/S,I., where I and ,I. are the measured and averaged intensities of multiple measurements of the same reflection, respectively. The summation is over all the observed reflections. DR5S|Fo2Fc|/S|Fo| calculated for all observed data. Rfree5S|Fo2Fc|/S|Fo| calculated for a specified number of randomly chosen reflections that were excluded from the refinement. dCalculated using PROCHECK (Laskowski et al., 1993).

Plasmids encoding each HA gene were amplified in E. coli DH5a and used to co-transfect Sf9 cells with linearized baculovirus chromosomal DNA (BaculoGold; BD Biosciences) by the calcium transfection method. The recombinant baculovirus was harvested from the cell supernatant on day 5. Protein expression and purification. Baculovirus containing HA

gene was used to infect suspension cultures of Hi5 cells. After 3–4 days at 28 uC, the culture medium was harvested and applied to a Ni-NTA column equilibrated with 20 mM Tris/HCl, pH 8.0, 200 mM NaCl. After washing with 50 mM imidazole, precursor HA protein was eluted in an imidazole gradient. It was then dialysed against 10 mM Tris/ HCl (pH 8.0) and 50 mM NaCl, and hydrolysed by TPCK-treated trypsin for 3 h for cleavage of HA0 into HA1 and HA2 and by thrombin for removal of the foldon region and 6xHis-tag. The reaction was halted by the addition of 1 mM phenylmethylsulphonyl fluoride, and the active form of HA was purified by Mono Q ionexchange chromatography and Superdex 200HR size-exclusion chromatography. Reductive methylation was carried out as previously described (Shaw et al., 2007), and the protein was subjected to size-exclusion chromatography to remove the methylation agents and to exchange the buffer. Immunization and production of a monoclonal antibody GC0757 are described in the supplementary material. All experiments were approved by the Gyeong-gi state authorities and complied with International Animal Care and Use Committee (IACUC MG-10121) requirements for the care and use of laboratory animals. A neutralizing antibody with activity against H1 subtype influenza 1720

viruses, GC0757, was treated with papain at a ratio of 1 : 50 (w/w) for 2 h in PBS buffer (10 mM Na2HPO4, 1.8 mM KH2PO4, 137 mM NaCl, 2.7 mM KCl, and pH 7.4) with 5 mM EDTA and 5 mM Lcysteine, and the reaction was stopped with 30 mM crystalline iodoacetamide. The solution containing Fab fragments was dialysed against the PBS buffer and applied to a Protein A column. The unbound fraction was purified by Superdex 200HR gel-filtration chromatography. It was then mixed with HA at a 1 : 1.5 molar ratio for 12 h incubation, and the complex was further purified by Superdex 200HR gel-filtration chromatography. Biochemical characterization of recombinant HA proteins.

MALDI-TOF mass spectrometry and deglycosylation of HA proteins are described in the supplementary material. Sialic acid-binding activity of HA proteins was assessed using a fetuin solid-phase assay. 1 mg ml21 fetuin and asialofetuin (Sigma-Aldrich) per well was used to coat 96-well Nunc MaxiSorp plates. After three washing steps, plates were blocked with PBS buffer containing 1 % BSA. HA protein was pre-complexed with anti-His-tag antibody and horseradish peroxidase-linked anti-mouse antibody (4 : 2: 1 molar ratio) for 30 min at 4 uC prior to incubation of limiting dilutions on the fetuin- or asialofetuin-coated plates (60 min, room temperature). Plates were successively rinsed with PBS containing 1 % BSA and 0.05 % Tween-20. HA binding was subsequently detected using tetramethylbenzidine substrate in an ELISA reader (Bio-Rad), reading the OD at 450 nm. For protein stability experiments, 5 mg of HA in 50 mM Tris/HCl, pH 8.0, and 100 mM NaCl was added into a low tube strip (Bio-Rad).

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Journal of General Virology 94

Structural diversity of influenza virus haemagglutinin To this was added 500-fold SYPRO Orange and it was incubated at 25 uC for 30 s, after which the temperature was increased by 0.5 uC every 30 s for 50 min. Relative fluorescence units were recorded using Mx3005P differential scanning fluorimetry (Stratagene). The excitation and emission wavelengths were 492 nm and 610 nm, respectively. Tm was calculated using MxPro QPCR Software. The pH was adjusted using 1 M citric acid solution and neutralization with 2 M Tris. For proteolytic cleavage analysis, HA proteins were treated with TPCKtrypsin, thrombin, chymotrypsin, or human airway trypsin-like protease (HAT) at 25 uC. Aliquots were analysed by SDS-PAGE at periodic time intervals. The concentration of HA was 0.2 mg ml21 and the concentrations of enzymes were 0.01, 0.1 and 1 mg ml21 (in the case of HAT, 10, 50 and 100 mg ml21 at 37 uC). At concentrations from 1 to 10 mg ml21, trypsin cleaved HA0 into HA1 and HA2. Crystallization and data collection. Crystals of KR01, CU44, BR59,

and Gy684 HAs were screened by the hanging drop vapour diffusion method using commercial kits (Emerald Biosystems). 1 ml of HA (15 mg ml21) was mixed with 1 ml of screening solution at 4 uC and 22 uC. Small single crystals of KR01 and CU44 HAs were obtained in 100 mM HEPES (pH 7.5), 20 % PEG 3350 and 0.2 M NaCl at 4 uC, and in 100 mM Tris/HCl (pH 7.0), 22 % PEG and 0.2 M calcium acetate at 24 uC, respectively, that diffracted to sufficiently high resolution. In the case of the complex of KR01 HA and Fab0757, 1 ml of the complex (10 mg ml21) was mixed with 1 ml of screening solution of 20 % PEG3350 and 200 mM potassium iodide, and incubated at 4 uC. Diffraction data were collected with the crystals flash-cooled at 100 K in a stream of liquid N2 in the mother liquor containing 22 % glycerol using synchrotron radiation sources. The microcrystals of KR01 HA, CU44 HA and KR01 HA-Fab0757 diffracted to 2.7 A˚, 2.5 A˚ and 2.8 A˚ resolutions, respectively, using beamline BL-17A at the Photon factory. All data were processed and scaled using the HKL2000 program (Otwinowski & Minor, 1997) and unit cell parameters and data statistics are listed in Table 1. Structure solution and refinement. The crystal structures of KR01

HA, CU44 HA and KR01 HA-Fab0757 were solved by molecular replacement using the HA structure of WDK/JX/12416/2005 (PDB ID 3HTT) as the template. Molecular replacement using the CCP4 (1994) or PHENIX (Adams et al., 2010) with a monomer gave a single prominent solution. For KR01 HA-Fab0757 complex, a head region of HA was used as an initial template and a Fab fragment (3LZF) was subsequently used as a template after fixing the solution of the head region. The solvent contents of each crystal indicated that two protomers of KR01 HA, one trimer of CU44 HA and four HAFab0757 complexes existed in an asymmetrical unit, respectively. After the substitution of the Fab sequences with those of Fab0757, the initial solution was optimized by rigid body refinement, which produced an interpretable electron density for the overall structure. Manual adjustment of the backbone and side chains was conducted in Coot (Emsley & Cowtan, 2004). Crystallographic refinement was carried out using the program refmac5 (Murshudov et al., 1997). Difference Fourier maps, 2|Fo|-|Fc| and |Fo|-|Fc|, have been used to model the active site or loop regions. After a few rounds of model rebuilding, water molecules were added using the |Fo| 2 |Fc| difference map peaks above 3.0s, if the B factors were below 50 A˚ after refinement. The Rfree value was used as an indicator to validate the water picking and refinement procedure and to guard against possible overfitting of the data (Bru¨nger, 1992). R factors and Rfree values are in the range 0.23–0.24 and 0.27–0.29, respectively (Table 1). Stereochemical analysis of all refined structures using PROCHECK (Laskowski et al., 1993) showed that there was one outlier in the Ramachandran plot with 91.4 % in the favoured region of 875 residues for KR01 HA, 62 outliers with 81.4 % in the favoured region of 1437 residues for CU44 HA and 27 outliers with 83.4 % in the

favoured region of 2530 residues for the HA-Fab0757 complex. These outliers were located in the disordered loop region.

ACKNOWLEDGEMENTS We wish to acknowledge the technical support from the staff at the beamlines of Pohang Light Source (5C), PF (BL-17A) and Spring-8 (BL38B1) synchrotrons and by PAL through the Abroad Beamtime Program under MEST performed under the approval of the PF Program Advisory Committee (proposal no. 2012G618). This work was supported by grants from the Mid-career Researcher (KHK) and Basic Research (KJC) Programs through the NRF funded by the MEST (2010-0029242 & 2012-044524), the Transgovernmental Enterprise for Pandemic Influenza in Korea (KHK) (TEPIK, 2011A103001), and Korea University (KJC). Data deposition: the atomic coordinate and structure factors have been deposited in the Protein Data Bank, (PDB ID codes 4EDA, 4F15 and 4EDB for KR01 HA, KR01 HA-Fab0757 and CU44 HA structures, respectively).

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Journal of General Virology 94


Insight into structural diversity of influenza virus haemagglutinin

Journal of General Virology (2013), 94, 1712–1722 DOI 10.1099/vir.0.051136-0 Insight into structural diversity of influenza virus haemagglutinin Ki ...

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