Structural Stability of Influenza A(H1N1)pdm09 Virus Hemagglutinins

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Hemagglutinins

Hua Yang, Jessie C. Chang, Zhu Guo, Paul J. Carney, David A. Shore, Ruben O. Donis, Nancy J. Cox, Julie M. Villanueva, Alexander I. Klimov,† James Stevens

Influenza Division, National Center for Immunization and Respiratory Diseases, Centers for Disease Control and Prevention, Atlanta, Georgia, USA

ABSTRACT

The noncovalent interactions that mediate trimerization of the influenza hemagglutinin (HA) are important determinants of its biological activities. Recent studies have demonstrated that mutations in the HA trimer interface affect the thermal and pH sen-sitivities of HA, suggesting a possible impact on vaccine stability (Farnsworth et al., Vaccine 29:1529 –1533, 2011, doi:10.1016/ j.vaccine.2010.12.120). We used size exclusion chromatography analysis of recombinant HA ectodomain to compare the differ-ences among recombinant trimeric HA proteins from early 2009 pandemic H1N1 viruses, which dissociate to monomers, with those of more recent virus HAs that can be expressed as trimers. We analyzed differences among the HA sequences and identified intermolecular interactions mediated by the residue at position 374 (HA0 numbering) of the HA2 subdomain as critical for HA trimer stability. Crystallographic analyses of HA from the recent H1N1 virus A/Washington/5/2011 highlight the structural basis for this observed phenotype. It remains to be seen whether more recent viruses with this mutation will yield more stable vaccines in the future.

IMPORTANCE

Hemagglutinins from the early 2009 H1N1 pandemic viruses are unable to maintain a trimeric complex when expressed in a re-combinant system. However, HAs from 2010 and 2011 strains are more stable, and our work highlights that the improvement in stability can be attributed to an E374K substitution in the HA2 subunit of the stalk that emerged naturally in the circulating vi-ruses.

T

he first influenza pandemic of the 21st century was identified

in April 2009, when a new H1N1 influenza virus, (H1N1) pdm09, found in patients in Mexico and the United States, rapidly spread globally by human-to-human transmission, resulting in the World Health Organization declaring a global pandemic on 11

June 2009 (1). As soon as it was recognized that this novel

A(H1N1)pdm09 virus was spreading from person to person, the laboratories within the WHO Global Influenza Program began the propagation of these viruses in eggs in order to obtain a virus suitable for vaccine production. However, even though vaccines were available in early October, the second wave of A(H1N1) pdm09 circulation peaked in late October, when vaccine coverage was extremely low, reducing the impact of vaccine interventions

to mitigate the pandemic (2,3).

It is interesting that almost 5 years after the first A(H1N1) pdm09 viruses were isolated, currently circulating viruses are still

antigenically homogeneous (4). However, during this time a

number of hemagglutinin (HA) mutations have emerged at sites that are within or close to the trimer’s monomer-monomer

inter-face (5). This raises the possibility that this reduced HA trimer

stability might have been detrimental to the fitness of the virus in the human host and these recent mutations are being maintained within the HA to improve its stability. To study the effect of some of these mutations, we have cloned and recombinantly expressed recently circulating A(H1N1)pdm09 virus HAs (recHAs) as solu-ble trimeric ectodomains and assessed their stability using bio-chemical and structural techniques. Results show that more recent HAs are stable compared to their 2009 counterparts, and residue 374 (HA0 numbering from the mature protein) was identified as being responsible for the increased trimer stability.

MATERIALS AND METHODS

Recombinant HA cloning and expression.All proteins described here

(Table 1) were generated by mutagenizing a previously described

baculo-virus transfer vector containing a codon-optimized HA gene of baculo-virus A/Texas/05/2009 (Tx09), using the QuikChange Lightning site-directed mutagenesis kit (Agilent Technologies) (6). Transfection and virus am-plification and protein expression were carried out as described previ-ously (6–8). Recombinant HAs, recovered from the culture supernatant and purified by sequential metal affinity and size exclusion chromatogra-phy (SEC), all contained a thrombin site at the C terminus followed by a trimerizing sequence (foldon) from the bacteriophage T4 fibritin for gen-erating functional trimers (9) and a His tag to aid purification. At this stage, the recHA protein was in the HA0 form, and this protein was used in all subsequent analyses.

Ability to maintain functional trimers in solution.Recombinant protein (300␮l at 1 mg/ml) in 50 mM Tris–100 mM NaCl, pH 8 (Tris buffer), was divided into 2 tubes. One tube received 17␮l 10⫻thrombin buffer, 0.5␮l thrombin (0.33 U/␮l activity), and 2.5␮l Tris buffer, while the other tube received 17␮l 10⫻thrombin buffer and 3␮l Tris buffer. Samples were incubated at room temperature and then subjected to SEC

Received12 August 2013 Accepted7 February 2014

Published ahead of print12 February 2014

Editor:W. I. Sundquist

Address correspondence to James Stevens, fwb4@cdc.gov.

† Deceased 5 February 2013.

Supplemental material for this article may be found athttp://dx.doi.org/10.1128 /JVI.02278-13.

Copyright © 2014, American Society for Microbiology. All Rights Reserved.

doi:10.1128/JVI.02278-13

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using a suitable Superdex-200 column (GE Healthcare) with 50 mM Tris-HCl–150 mM NaCl, pH 8, as running buffer. To ensure equivalent injec-tion volumes, the entire 170␮l of each sample was loaded through a 100-␮l sample loop.

DLS.Each recombinant protein (100␮g in 90␮l of 50 mM Tris and 100 mM NaCl, pH 8, was aliquoted into two tubes. In one, 5␮l of a trypsin solution (10␮g/ml in 50 mM Tris and 100 mM NaCl, pH 8) was added, while the other received an equivalent volume of buffer only. The final recHA/trypsin ratio was 1,000:1 (wt/wt). Tubes were incubated overnight at 20°C, after which samples were analyzed by SDS-PAGE and dynamic light scattering (DLS) using a Dynapro plate reader and Dynamics 7 soft-ware (Wyatt Technology Corp., Santa Barbara, CA). Results are presented as the estimated molecular mass of species in the solution, as calculated by the software.

Thermal stability analyses.Recombinant HA proteins (0.5 mg/ml) in 50 mM Tris-HCl and 150 mM NaCl, pH 8, were incubated at 37°C or 50°C for 2 h prior to protease digestion using trypsin at a ratio of 30:1 (mass protein to mass enzyme) in a final volume of 30␮l. Digestion mixtures were subsequently incubated at 17°C for 16 h prior to analysis by SDS-PAGE under reducing conditions. For protein-melting experiments, each recombinant protein (150␮g of each HA at 1 mg/ml in phosphate-buff-ered saline [PBS]) was added to a 96-well black quartz microplate (Hellma USA, Plainview, NY) and overlaid with 30␮l of paraffin oil (Hampton Research, CA). Samples were analyzed by DLS using a Dynapro plate reader and Dynamics 7 software (Wyatt Technology Corp.). The hydro-dynamic radius of each protein in each well was measured as the temper-ature of the entire plate was increased from 25°C to 80°C, at a rate of 0.33°C/min.

SEC-MALS. Size exclusion chromatography-coupled multiangle static light scattering (SEC-MALS) experiments were performed using an Agilent autosampler and pump (Agilent, Santa Clara, CA) connected to a Wyatt SEC column (WTC-030S5) and a Wyatt DAWN HELEOS II 18-angle MALS detector. The MALS detector was equipped with a gallium-arsenic laser (658 nm), and measurements were obtained at 25°C by de-tectors situated at angles of 44°, 50°, 57°, 64°, 72°, 81°, 90°, 99°, 108°, 117°, 126°, 134°, and 144° to the incident beam. Purified HA proteins (100␮l of Tex09, Tex09-Glu374Lys, or Wash11) at 0.88 mg/ml treated with or with-out thrombin were injected and eluted through the system at a flow rate of 0.5 ml/min in 50 mM Tris-HCl and 150 mM NaCl, pH 8. Protein fractions were detected by the UV spectrophotometer, and the molar mass of each fraction was measured by the MALS detector. The molecular mass of each fraction was calculated using Astra V software (Wyatt Technology Corp.). The system was calibrated using bovine serum albumin as a standard (Thermo Scientific, Rockford, IL) according to the manufacturer’s pro-tocol.

pH stability.Recombinant HA proteins (0.5 mg/ml) were incubated in buffers at different pHs ranging from 4.6 to 5.4 for 2 h and then brought to pH 8.0 prior to protease digestion using trypsin at a ratio of 30:1 (mass of protein to mass of enzyme). Digestion mixtures, in a final volume of 30 ␮l, were subsequently incubated at 17°C for 16 h (overnight) prior to analysis by SDS-PAGE under reducing conditions.

Glycan microarray analysis.Microarray printing and recHA analyses have been described previously (6). Briefly, HA-antibody precomplexes were prepared by mixing HA (10␮l, 1 mg/ml), mouse anti-penta-His-Alexa Fluor 488 (17.5␮l, 0.2␮g/ml; Qiagen Inc.), and anti-mouse-IgG-Alexa Fluor 488 (1.2␮l, 2 mg/ml; Life Technologies) in a molar ratio of 4:2:1, respectively. Mixtures were incubated for 60 min on ice and then diluted with 500␮l PBS buffer containing 2% (wt/vol) bovine serum albumin and streptavidin-Alexa Fluor 488 (1:1,000, vol/vol; Life Technol-ogies) and incubated on the microarray slide in a humidified chamber on ice for 90 min. Slides were subsequently washed by successive rinses in PBS with 0.05% Tween 20 (vol/vol), PBS, and deionized water and then dried. Fluorescence intensities were detected using a ProScanArray HT microarray scanner (Perkin-Elmer). Image analyses were carried out us-ing ImaGene 8 image analysis software (BioDiscovery, El Segundo, CA). Please refer to Table S1 in the supplemental material for the specific gly-cans on the arrays.

Crystallization and data collection.Wash11 protein was subjected to thrombin cleavage and SEC (10). Purified trimeric protein was buffer exchanged into 10 mM Tris-HCl and 50 mM NaCl, pH 8.0, and concen-trated to 14 mg/ml for crystallization trials. Initial crystallization trials were set up using a Topaz free interface diffusion (FID) crystallizer system (Fluidigm Corporation, San Francisco, CA). Following optimization, dif-fraction quality crystals for Wash11 were obtained at 20°C using a sitting drop method with reservoir solution containing 0.2 M ammonium tar-trate dibasic and 20% (wt/vol) polyethylene glycol (PEG) 3350. Crystals were flash-cooled at 100 K, and data were collected at the Advanced Pho-ton Source (APS) beamline 22-ID at 100 K and processed with the Denzo-Scalepack suite (11). Statistics for data collection are presented inTable 2. Structure determination and refinement.The structure of Wash11 was determined by molecular replacement with Phaser (12) using the HA structure from influenza virus A/Darwin/2004/2009, PDB3M6S(HA1, 98% identity; HA2, 98% identity) (6) as the search model. Two noncrys-tallographic trimers occupy the asymmetric unit with an estimated sol-vent content of 62% based on a Matthews’ coefficient (Vm) of 3.29 Å3/Da. Rigid body refinement led to an overall R/Rfree of 21.1%/23.5%. The model was then “mutated” to the correct sequence, rebuilt by Coot (13), and refined with REFMAC using TLS (translation/libration/screw ro-tation) refinement (14). The final models were assessed using MolPro-bity (15). Statistics for data processing and refinement are presented in

Table 2.

Protein structure accession number.The atomic coordinates and structure factors of Wash11 HA are available from the RCSB PDB under accession code4LXV.

RESULTS

Stability of recombinant A(H1N1)pdm09 HAs isolated in 2009 and 2011.Our baculovirus expression system produces soluble recombinant HA (recHA) ectodomains. In order to achieve trim-erization in the absence of the transmembrane domain, the HA ectodomains are fused to a C-terminal cassette containing a

TABLE 1Sequence differences between the recombinant HA proteins used in this study

Strain/recHA GISAID accession no. Abbreviated name

Amino acid at residue no.:

83 97 143 185 197 203 321 374aor 47b 451aor 124b

A/California/7/2009 EPI177294 CA709 P D S S A S I E S

A/Texas/5/2009 EPI179187 Tex09 S V

A/Sydney/DD3-25/2010 EPI321067 Syd10 S T T V K N

A/Singapore/GP4369/2010 EPI385769 Sing10 S G T T V K N

A/Ontario/720545/2010 EPI307338 Ont10 S T T T V K N

A/Texas/1/2011 EPI310124 Tex11 S N T T V K N

A/Washington/5/2011 EPI310164 Wash11 S G T T T V K N

aHA2 position number in the HA0 form of HA.

b

HA2 position number in the HA1/HA2 form of HA.

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thrombin cleavage site, a trimerization domain (foldon) (9), and a His tag at the extreme C terminus to enable protein purification (10) (Fig. 1A). When HAs of seasonal as well as potentially

pan-demic viruses (7,8,16) are expressed in this system and analyzed

by SEC, they usually elute as high-molecular-mass species (⬃240

kDa based on the molecular mass standards with a retention

vol-ume of⬃11.6 ml), and their retention volume increases slightly

upon removal of the trimerization domain (foldon) and His tag following proteolytic cleavage with thrombin. This was demon-strated when the recHA from the pre-2009 seasonal H1N1 virus A/Brisbane/59/2007 (Brisb07) was analyzed. Following proteo-lytic digestion with thrombin, the Brisb07 recHA maintained a

trimeric state (Fig. 1B). In contrast, recHA from the A(H1N1)

pdm09 virus A/California/7/2009 (CA709) eluted as a species with

a much higher estimated molecular mass of⬃340 kDa. In

addi-tion, when its foldon was removed by thrombin cleavage, the

re-sulting species eluted as an⬃80-kDa monomer (with a retention

volume of⬃13.9 ml) (Fig. 1C). This inability to maintain a

trim-eric state was also echoed in previous structural work with a 2009 A(H1N1)pdm09 virus recHA from A/Darwin/2004/2009, and while the protein dissociated to monomers, it was still able to reassemble as trimers to form the crystals that enabled structural

analysis (6). The variability in retention time pre- and

postcleav-age, as well as the difference between what is expected from the protein sequence alone (186 kDa) and what is estimated by SEC, is dependent on the HA under study, and differences have been observed in other studies (data not shown). Results can be affected by the structural features of these proteins such as variations in glycosylation, different conformations in solution, and the

non-globular nature of the HA (Fig. 1A).

To address whether this structural instability is maintained for more recent viruses, we analyzed a number of recHA proteins

from recent A(H1N1)pdm09 viruses from 2010 and 2011, A/Syd-ney/DD3-25/2010 (Syd10), A/Singapore/GP4369/2010 (Sing10), A/Ontario/720545/2010 (Ont10), A/Washington/5/2011 (Wash11), and A/Texas/1/2011 (Tex11). Recombinant proteins were pro-duced in our baculovirus expression system, purified, and sub-jected to thrombin cleavage. Notably, untreated proteins eluted with a retention time similar to that of Brisb07; protease-treated

recHAs from both 2010 and 2011 eluted as trimers by SEC (Fig. 2).

Proteins had only minor increases in their retention volume after protease treatment. Proteins were also analyzed by dynamic light scattering (DLS) using a Dynapro plate reader. Results showed that while the 2009 strain CA709 had species with an estimated molecular mass almost twice that of what was expected following

TABLE 2Data collection and refinement statistics for the Wash11 crystal structure

Characteristic or statistic Resulta

Space group P21 21 21

Cell dimensions (Å) 73.24, 226.0, 271.0

Cell angles (°) 90, 90, 90

Resolution (Å) 50–3.0 (3.08–3.0)

Rsymor Rmerge 13.7 (59.7)

I/␴ 9.1 (1.3)

Completeness (%) 98.2 (97.8)

Redundancy 3.8 (3.5)

Refinement

Resolution (Å) 50–3.0 (3.08–3.0)

No. of reflections (total) 85,184

No. of reflections (test) 4,494

Rwork/Rfree 20.5/23.0

No. of atoms 23,736

RMSD, bond length (Å) 0.011

RMSD, bond angle (°) 1.521

MolProbitybscores

Favored (%) 95.5

Outliers (%); no. of residues 0.2; 6/2,940

PDB accession no. 4LXV

aNumbers in parentheses refer to the highest-resolution shell.

b

Data from reference15.

FIG 1RecHA stability as judged by SEC. (A) The HA ectodomain is expressed with a foldon trimerization domain at its C terminus and a thrombin cleavage site. After cleavage to remove the foldon, the ability of the recHA to maintain a trimer is assessed. (B and C) Pre-2009 H1 recHA (Brisb07) (B) was compared with an early A(H1N1)pdm09 CA709 protein (C). Graphs represent the elu-tion profiles of pretreated (green) and post-protease-treated (red) HAs from the SEC column, plotted as milliabsorbance units (mAU) at 280 nm against retention volume (ml).

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exposure to trypsin, all 2010 and 2011 A(H1N1)pdm09 recHAs resulted in species as trimers, while the 2009 strains (CA709 and Tex09) resulted in species that had a calculated molecular mass

close to that expected for a monomer (Fig. 3A). SDS-PAGE also

revealed a band smaller than the expected size for an HA2 (Fig.

3B), in agreement with increased protease susceptibility, as

re-ported recently for recombinant pandemic HA vaccines (17).

These findings highlight the fact that more recent A(H1N1) pdm09 recHA trimers are more stable than their early 2009 coun-terparts.

Sequence differences between early 2009 and recent 2010-2011 recombinant A(H1N1)pdm09 virus HAs.To identify the residues responsible for the improved stability of recent HA trim-ers, amino acid sequences for those HAs screened were aligned

and compared to CA709, the current vaccine strain (Table 1; see

Fig. S1 in the supplemental material). Results revealed a total of nine residue differences among the five strains analyzed, with six of the nine sequence differences (Pro83Ser, Ser185Thr, Ser203Thr, Ile321Val, Glu374Lys, and Ser451Asn) being con-served between all the 2010 and 2011 recHAs studied. The other three differences were unique to specific virus recHAs (Asp97Asn in Tex11; Ser143Gly in Sing10 and Wash11; and Ala197Thr in Ont10 and Wash11) (see Fig. S1 in the supplemental material).

From the SEC results (Fig. 2), only the Asp97Asn unique

differ-ence of Tex11 (compared to Syd10) appeared to produce more monomeric species when the foldon was removed and thus may have a negative contribution to trimer stability.

Indeed, since 2009, mutations at various locations in the HA have been under different selective pressures. Three changes, Pro83Ser, Ser203Thr, and Ile321Val, were introduced very early

on in the pandemic and have been present in⬎97% of all sequences

deposited in the GISAID EpiFlu Database (http://platform.gisaid

.org) (Table 3). The three substitutions, Asp97Asn, Ser143Gly, and Ala197Thr, have all increased with time but were still present in

⬍50% (49%, 45%, and 46%, respectively) of all A(H1N1)pdm09

HA sequences deposited in 2012. The remaining three changes (Ser185Thr, Glu374Lys, and Ser451Asn) were present in 2009 at low levels (0, 14, and 1%, respectively) but have increased with

time to be present in ⬎93% of all sequences. Indeed, for

FIG 2Stability of recombinant HAs from recent A(H1N1)pdm09 viruses assessed by SEC. Proteins were digested with thrombin, and their ability to maintain trimeric structures in solution was assessed by SEC. On the top left chromatogram, the profile for molecular mass standards (MM Stds; in kDa) is shown in black, and the regions where trimers and monomers elute are indicated. Graphs are plotted as forFig. 1, and retention volumes for peak maxima are also indicated for pretreatment (green) and post-protease treat-ment (red).

FIG 3Stability of recombinant HAs from recent A(H1N1)pdm09 viruses assessed by dynamic light scattering and SDS-PAGE. (A) The calculated mo-lecular mass of each recHA in solution after overnight incubation at room temperature with (light gray) and without (dark gray) trypsin. Results are based on the estimated hydrodynamic radius, particle density, and conforma-tion model from the regularizaconforma-tion fit calculated by Dynamics 7.0 software. Molecular mass estimates were calculated using intensity-weighted size distri-bution analysis. The upper dashed lines denote the theoretical molecular mass of the Wash11 trimer before/after trypsin treatment, while the lower dashed lines denote the theoretical molecular mass of a monomer before/after trypsin. Theoretical molecular masses were calculated based on all six potential glyco-sylation sites being occupied with paucimannose (⬃911 Da per glycan). (B) Pre- and post-trypsin-treated samples were also analyzed by SDS-PAGE. Re-sults highlight an increased protease sensitivity of the HA2 domain from 2009 strains compared to the later 2010-2011 strains.

TABLE 3Evolution of residue differences highlighted in this study since 2009

Mutation

% of sequenced H1pdm HAs with the specific mutation in the GISAID database for yr:

2009 2010 2011 2012

Pro83Ser 99 98 99 99

Asp97Asna 3 16 46 49

Ser143Gly 1 2 27 45

Ser185Thr 0 15 66 94

Ala197Thr 2 7 32 46

Ser203Thr 81 98 99 99

Ile321Val 96 97 99 98

Glu374Lys 14 60 92 100

Ser451Asn 1 15 65 93

aAmong the virus HAs studied here, Asp97Asn was present only in Tex11.

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Glu374Lys, this position is currently a lysine in 100% of all

se-quences deposited in 2012 (Table 3).

Structural analysis of Wash11 recHA and the effect of HA1 residue differences on RBS function.Wash11 was selected for structural analysis to provide a structural explanation for why these recent recHAs are more stable. Using X-ray crystallography, the structure of the recHA trimer from the Wash11 A(H1N1)

pdm09 virus was determined to 3.0-Å resolution (Table 2). The

overall structure of Wash11 is similar to previously reported 2009 pandemic recHA structures, with a globular head containing the receptor binding site (RBS) and a vestigial esterase domain and a membrane-proximal domain with its distinctive, central helical

stalk and HA1/HA2 cleavage site (Fig. 4A). Superimposition of the

Wash11 HA1/HA2 heterodimer onto the heterodimer of

A/Dar-win/2001/2009 (PDB accession number3M6S) gave a root mean

square deviation (RMSD) of only 0.41 Å for all C␣atoms in each

structure. Although six asparagine-linked glycosylation sequons are present in the Wash11 recHA monomer, interpretable

elec-tron density for only one or two N-acetyl glucosamines was ob-served at three of these sites (Asn11, Asn87, and Asn276).

As with other HAs, the Wash11 RBS is composed of three structural elements: a 180 helix (residues 183 to 193), a 220 loop (residues 219 to 227), and a 130 loop (residues 131 to 136), while other highly conserved residues, Tyr91, Trp150, His180, and

Tyr192, form the base of the pocket (Fig. 4B). Although not all

within the RBS, sequence differences (residues 143, 185, 197, and 203) between early 2009 and recent 2010-2011 A(H1N1)pdm09 virus HAs are in close proximity. To rule out any effect on receptor binding, we used glycan microarrays to compare the receptor specificity of the CA709 vaccine strain HA with that of the more recent Wash11 HA, which has amino acid substitutions at all four

of these positions (Fig. 4CandD; see Table S1 in the supplemental

material). Both recombinant HAs bound to an␣2-6

sialylated-sulfated N-acetyllactosamine structure (glycan 41),␣2-6

sialy-lated biantennary glycans (glycans 44 to 47), which are typically

found on membrane glycoproteins (18), an␣2-6 sialylated

tri-N-FIG 4Structural overview and receptor binding properties of Wash11 HA trimer. (A) One monomer is highlighted, with the HA1 chain in green and the HA2 chain in cyan. The locations of the glycosylation sites are labeled. (B) RBS of Wash11 HA with the three structural elements comprising this binding site, the 220 loop, the 130 loop, and the 180 helix, colored blue, purple, and yellow, respectively. Glycan microarray analysis of recombinant CA709 HA (C) and Wash11 HA (D) reveal similar binding profiles. Colored bars highlight glycans that contain2-3 sialosides (blue) and2-6 sialosides (red),2– 6/2-3 mixed sialosides (purple), N-glycolyl sialosides (green),␣2-8 sialosides (brown),␤2-6 and 9-O-acetyl sialosides (yellow), and nonsialoside glycans (gray). Error bars reflect the standard errors in the signal for six independent replicates on the array. Structures of each of the numbered glycans can be found in Table S1 in the supplemental material.

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acetyllactosamine glycan in which the two lactosamines proximal to the reducing end are fucosylated (glycan 59 in Table S1 in the

supplemental material), as well as to long, linear␣2-6 sialylated

mono-, di-, and tri-N-acetyllactosamines (glycans 52 and 53, 56 to 59, and 62 and 63), some of which were detected in N-glycans of

cultured human bronchial epithelial cells (19). In summary, the

two recHAs had equivalent glycan binding profiles, and thus, res-idues 143, 185, 197, and 203 do not influence the receptor binding characteristics of these recHAs.

Structural analyses and effects of HA2 mutations Glu374Lys and Ser451Asn on the stability of A(H1N1)pdm09 virus HA trimers. Structural analysis of nine amino acid substitu-tions (Pro83Ser, Asp97Asn, Ser143Gly, Ser185Thr, Ala197Thr, Ser203Thr, Ile321Val, Glu374Lys, and Ser451Asn) also revealed interesting features. Only 5 of these substitutions (Pro83Ser, Ser143Gly, Ser185Thr, Ile321Val, and Glu374Lys) have solvent-exposed side chains on the trimer surface, while only one, Ser185Thr, is in one of the four H1N1 antigenic sites (Ca, Cb, Sa,

and Sb) (20–23), although this number increases to 3 (Pro83Ser,

Ser143Gly, and Ser185Thr) if one considers sites complementary to those identified for H3N2 viruses (see Fig. S1 in the

supplemen-tal material) (23). Indeed, analysis of these residue differences on

the HA trimer structure revealed that 5 of these substitutions (Asp97Asn, Ala197Thr, Ser203Thr, Glu374Lys, and Ser451Asn) were either buried within the trimer or close to the interface be-tween monomers in the trimer (data not shown).

Further analyses (Fig. 5) also revealed that four of the nine

muta-tions (Asp97Asn, Ser143Gly, Ser185Thr, and Ile321Val) conferred no additional side chain interactions in the Wash11 structure, while an-other four (Pro83Ser, Ala197Thr, Ser203Thr, and Ser451Asn) intro-duced only intramolecular interactions, resulting in new potential hydrogen bonds and/or salt bridges to residues within the same monomer. For the adaptation at 451 (Ser451Asn), this was a little surprising since modeling suggested that the longer carboxamide side chain could allow for a possible intermolecular interaction with Glu459 on the adjacent monomer. However, although this is re-garded as an interface residue by the structural analysis software PISA

(24), there was no apparent intermolecular hydrogen bond identified

from the Wash11 structure. Thus, only one position, Glu374Lys, was identified that introduced a basic side chain, potentially forming a new salt bridge across the monomer interface to Glu21 on the

adja-cent chain (Fig. 5).

To study the effect of both HA2 mutations, Glu374Lys and Ser451Asn mutations were introduced onto the framework of the

2009 Tex09 HA backbone, which, like CA709, is unstable (Fig. 6).

Comparison of wild-type Tex09 recHA with the single and double mutations by SEC agreed with our structural analyses in that only the Glu374Lys mutation appeared to significantly stabilize and

maintain trimeric species following thrombin treatment (Fig. 6).

These findings were also confirmed by assessing the protease sen-sitivity of each recHA following thermal treatment. Results sug-gested that the Glu374Lys mutation specifically conferred confor-mational stability of the recHA trimer to protease sensitivity, even at 50°C, while the Ser451Asn mutation showed no improvement

to the overall stability of Tex09 (Fig. 7A).

RecHA stability was also assessed by thermal denaturation, pH/protease sensitivity assays, DLS, and SEC-MALS. In PBS, Tex09 recHA clearly aggregates into large complexes at a lower

temperature than either Tx09-Glu374Lys or Wash11 (Fig. 7B).

Protease sensitivity assays following exposure to increasing acidity

were also used to assess the conformational stability of these re-cHAs at different pH; while the Wash11 recHA was stable at pH 5.0, the Tex09 recHA was unstable below pH 5.2, and only the Glu374Lys mutation was able to improve stability at the lower pH (Fig. 7C). DLS analysis was also consistent with other results in that only post-trypsin-treated samples with a substitution at resi-due 374 yielded material with a calculated molecular mass

approx-imating that of a trimer (Table 4). While DLS results were

com-parable to SEC-fast protein liquid chromatography (SEC-FPLC) results, molecular mass calculations were higher than the pre-dicted values. In order to dismiss the possibility that the recHAs were higher-order multimers rather than true trimers, SEC-MALS was performed to determine absolute molecular mass measure-ments. Results for SEC-MALS yielded values closer to the

ex-pected values based on sequence alone (Table 4).

From these results, it is possible that the Tex09 recHA protein, in solution, is a loose trimer held together primarily by the trim-erization foldon domain. This could lead to a species with a higher molecular mass by SEC and a larger hydrodynamic radius and hence a higher calculated molecular mass by DLS. When the

FIG 5Molecular interactions of the Wash11 sequence differences, compared to the CA709 vaccine strain. Inter- and intramolecular interactions of the nine sequence differences are highlighted. Positions that have no interactions (res-idues Asn97, Gly143, Thr185, and Val321) have no border. Those that intro-duce intramolecular interactions (Ser83, Thr197, Thr203, and Asn451) are highlighted with brown borders, and those that have intermolecular interac-tions (Lys374) have red borders. Posiinterac-tions with residue differences between CA709 and Wash11 are listed with the CA709 residue listed first. The position highlighted with an asterisk is the difference between CA709 and Tex11. All the figures were generated and rendered with the use of MacPyMOL (38).

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Glu374Lys substitution is introduced, it improves interaction be-tween the HA2 stalk, thus reducing the measured hydrodynamic radius/molecular mass ratio. In summary, the presence of Lys374 enhanced the ability of the mutant Tex09 ectodomain trimer to withstand changes in both heat and acidity to levels equivalent to those of the Wash11 recHA. This residue was, therefore, identified as the predominant factor in mediating the stability of the A(H1N1)pdm09 virus HA trimer.

DISCUSSION

The 2009 A(H1N1)pdm09 pandemic demonstrated that while the public health response was robust, the virus itself caused

unfore-seen bottlenecks in the vaccine production pipeline. Although an unstable HA may not fully explain poor virus growth, it may have contributed to other observed problems, such as delays in the availability of potency reagents and a lower HA antigen yield dur-ing vaccine production, as well as reduced shelf lives of some

com-mercial A(H1N1)pdm09 monovalent pandemic vaccines (25–

29). Interestingly, recent studies reported mutations in A(H1N1)

pdm09 vaccine candidate viruses that affected antigen stability

and yield (29,30). The mutations identified were all in the HA1

globular head, and although differences in yield/stability were ob-served in the viruses studied, it was difficult to draw any firm conclusions as to what exact effect these mutations were having, since changes upon egg adaptation, stability, and/or glycosylation were all potential mechanisms. The HA trimer is important for virus entry and infection, and in order to function optimally, it must remain in trimeric form. Recent structural studies with the H5N1 HA from a ferret-adapted airborne transmissible virus illustrates the importance of the trimer interface. Zhang et al. showed that an H5 Tyr110 substitution generates a stabilizing hydrogen bond with the Asn413 of the adjacent monomer,

whereas the wild-type His110 cannot do so (31).

To focus specifically on an HA’s inability to maintain a trimeric form, we cloned/expressed a number of A(H1N1)pdm09 virus HAs using a baculovirus expression system. The results presented here highlight the fact that more recent A(H1N1)pdm09 virus HAs appear significantly more stable as a trimer than those from 2009. Amino acid sequence and structural analyses identified a natural mutation at position 374 as a major explanatory factor for this phenotypic change. Interestingly, the Glu374Lys substitution had already been highlighted in a number of reports but was not

reported to have any phenotypic or clinical relevance (6,32–34).

In addition, while this paper was under review, a report that also highlighted this substitution as important for HA stability in

A(H1N1)pdm09 viruses was published (35). From the structure

of the Wash11 HA, this HA2 residue is positioned in an alpha helix at the monomer-monomer interface in the trimeric structure. The nearby Asn377 helps form a network of hydrogen bonds with the HA1, 15-25-loop, of the neighboring monomer. As a Glu, position

374 does not appear to interact at this interface (Fig. 8A).

How-ever, when mutated to a Lys, the amino group of the side chain can now extend over to form a potential salt bridge with Glu21 in the

neighboring HA1 loop (Fig. 8B). While this single change appears

to be innocuous, it has a profound effect on maintaining the HA trimer, as shown by its introduction on the framework of an early

2009 HA (Fig. 6). A comparison of this region to existing

struc-tures of other human virus subtypes highlights that this specific trimer stabilization mechanism appears to be unique to A(H1N1) pdm09 virus HAs. Indeed, the structurally equivalent residue at position 374 in the majority of pre-2009 H1N1, H2, and H5

se-quences is a glycine, while H3 HAs have a Gln (Fig. 9).

Interestingly, a comparison of salt bridges at the monomer-monomer interface of A(H1N1)pdm09 HAs with those from other human virus subtypes reveals a number of interesting

fea-tures (Fig. 10). Each HA subtype has a network of salt bridges

positioned at the top of the long parallel␣-helices that make up

the distinctive HA2 triple-stranded coiled coil. These complex salt bridges link together two neighboring HA2 domains with an HA1 that helps maintain the trimeric, metastable form. For H1 and H2 HAs, there are another 4 or 5 simple salt bridges that are

posi-tioned almost at regular intervals (Fig. 10) and extend over from

FIG 6Stability of recombinant Tex09 mutants. Mutations at positions 374 (E374K) and 451 (S451N) were introduced onto an unstable Tex09 framework to assess their ability to stabilize the HA trimer. Mutants with the E374K substitution are comparable to the Wash11 HA shown inFig. 2. The top chromatogram pro-files molecular mass standards of known sizes (in kDa) and highlights the regions where trimers and monomers elute. Graphs were plotted as forFig. 1.

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the long HA2␣-helix to its neighboring monomer in the trimeric structure. Three of these bonds are conserved across the HA struc-tures. It has been hypothesized that both intramonomer and in-termonomer salt bridges in the membrane-distal domain of the HA may contribute to HA pH-dependent stability during

infec-tion (36). Indeed, for pre-2009 H1 HAs, there are an additional

two salt bridges in the globular head that extend across the HA1 interface. However, for H2 and H3 subtype HAs, additional salt

bridges are instead situated at the base of the HA2, below the fusion peptide pocket.

From our studies (Fig. 8AandB) and those of others (17), the

ability of an recHA ectodomain to maintain a trimeric state has a

FIG 7Effects of temperature and low pH on the stability of recHA mutants. The stability of Tex09 mutants was assessed by protease sensitivity at 37°C and 50°C (A), aggregation with increasing temperature (B), and low pH (C). Results were compared to those obtained with wild-type Tex09 and Wash11 proteins.

TABLE 4Molecular mass determinations for recHAs from different analysis methods used in this study (SEC-FPLC, DLS, and SEC-MALS)

Protein

Protease treatment

Molecular mass (kDa)

Predicteda SEC-FPLC DLS SEC-MALS

Tex09 ⫺ 200 340 372 212

⫹ 187 84 98 65

Tex09-E374K ⫺ 200 216 258 205

⫹ 187 211 179 191

Wash11 ⫺ 202 187 272 183

⫹ 189 184 189 175

aValues for predicted molecular masses are based on the protein sequence of the

secreted recombinant protein and assuming that all 6 potential glycosylation sites per HA monomer are occupied by a paucimannose glycan.

FIG 8Intersubunit interactions around residue 374. Interactions around the region of residue 374 were structurally assessed using a previously published 2009 A(H1N1)pdm09 virus HA from A/California/04/2009 (PDB accession number3LZG) (A) and compared to those of our Wash11 model (B). The HA1 subunit is labeled as chain C, while neighboring HA2 subunits are labeled B and D. The numbering follows that of the HA0 form of the mature protein. Positions labeled with an asterisk (*) and colored yellow indicate potential glycosylation sites.

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profound effect on the protein’s stability to temperature, pH, and protease susceptibility. Results reveal the HA2 to be particularly susceptible to protease cleavage. Indeed, Feshchenko et al. identi-fied specific protease cleavage sites (Lys402, Arg403, and Lys411) that are positioned on the loop at the top of the long parallel

␣-helices that make up the HA2 triple-stranded coiled coil (17).

As a trimer, these sites should not be exposed to proteases such as trypsin. However, if reduced stability, as reported in these early A(H1N1)pdm09 virus HA trimers, resulted in a tendency of the monomers to separate, then these internal residues could become exposed. In the context of working with the virus in the laboratory and the vaccine-manufacturing pipeline, as well as the long-term storage of vaccines, having such a loosely held together HA trimer

could result in gradual HA degradation due to random protease cleavage. In addition, once extracted, concentrated, and processed to the rosette forms present in the vaccine, exposing the internal interfaces of the HA could expose hydrophobic patches that could also increase aggregation and thus reduce vaccine yield and effi-cacy with time.

With respect to A(H1N1)pdm09 virus function, one could ar-gue that this was not a major concern for viral fitness since this strain was the cause of the first pandemic in the 21st century. That being said, the virus may not have adapted optimally for transmis-sion within the human population. The stabilizing Glu374Lys HA mutation was first seen in a Moroccan virus in May 2009. In June, it was present in sequenced viruses from China, India, and

Eng-FIG 9The equivalent region around residue 374, as shown inFig. 8, was generated for other HA subtypes. (A) H1:A/South Carolina/1/1918 (PDB accession number1RD8); (B) H1:A/Solomon Islands/3/2006 (PDB accession number3SM5); (C) H2:A/Japan/305/1957 (PDB accession number2WRC); (D) H3:A/Hong Kong/19/1968 (PDB accession number2HMG); (E) H5:A/Vietnam/1023/2004 (PDB accession number2FK0). The HA1 subunit is labeled as chain C, while neighboring HA2 subunits are labeled B and D. The numbering follows that of the HA0 form of the mature protein. Positions labeled with an asterisk (*) and colored yellow indicate potential glycosylation sites.

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land, and by July, it was seen in viruses from such geographically diverse areas as Singapore, Latvia, Canada, Nicaragua, and the states of Washington and New York. Indeed, by October 2009, 24% of the deposited HA sequences that month possessed this mutation, and by 2012, 100% of sequenced virus HAs possessed a

Lys at this position (Table 3).

Almost 5 years after the first A(H1N1)pdm09 pandemic vi-ruses were identified in humans, currently circulating vivi-ruses are

still antigenically homogeneous (37). However, as the HA

contin-ues to circulate in the human population, its antigenic sites con-tinue to be targeted by the human antibody response. When the need arises to update the composition of the H1N1pdm09 vac-cine, an improved HA stability could potentially help mitigate the risk of manufacturing problems.

ACKNOWLEDGMENTS

This work was funded by the Centers for Disease Control and Prevention and the HHS Influenza Vaccine Manufacturing Improvement Initiative. We thank the WHO Global Influenza Surveillance and Response Sys-tem (GISRS) for providing the virus sequences used to generate the HA expression clones described here, as well as all submitting laboratories who have deposited their A(H1N1)pdm09 HA sequences to the GISAID database.

Use of the Advanced Photon Source at Argonne National Laboratory was supported by the U.S. Department of Energy, Office of Science, Office of Basic Energy Sciences, under Contract No. DE-AC02-06CH11357.

We thank the staff of SER-CAT sector 22 for their help with data collection.

Glycan microarray slides were produced under contract for the Cen-ters for Disease Control using a glycan library generously provided by the Consortium for Functional Glycomics funded by National Institute of General Medical Sciences Grant GM62116.

The findings and conclusions in this report are those of the authors and do not necessarily represent the views of the Centers for Disease Control and Prevention or the Agency for Toxic Substances and Disease Registry.

Alexander Klimov passed away on 5 February 2013.

Author contributions: H.Y., D.A.S., and J.S. conceived and designed the experiments. H.Y., J.C.C., Z.G., D.A.S., and P.J.C. performed the

ex-periments. H.Y., J.C.C., Z.G., P.J.C., D.A.S., R.O.D., N.J.C., J.M.V., and J.S. analyzed the data.

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Figure

TABLE 1 Sequence differences between the recombinant HA proteins used in this study

TABLE 1

Sequence differences between the recombinant HA proteins used in this study p.2
TABLE 2 Data collection and refinement statistics for the Wash11crystal structure

TABLE 2

Data collection and refinement statistics for the Wash11crystal structure p.3
FIG 1 RecHA stability as judged by SEC. (A) The HA ectodomain is expressedwith a foldon trimerization domain at its C terminus and a thrombin cleavagesite

FIG 1

RecHA stability as judged by SEC. (A) The HA ectodomain is expressedwith a foldon trimerization domain at its C terminus and a thrombin cleavagesite p.3
FIG 3 Stability of recombinant HAs from recent A(H1N1)pdm09 virusesassessed by dynamic light scattering and SDS-PAGE

FIG 3

Stability of recombinant HAs from recent A(H1N1)pdm09 virusesassessed by dynamic light scattering and SDS-PAGE p.4
FIG 2 Stability of recombinant HAs from recent A(H1N1)pdm09 virusesassessed by SEC. Proteins were digested with thrombin, and their ability tomaintain trimeric structures in solution was assessed by SEC

FIG 2

Stability of recombinant HAs from recent A(H1N1)pdm09 virusesassessed by SEC. Proteins were digested with thrombin, and their ability tomaintain trimeric structures in solution was assessed by SEC p.4
TABLE 3 Evolution of residue differences highlighted in this study since2009

TABLE 3

Evolution of residue differences highlighted in this study since2009 p.4
FIG 4 Structural overview and receptor binding properties of Wash11 HA trimer. (A) One monomer is highlighted, with the HA1 chain in green and the HA2(purple), N-glycolyl sialosides (green),standard errors in the signal for six independent replicates on th

FIG 4

Structural overview and receptor binding properties of Wash11 HA trimer. (A) One monomer is highlighted, with the HA1 chain in green and the HA2(purple), N-glycolyl sialosides (green),standard errors in the signal for six independent replicates on th p.5
FIG 5 Molecular interactions of the Wash11 sequence differences, comparedto the CA709 vaccine strain

FIG 5

Molecular interactions of the Wash11 sequence differences, comparedto the CA709 vaccine strain p.6
FIG 6 Stability of recombinant Tex09 mutants. Mutations at positions 374where trimers and monomers elute

FIG 6

Stability of recombinant Tex09 mutants. Mutations at positions 374where trimers and monomers elute p.7
FIG 8 Intersubunit interactions around residue 374. Interactions around theHA1 subunit is labeled as chain C, while neighboring HA2 subunits are labeledB and D

FIG 8

Intersubunit interactions around residue 374. Interactions around theHA1 subunit is labeled as chain C, while neighboring HA2 subunits are labeledB and D p.8
FIG 7 Effects of temperature and low pH on the stability of recHA mutants. The stability of Tex09 mutants was assessed by protease sensitivity at 37°C and 50°C(A), aggregation with increasing temperature (B), and low pH (C)

FIG 7

Effects of temperature and low pH on the stability of recHA mutants. The stability of Tex09 mutants was assessed by protease sensitivity at 37°C and 50°C(A), aggregation with increasing temperature (B), and low pH (C) p.8
TABLE 4 Molecular mass determinations for recHAs from differentanalysis methods used in this study (SEC-FPLC, DLS, and SEC-MALS)

TABLE 4

Molecular mass determinations for recHAs from differentanalysis methods used in this study (SEC-FPLC, DLS, and SEC-MALS) p.8
FIG 9 The equivalent region around residue 374, as shown innumber Fig. 8, was generated for other HA subtypes

FIG 9

The equivalent region around residue 374, as shown innumber Fig. 8, was generated for other HA subtypes p.9
FIG 10 Interface analysis of published virus HA structures. Analyses on Wash11 and early California/4/2009 (PDB accession number 3LZG) (H1N1)pdm09and H3:A/Hong Kong/4443/2005 (PDBvirus HAs were performed using the Protein Interfaces, Surfaces and Assemblie

FIG 10

Interface analysis of published virus HA structures. Analyses on Wash11 and early California/4/2009 (PDB accession number 3LZG) (H1N1)pdm09and H3:A/Hong Kong/4443/2005 (PDBvirus HAs were performed using the Protein Interfaces, Surfaces and Assemblie p.10

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