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. Author manuscript; available in PMC: 2015 Feb 1.
Published in final edited form as: Virology. 2013 Dec 22;0:71–83. doi: 10.1016/j.virol.2013.11.038

The Roles of Hemagglutinin Phe-95 in Receptor Binding and Pathogenicity of Influenza B Virus

Fengyun Ni 1,2, Innocent Nnadi Mbawuike 3, Elena Kondrashkina 4, Qinghua Wang 1,*
PMCID: PMC4002292  NIHMSID: NIHMS552187  PMID: 24503069

Abstract

Diverged ~4,000 years ago, influenza B virus has several important differences from influenza A virus, including lower receptor-binding affinity and highly restricted host range. Based on our prior structural studies, we hypothesized that a single-residue difference in the receptor-binding site of hemagglutinin (HA), Phe-95 in influenza B virus versus Tyr-98 in influenza A/H1~H15, is possibly a key determinant for the low receptor-binding affinity. Here we demonstrate that the mutation Phe95→Tyr in influenza B virus HA restores all three hydrogen bonds made by Tyr-98 in influenza A/H3 HA and has the potential to enhance receptor binding. However, the full realization of this potential is influenced by the local environment into which the mutation is introduced. The binding and replication of the recombinant viruses correlate well with the receptor-binding capabilities of HA. These results are discussed in relation to the roles of Phe-95 in receptor binding and pathogenicity of influenza B virus.

Keywords: hemagglutinin, host range, influenza virus, pathogenicity, receptor-binding affinity

Introduction

Influenza A and B viruses remain a major cause of morbidity and mortality in humans. The entry of influenza virus into host cells is mediated by hemagglutinin (HA), a major surface glycoprotein of influenza A or B virus, via two distinct steps. The fist step is the binding of HA to cell-surface sialic acid receptors at its distal globular domain that initiates the endocytosis process. The second step involves an extremely large-scale conformational change of HA at its proximal stem domain that causes the fusion of endosomal membrane with viral envelope, thus releasing the viral genetic materials into host cytosols for replication.

Influenza A virus infects a wide range of hosts including humans, avians, equines and swine (Skehel and Wiley, 2000; Steinhauer, 2010; Wiley and Skehel, 1987). Aquatic birds are the natural reservoir for influenza A virus where 16 serologically different HA subtypes have been discovered (Fouchier et al., 2005; WHO-MEMORANDUM, 1980), and a new subtype H17 has been recently found from little yellow-shouldered bats (Tong et al., 2012). Among them, only influenza A viruses of H1, H2, and H3 HA subtypes have been established in humans and caused seasonal influenza epidemics and all known pandemics, while other subtypes such as H5, H6, H7 and H9 are believed to have potential for human pandemics (Yen and Webster, 2009). The ability of influenza A virus to efficiently infect human population is correlated with the high affinity of HA to Neu5Acα(2,6)Gal sialic acid receptors, also known as human receptors, with a characteristic umbrella-like structural topology (Srinivasan et al., 2008; Viswanathan et al., 2010a). The other type of receptors, Neu5Acα(2,3)Gal, as frequently found in bird intestines, are referred to as avian receptors. The receptors bind to a shallow receptor-binding site at the globular domain of HA formed by three key residues at the base that are absolutely conserved among influenza A/H1~H15 HA glycoproteins: Tyr-98, Trp-153, and His-183 (A/H3 HA numbering) (Wilson, Skehel, and Wiley, 1981).

Different from influenza A virus, influenza B virus does not have subtypes. Instead, currently circulating influenza B virus belongs to either Yamagata lineage or Victoria lineage that diverged from each other in early 1970 (Chen et al., 2007; Shen et al., 2009). Most strikingly, influenza B virus has a very limited host range, circulating almost exclusively among humans and seals (Osterhaus et al., 2000). In addition, influenza B viruses were found to have overall lower receptor-binding affinities than influenza A viruses (Matrosovich et al., 1993).

Our group has determined the crystal structures of influenza B virus HA in various states (Ni, Kondrashkina, and Wang, 2013; Wang et al., 2008; Wang et al., 2007). In marked contrast to influenza A/H1~H15 HA that have Tyr-98, influenza B virus HA has Phe-95 instead (based on the numbering of influenza B/Hong Kong/8/73 (B/HK/73) HA) (Wang, 2010; Wang et al., 2008; Wang et al., 2007). This is similar to the Phe-98 residue observed in influenza A/H16~H17 HA proteins (Fouchier et al., 2005; Sun et al., 2013; Tong et al., 2012; Zhu et al., 2013). When Tyr-98 is present, the hydroxyl oxygen atom on its side chain makes one hydrogen bond with His-183 to stabilize the base of the receptor-binding site and two additional hydrogen bonds with the Sia-1 moiety of the bound receptors (Weis et al., 1988). The absence of the hydroxyl oxygen atom on the side chain of Phe-95 in influenza B virus HA results in the loss of all three hydrogen bonds (Wang et al., 2007), and is likely responsible for the low binding affinity of influenza B virus.

In this study, we examined the effects of the Phe95→Tyr mutation at the levels of both recombinant protein and virus. Although naturally occurring influenza B viruses generally have Asn and Ser at HA1 194 and 196 respectively, making a glycosylation site at Asn194, this glycosylation site was frequently abolished in egg-adapted variants (Gambaryan, Robertson, and Matrosovich, 1999; Oxford et al., 1991; Oxford et al., 1990; Robertson et al., 1990; Robertson et al., 1985; Saito et al., 2004; Schild et al., 1983), and sporadic field isolates (Ikonen et al., 2005; Nakagawa et al., 2000), as a result of mutations at HA1 194 or 196 or both. Since this glycosylation site is located on the 190-helix that forms the upper edge of the receptor-binding site (Ni, Kondrashkina, and Wang, 2013; Wang et al., 2007), we tested the mutation Phe95→Tyr on influenza B virus HA with a glycosylation at HA1 194 (wild type) or without (Asn194→Asp). The mutation was assessed on two different backgrounds (B/Yamagata/73 and B/Lee/40 HA). Our data demonstrate that the mutation Phe95→Tyr restores all three hydrogen bonds made by Tyr-98 in influenza A/H3 HA and has the potential to enhance receptor binding. However, the complete realization of this potential is context dependent. The binding and replication of the recombinant viruses correlate well with the receptor-binding capabilities of their corresponding HA proteins. These results are discussed in relation to the roles of HA Phe-95 in receptor binding and pathogenicity of influenza B virus.

Materials and Methods

Cells

All cells were maintained at 37°C in 5% CO2 incubator. 293T, A549, BHK21, CV-1 and Vero cells were maintained in Dulbecco’s Modified Eagle Medium (DMEM) with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin. MDCK and MDCK1 cells were maintained in Eagle’s minimal essential medium (EMEM) with 10% FBS and 1% penicillin-streptomycin. All cell lines were purchased from ATCC.

Cloning, expression and purification of influenza B virus HA and its mutants

The HA cDNA of influenza B/Yamagata/73 and B/Lee/40 viruses are kind gifts from Dr. Peter Palese and Dr. Yoshihiro Kawaoka, respectively. Their ectodomains (HA1 1~342 with HA2 1~176) were separately cloned to pRB21 plasmid (a kind gift from Dr. Bernard Moss) to allow the expression in vaccinia systems (Blasco and Moss, 1995). Site-directed mutagenesis was used to make the Phe95→Tyr, Asn194→Asp and Phe95→Tyr/Asn194→Asp mutants (Agilent Technologies). All the mutant constructs were fully sequenced. The recombinant vaccinia viruses were generated by infecting CV-1 cells with vRB12 and then transfected with pRB21 vector containing HA or its mutants (Blasco and Moss, 1995). Protein expression and purification followed the same protocol as previously published (Ni, Kondrashkina, and Wang, 2013).

Deglycosylation of HA

Purified HA protein was first denatured in glycoprotein denaturing buffer at 95°C for 10 minutes. The denatured protein was treated with peptide-N-glycosidase F (PNGase F) (New England Biolabs) with 1% NP-40 at 37°C for 1 hour to cleave the N-linked glycans. The reaction products were visualized by SDS-PAGE.

Glycan microarray analysis

The binding profiles of purified HA and its mutants to different glycans were determined by the Consortium of Functional Glycomics (www.functionalglycomics.org) using version 4.1 glycan microarray, which contains 465 glycan structures in replicates of six. The HA proteins were first incubated with anti-penta His antibody (Qiagen) and Alexa488-labeled anti-mouse IgG (Invitrogen) on ice for 30 minutes. The final concentration of HA proteins used in the assay was 200 μg/mL. The protein complex was then applied to a freshly hydrated glycan microarray under a cover slip and the fluorescence signals were recorded. As previously demonstrated (Stevens et al., 2006), the success of the glycan array depends on the use of the secondary fluroscent anti-mouse IgG antibody that can bind up to four HA trimers (thus having up to 12 glycan binding sites). This cooperative nature of the interaction enhances the binding avidity and the binding signals. The glycans with a signal of 5,000 relative fluorescence unit (RFU) or higher for any of HA or its mutants are shown in Fig. 2 and listed in Supplemental Table 1.

Fig. 2. The mutation Phe95→Tyr promotes the binding of influenza B/Yamagata/73 HA protein to synthetic glycans in glycan microarray assays.

Fig. 2

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The results of glycan microarray assays are shown for (a) wild type, (b) Asn194→Asp, (c) Phe95→Tyr and (d) Phe95→Tyr/Asn194→Asp. Please refer to Supplemental Table 1 for the full name of each glycan.

Dose-dependent glycan binding assay

To characterize the receptor binding affinities of HA and its mutants, biotinylated 3′SLN-LN and 6′SLN-LN from the Consortium of Functional Glycomics were used in the dose-dependent glycan binding assay (Srinivasan et al., 2008). LN represents lactosamine (Galβ1-4GlcNAc), 3′SLN and 6′SLN represent Neu5Acα(2,3) and Neu5Acα(2,6) linked to LN, respectively. The different glycans were first diluted to 2.4 μM and loaded to the streptavidin-coated high binding capacity 384-well plates (Pierce), followed by incubation at 4°C overnight. Excessive glycans were removed by washing with phosphate-buffered saline (PBS) for three times. The pre-complex of HA, mouse anti-His antibody (0.2 mg/mL, Sigma) and anti-mouse-IgG antibody (2 mg/mL, Sigma) was prepared at a molar ratio of 4:2:1. The mixture was incubated on ice for 20 minutes, and the pre-complex was diluted to the expected concentration with 1% bovine serum albumin (BSA) in PBS. Each glycan-coated well was then loaded with 50 μL of the HA pre-complex and incubated at room temperature for 2 hours followed by wash with 0.05% Tween-20 in PBS to remove any unbound pre-complex. The Amplex Red Peroxidase Assay kit (Invitrogen) was used to measure the binding signal based on the horseradish peroxidase (HRP) activity. Negative controls, where no pre-complexes were loaded in those wells, were included for each glycan and the assays were performed in triplicates. The linearized Hill equation was used to fit the data (Srinivasan et al., 2008).

Cell-based ELISA assay

In order to compare the binding of recombinant HA and its various mutants to different types of cells, cell-based ELISA assay was carried out with three cultured cell lines: MDCK1, BHK21 and Vero. The confluent cell monolayer in white 96-well Falcon plate (BD Biosciences) was first washed with Dulbecco’s PBS (DPBS, Invitrogen) and various amounts of HA or its mutants diluted with 1% BSA in DPBS was added to different wells, complete DMEM (0.2% BSA, 25 mM Hepes in DMEM) was added to bring all the wells to the same volume. The plate was incubated at room temperature for 1 hour. The supernatant was removed and each well was washed with DPBS for three times and then 100 μL 3.7% formaldehyde diluted in DPBS was added and incubated at room temperature for 10 minutes. The fixative was removed and wells were washed with PBST (0.1% Triton X-100 in PBS) for three times. The wells were then blocked with 10% FBS for 1 hour at room temperature followed by incubation with primary antibody (mouse anti-His antibody, Sigma, 1:4,000 dilution with 1% BSA in PBST) for 1 hour at room temperature. The wells were then washed three times with PBST and finally incubated with secondary antibody (HRP-conjugated anti-mouse-IgG, Sigma, dilution 1:40,000) for 1 hour at room temperature. After washing with PBST, the binding signal was measured with ECL reagent (Amersham). All the assays were performed in triplicates.

Binding inhibition assay in MDCK1 cells

MDCK1 cells were seeded at appropriate concentration in 96-well plate one day before to achieve 100% confluency when the assays were performed. The cell monolayer was washed with DPBS twice and then with complete DMEM once. Various amounts of HA were loaded into different wells and complete DMEM was added to bring all the wells to the same volume. After the plate was incubated at room temperature for 1 hour, 5 hemagglutination units (HAU) of influenza A/Brisbane/10/2007 – like isolate TX 419 virus and TPCK-trypsin (final concentration of 2 μg/mL, Worthington) were added to wells and the plate was incubated at 37 °C for an additional hour (with 5% CO2). The supernatant was removed and the wells were washed twice with complete DMEM. Finally, each well was loaded with 100 μL complete DMEM with 2 μg/mL TPCK-trypsin. After 24 hours of infection, the supernatant was centrifuged (300 g, 15 minute) to pellet the cellular debris and used for the standard hemagglutination assay with 1% human red blood cells. All the assays were performed in triplicates.

Reverse genetics and virus stock preparation

The plasmids for reverse genetics of influenza virus B/Lee/40 were generous gifts from Dr. Yoshihiro Kawaoka. Site-directed mutagenesis was used to introduce the Phe95→Tyr, Asn194→Asp and Phe95→Tyr/Asn194→Asp mutants into the gene of B/Lee/40 HA (Agilent Technologies). Co-cultures of 293T and MDCK in DMEM with 10% FBS were plated one day before. 2 μL of TransIT-LT1 (Mirus Bio LLC) was used per 1 μg plasmid to make the DNA-lipid complex. The DNA-lipid mixture was added to the 293T-MDCK co-culture and the plate was incubated at 33°C. At 16 hours post-transfection, the medium was removed and cell monolayer was washed twice with DMEM. Fresh DMEM with 1 μg/mL trypsin was added and the plate was further incubated for 48 hours. For virus with Phe95→Tyr or Phe95→Tyr/Asn194→Asp in HA, 1 μg/mL neuraminiase (NA) from Clostridium perfringens (Sigma) was added to accommodate the increased binding affinity of HA.

Viruses prepared by reverse genetic were then plaque-purified and propagated twice in MDCK cells as follows. MDCK monolayer in T-75 flask after overnight incubation was washed with EMEM for three times, followed by infection of 1 mL virus at room temperature with occasional gentle rocking. After 1 hour, the innoculum was removed and 30 mL EMEM containing 1 μg/mL TPCK-trypsin was added. The cells were incubated at 33°C for 72 hours until at least 70% cells showed cytopathic effect. The supernatant was clarified at 2,600 g for 5 minutes, and laid over a 25% sucrose cushion in NTE buffer (100 mM NaCl, 1 mM EDTA, 10 mM Tris-HCl, pH 7.4). The virus was concentrated by ultracentrifugation at 30,000 rpm for 3 hours at 4°C. The virus pellet was re-suspended in NTE buffer, aliquoted, and stored at −80°C. The purified viruses were thawed before each use, and the titer was determined with standard techniques.

The work on recombinant influenza B viruses was carried out in Biosafety Level 2 plus facility located in Room 244E at Baylor College of Medicine.

Sequencing of virus stocks

The virus stocks were sequenced to confirm the desired mutation in HA gene. QIAamp viral RNA kit (Qiagen) was used to extract the viral RNA. The HA gene was amplified with primers using Onestep RT-PCR kit (Qiagen) and sequenced. The viruses with Phe95→Tyr, Asn194→Asp and Phe95→Tyr/Asn194→Asp all had the expected sequences in HA. However, the wild-type virus exhibited different behaviors. It had the expected Asn194 after one passage in MDCK cells, but the glycosylation site at HA1 194~196 was quickly lost after the second passage in MDCK cells (Asn194 was mutated to Asp194 in five out of six sequenced viruses, and to Ser194 in the other sequenced virus). Because Asn194 was not stable in MDCK cells, only viruses with Asn194→Asp and Phe95→Tyr/Asn194→Asp in HA were used in our subsequent studies.

Binding assay of recombinant viruses

The binding of fluorescently labeled viruses to different cell lines was performed following a previous report (Bradley et al., 2011). First, 50 μL virus was incubated with 25 μg Alexa 488 (Invitrogen) in the presence of 0.1 M NaHCO3 (pH 9.0) for 1 hour at 37°C. The excessive Alexa 488 was then removed by dialysis against PBS with 1 mM EDTA overnight at 4°C using Slide-A-Lyzer MINI Dialysis Devices (7K MWCO, Thermo Scientific). The labeled viruses were then used to bind confluent BHK21, Vero, A549 and MDCK cells on 96-well plates that were pre-chilled at 4°C for 1 hour. The cell monolayer was overlaid with labeled viruses for 1 hour at 4°C, washed with PBS for three times, and read by FLUOstar Omega (BMG LABTECH) using excitation and emission at 485 nm and 528 nm, respectively. The binding assays were performed in triplicates.

Replication of recombinant viruses in MDCK and mice

We compared the replication fitness of recombinant viruses in MDCK and mice. For replication of recombinant viruses in MDCK, cell monolayer was infected with viruses at an MOI of 0.1. The supernatant was harvested at 48 hours post-infection.

For the replication in mice, six to eight-week old female BALB/C mice (Charles River Laboratories) were administered intranasally with 104 PFU recombinant viruses harboring Asn194→Asp or Phe95→Tyr/Asn194→Asp HA, or with sterile PBS as negative controls (four mice per group). At 72 hours post-infection, mice from each group were euthanatized; lungs were collected and immediately frozen on dry ice, and stored at −80°C. The frozen lungs were later thawed, homogenized in 1 mL cold PBS, and clarified by centrifugation at 4°C. Virus titers in the clarified lung lysates were determined by plaque assay using MDCK cells. For plaque assay with Phe95→Tyr/Asn194→Asp virus, the plaques were of rod-like shape. To get round-shaped plaques, 1 μg/mL Clostridium perfringens NA was added.

The mice work was carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. The protocol was approved by the Institutional Animal Care and Use Committee of Baylor College of Medicine (Protocol Number: AN-856). All efforts were made to minimize suffering.

Expression, purification, crystallization and structure determination of Phe95→Tyr/Asn194→Asp mutant of B/Lee/40 HA

The gene corresponding to the ectodomain of the Phe95→Tyr/Asn194→Asp mutant of influenza B/Lee/40 HA was amplified and inserted into pRB21 vector for expression. The purified protein was concentrated to 15 mg/mL for crystallization. Crystals were obtained by hanging drop vapor diffusion method at 290 K in the reservior solution of 0.1 M succinic acid (pH 7.0) and 12% PEG3350. A final concentration of 25% glycerol was included in the reservior solution as the cryoprotectant. The crystals were also soaked in 10 mM avian-like receptor analogue LSTa or human-like receptor analogue LSTc (Carbosynth) overnight. The crystals were flash-frozen in liquid nitrogen and X-ray diffraction data collection was carried out at 100 K at Sector 21 (LS-CAT) at the Advanced Photon Source, Argonne National Laboratory, U.S.A. The diffraction data were processed as previously described (Ni, Kondrashkina, and Wang, 2013). Five percents of unique reflections were randomly selected as the test set for monitoring the Rfree-factors in the refinement. The influenza B/Yamanashi/98 HA structure (Protein Data Bank accession code: 4M40) was used as the search model for molecular replacement, and one copy of the HA trimer was found by PHENIX (Adams et al., 2010). The molecular replacement solution was further built and refined by PHENIX. The model and map were viewed in COOT (Emsley and Cowtan, 2004) for manual adjustment.

Results

Expression of recombinant wild-type and mutant influenza B/Yamagata/73 HA

The ectodomains of the recombinant wild-type influenza B/Yamagata/73 HA and its mutants (single mutants: Phe95→Tyr, Asn194→Asp and double mutant Phe95→Tyr/Asn194→Asp) were expressed in mammalian cell CV-1 using vaccinia virus system (Blasco and Moss, 1995) and purified to high purity. For the Asn194→Asp and Phe95→Tyr/Asn194→Asp mutants, the glycosylation at HA1 194 was depleted in order to mimic the situation in some field isolates (Ikonen et al., 2005; Nakagawa et al., 2000) or due to egg adaptation (Gambaryan, Robertson, and Matrosovich, 1999; Oxford et al., 1991; Oxford et al., 1990; Robertson et al., 1990; Robertson et al., 1985; Saito et al., 2004; Schild et al., 1983). Consequently, the molecular weights of the HA0 monomer for these two mutants are about three kilodaltons smaller than that of wild-type HA as shown on SDS-PAGE (Fig. 1a). After using PNGase F to remove the N-linked glycans, all the recombinant proteins exhibit the same molecular weight on SDS-PAGE (Fig. 1b).

Fig. 1. SDS-PAGE analysis of recombinant proteins of wild-type influenza B/Yamagata/73 HA and its mutants.

Fig. 1

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(a). Purified recombinant proteins. (b). Purified proteins treated with PNGase F to remove the N-linked glycans. Shown are wild-type HA (Lane 1), mutant Phe95→Tyr (Lane 2), Asn194→Asp (Lane 3) and Phe95→Tyr/Asn194→Asp (Lane 4). The proteins are in uncleaved HA0 state.

The Phe95→Tyr mutation enhances receptor-binding affinity of influenza B/Yamagata/73 HA

To investigate the impact of the Phe95→Tyr mutation on the receptor binding capability of HA, we carried out a glycan microarray analysis on wild-type B/Yamagata/73 HA and its three mutants (Fig. 2). Out of the more than 400 glycans on the microarray chips, a total of 97 glycans showed significant binding (fluorescence intensity > 5,000) for at least one of the four protein samples (Fig. 2). Clearly, wild-type HA has an overall weak binding for the majority of glycans, however, it does exhibit an obvious preference towards human-like Neu5Acα(2,6)Gal receptors (Fig. 2a, in red color), which are abundantly found in human upper respiratory tracts, the entry point of wild-type influenza B virus. On the other hand, the mutation Asn194→Asp, with the loss of the glycosylation at HA1 194, results in a preferential binding to avian-like Neu5Acα(2,3)Gal receptors, with overall increased fluorescence intensities relative to the wild-type HA (Fig. 2b).

Compared to the wild-type HA, Phe95→Tyr HA shares the same preference for human-like Neu5Acα(2,6)Gal receptors, but with much higher fluorescence intensities (Fig. 2c). In addition, the binding is also significantly improved for avian-like Neu5Acα(2,3)Gal receptors (in green color), mixed Neu5Acα(2,3)/ α(2,6)Gal receptors (in blue color) and free sialic acids (in black color). Similarly, compared to Asn194→Asp, the Phe95→Tyr/Asn194→Asp mutant has significantly enhanced binding signals to all types of Neu5Ac-containing receptors (Fig. 2d). Furthermore, Phe95→Tyr/Asn194→Asp exhibits substantial binding signals even for Neu5Gcα(2,3)Gal (in dark green color), Neu5Gcα(2,6)Gal (in purple color) and Neu5Gcα(2,6)GlcNAc glycans (in cyan color) (Fig. 2d). Interestingly, it also binds to several other glycans (Fig. 2d, in grey color) including a Neu5Acα(2,8)-linked glycan (#3) and non-sialic-acid glycans such as Galβ1-4GlcNAcβ1-2Manα-Sp0 (#4) whose terminal residues resemble the cleavage products of influenza NA (Supplemental Table 1).

To obtain a quantitative comparison, we performed dose-dependent glycan binding assays using an avian-like Neu5Acα(2,3)Gal receptor (3′SLN-LN) and a human-like Neu5Acα(2,6)Gal receptor (6′SLN-LN) (Table 1). The calculated apparent dissociation constants of wild-type and Phe95→Tyr HA are 5.8 × 10−3 M and 3.5 × 10−6 M for the 3′SLN-LN, 2.3 × 10−4 M and 1.6 × 10−11 M for the 6′SLN-LN, respectively (Table 1). Thus, the wild-type HA binds to 6′SLN-LN with ~25 times higher affinity than 3′SLN-LN. Compared to the wild-type HA, the mutation Phe95→Tyr enhances the binding for 3′SLN-LN and 6′SLN-LN by 103 and 107 times, respectively (Table 1).

Table 1.

Equilibrium dissociation constants of recombinant wild-type influenza B virus HA and its mutants in binding to Neu5Acα(2,3)Gal and Neu5Acα(2,6)Gal receptor analogues

HA 3′SLN-LN*
6′SLN-LN*
n Kd′ R2 n Kd′ R2
B/Yamagata/73
Wild type 0.49±0.05 5.8±4.7×10−3 M 0.97±0.01 0.59±0.01 2.3±0.2×10−4 M 0.97±0.01
Phe95→Tyr 0.88±0.05 3.5±3.1×10−6 M 0.991±0.006 1.27±0.03 1.6±1.0×10−11 M 0.995±0.001
Asn194→Asp 1.50±0.02 4.5±1.7×10−12 M 0.997±0.001 0.81±0.01 7.5±1.1×10−5 M 0.987±0.001
Phe95→Tyr/Asn194→Asp 1.306±0.006 1.1±0.1×10−11 M 0.994±0.001 1.65±0.03 3.3±1.8×10−12 M 0.971±0.002
B/Lee/40
Asn194→Asp 1.51±0.06 8.7±5.5×10−11 M 0.98±0.01 0.976±0.009 4.74±0.01× 10−6 M 0.978±0.001
Phe95→Tyr/Asn194→Asp 0.99±0.06 2.4±1.7×10−8 M 0.989±0.006 1.139±0.009 2.4±0.4×10−9 M 0.99±0.01
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*

The apparent dissociation constant (Kd′), co-operativity factor (n) and the R-square (R2) were obtained by fitting the data to the linearized Hill equation in order to quantitatively determine the relative binding affinities of HA and its mutants. Their absolute values should be compared only in this context.

The mutation Asn194→Asp has an apparent dissociation constant of 4.5 × 10−12 M for 3′SLN-LN, in contrast to the 7.5 × 10−5 M dissociation constant for 6′SLN-LN (Table 1). The loss of glycosylation at HA1 194~196 is frequently observed in egg-adapted variants, indicating an advantage of doing so in order to adapt to a Neu5Acα(2,3)Gal-enriched environment. Our quantitative data reported here provide a molecular basis for this advantage: the Asn194→Asp mutant has an ~109 times higher affinity for 3′SLN-LN than the wild-type HA, while they share a similar affinity for 6′SLN-LN (Table 1).

As seen for the single mutant Phe95→Tyr, the double mutant Phe95→Tyr/Asn194→Asp improves the binding for 6′SLN-LN receptors by about 107 times compared to Asn194→Asp. Thus, regardless of whether there is a glycosylation at HA1 194 (in the case of Phe95→Tyr) or not (in the case of Phe95→Tyr/Asn194→Asp), the mutation Phe95→Tyr consistently enhances the binding of HA for 6′SLN-LN receptors by about 107 times. In contrast, while the loss of the glycosylation at HA1 194 increases the affinity for 3′SLN-LN by 109 times (Asn194→Asp vs. wild type), no further increase was seen for Phe95→Tyr/Asn194→Asp (Table 1). In other words, in the double mutant Phe95→Tyr/Asn194→Asp, the enhanced binding for 3′SLN-LN is mostly brought about by Asn194→Asp, while the tighter binding for 6′SLN-LN is largely the result of Phe95→Tyr. Our structural studies as reported in the later section provide an explanation for this observation.

In fitting the dose-dependent glycan binding assay data using the linearized Hill equation, besides the apparent dissociation constants, the values of the Hill coefficient (n) were also obtained (Table 1). A value of n >1.0 suggests a positive cooperativity in the HA-glycan interactions, i.e., the binding of one receptor to the receptor-binding site of one HA subunit may make it easier for the binding of the second receptor to another HA subunit. On the other hand, a value of n < 1.0 indicates a negative cooperativity. It is interesting to notice that comparing to the wild-type HA, the Hill coefficient values of Phe95→Tyr are almost doubled for both 3′SLN-LN and 6′SLN-LN (Table 1). Similarly, relative to Asn194→Asp, Phe95→Tyr/Asn194→Asp has also doubled its Hill coefficient for 6′SLN-LN. Consistent to their similar affinity for 3′SLN-LN, Asn194→Asp and Phe95→Tyr/Asn194→Asp have a similar Hill coefficient.

The mutation Phe95→Tyr improves the binding of influenza B/Yamagata/73 HA to cultured cell lines

We next tested whether the enhanced receptor-binding affinity of the Phe95→Tyr and Phe95→Tyr/Asn194→Asp mutants allows influenza B/Yamagata/73 HA to bind host cells significantly better. To explore this, we developed a whole-cell-based ELISA assay that quantitatively measures the amount of HA proteins immobilized on cell culture monolayers through their association with cell-surface receptors. Three established cell lines were used: MDCK1, Baby hamster kidney (BHK21), and African green monkey kidney (Vero). Although MDCK (ATCC catalog number: CCL-34) is a well-established cell line for influenza A and B viruses, its derivative MDCK1 (ATCC catalog number: CRL-2935) binds poorly to influenza B virus. Moreover, Vero cells have a high level of Neu5Acα(2,3)Gal receptors and a low level of Neu5Acα(2,6)Gal receptors, but still allow efficient infection by influenza A or B virus (Govorkova et al., 1996). On the other hand, BHK21 cells barely have any detectable Neu5Acα(2,6)Gal receptors, and supports poorly the growth of human influenza A and B viruses (Govorkova et al., 1999; Milliken, 1967).

Overall, the wild-type influenza B virus HA has a very weak binding to all three cell lines, with the strongest for Vero and the weakest for BHK21 (Fig. 3a). The Asn194→Asp mutant has a stronger binding to all three cell lines, with the strongest being Vero cells (Fig. 3b). Phe95→Tyr, and especially Phe95→Tyr/Asn194→Asp, significantly improve the binding to all three cell lines (Fig. 3c, 3d).

Fig. 3. The mutation Phe95→Tyr enhances the binding of influenza B/Yamagata/73 HA protein to cultured cell lines MDCK1, BHK21 and Vero.

Fig. 3

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(a) Wild type, (b) Asn194→Asp, (c) Phe95→Tyr and (d) Phe95→Tyr/Asn194→Asp. The mutant Phe95→Tyr/Asn194→Asp showed the highest binding signals in all three cell lines. Its maximal (saturated) chemiluminescence intensity with each cell line serves as the maximum for that cell line, and the binding signals are expressed as the percentage of the maximum.

The B/Yamagata/73 HA Phe95→Tyr mutant competitively blocks the binding and infection of influenza A virus

Since binding to cell-surface receptors is a prerequisite for infecting host cells by influenza virus, we asked whether the higher binding affinity of the Phe95→Tyr and Phe95→Tyr/Asn194→Asp mutants would allow them to compete more effectively against infection caused by influenza virus. As a stringent test, we used the A/Brisbane/10/2007 - like isolate TX-419 strain belonging to influenza A/H3N2 virus that generally has a higher binding affinity for cell-surface receptors than influenza B virus (Matrosovich et al., 1993). The test also included a “blank” where no influenza A virus was added and a “positive control” where only influenza A virus was added (Fig. 4). If the binding affinity of influenza B virus HA and/or its mutants is strong enough to compete against influenza A virus for binding to cultured cells, we expect to see a lower virus titer in the presence of these recombinant proteins. Again we used MDCK1 for the assay, which binds poorly by influenza B virus. Using five HAU of influenza A virus as input to infect MDCK1 cells, at 24 hours post-infection, the “positive control” has a virus titer of about 64 HAU. With 400 μg recombinant proteins, the wild-type has about twofold inhibition of influenza A virus over the “positive control” (Fig. 4), and the Phe95→Tyr mutant displays eight-fold inhibition. Most strikingly, while the Asn194→Asp single mutant exhibits very weak inhibition against the binding of influenza A virus, the mutant Phe95→Tyr/Asn194→Asp completely blocks the infection of influenza A virus with 400 μg (Fig. 4), 200 μg or even as low as 100 μg recombinant protein. In conclusion, the higher binding affinity of the Phe95→Tyr and Phe95→Tyr/Asn194→Asp mutants for sialic acid receptors does allow a much stronger competition against influenza A virus infection.

Fig. 4. The mutation Phe95→Tyr on influenza B/Yamagata/73 HA protein efficiently inhibits the binding of influenza A virus to MDCK1 cells.

Fig. 4

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(a) and (b) Comparison of inhibition efficiency by recombinant influenza B virus HA. The “Blank” sample is the negative control where the MDCK1 cells grow under normal condition in the binding inhibition assay. The “Positive” sample is the positive control where the MDCK1 cells were infected with 5 HAU influenza A virus without the addition of any recombinant influenza B virus HA proteins. The “Wild type”, “Phe95→Tyr”, “Asn194→Asp” and “Phe95→Tyr/Asn194→Asp” samples represent those wells where the MDCK1 cells were first bound with 400 μg recombinant proteins before the infection of influenza A virus. The titer was read as the highest dilution where the red button was observed.

Production of recombinant influenza B/Lee/40 viruses by reverse genetics

In order to examine the impact of the Phe95→Tyr mutation on influenza virus infection and replication, we generated recombinant influenza B viruses by reverse genetics using all genes from influenza B/Lee/40 virus (generous gifts from Dr. Yoshihiro Kawaoka) following the previously reported protocol (Hatta and Kawaoka, 2003). The mutations of Asn194→Asp and Phe95→Tyr were introduced into the coding region of B/Lee/40 HA by site-directed mutagenesis. All the viruses had correct sequences after one passage in MDCK cells. However, in recombinant wild-type B/Lee/40 virus, Asn194 was mutated to Asp194 or Ser194 after the second passage in MDCK cells. The instability of Asn194 of recombinant wild-type B/Lee/40 virus in MDCK cells precluded a study of the wild-type and Phe95→Tyr viruses. Consequently, the following studies were only conducted on recombinant B/Lee/40 viruses harboring Asn194→Asp or Phe95→Tyr/Asn194→Asp in the HA sequence.

The Phe95→Tyr mutation introduced into B/Lee/40 HA enhances binding for human receptors but decreases binding for avian receptors

The recombinant proteins in earlier sections of this study used B/Yamagata/73 HA as the background. However, the recombinant viruses generated by reverse genetics have B/Lee/40 HA instead. These proteins share ~94% sequence identity. There are two noticeable differences between them: one is a glycosylation at HA1 163 in B/Yamagata/73 HA but not in B/Lee/40 HA, while the other is a glycosylation at HA1 230 in B/Lee/40 HA, but not in B/Yamagata/73 HA (following B/HK/73 HA numbering). These glycosylation sites are not at the receptor-binding site per se, but are nearby. Therefore, we also expressed and purified recombinant proteins of B/Lee/40 HA containing Asn194→Asp or Phe95→Tyr/Asn194→Asp and tested their binding affinity for synthetic glycans (Table 1). Similar to the results of B/Yamagata/73 HA, the Asn194→Asp mutation in B/Lee/40 HA exhibits ~105 times stronger binding for 3′SLN-LN than for 6′SLN-LN. However, the introduction of Phe95→Tyr into B/Lee/40 HA containing the Asn194→Asp mutation enhances the binding affinity for 6′SLN-LN by ~2,000 times, but at the same time decreases the binding for 3′SLN-LN by 276 folds (Table 1).

Binding of recombinant B/Lee/40 viruses to different cell lines

To compare the binding capability of the recombinant viruses to different cell lines, we first labeled the viruses using Alexa 488 (Invitrogen), which were then used to bind to confluent BHK21, Vero, A549 and MDCK cells on 96-well plates that were pre-chilled at 4°C. The cell monolayer was overlaid with labeled viruses at a multiplicity of infection (MOI) of 3 for 1 hour at 4°C. The incubation at 4°C was to ensure that no NA activity was present, which was confirmed by using the substrate 2′-(4-methylumbelliferyl)-a-D-N-acetylneuraminic acid (MUNANA) at 4°C. The fluorescence obtained for Asn194→Asp virus to each cell line was expressed as 100%. The Phe95→Tyr/Asn194→Asp virus exhibits 60~80% binding signals relative to the Asn194→Asp virus (Fig. 5). The about 20% higher binding of the Phe95→Tyr/Asn194→Asp virus to MDCK cells, compared to Vero and BHK21 cells, agrees well with the higher content of Neu5Acα(2,6)Gal receptors on MDCK cells (Govorkova et al., 1996; Ito et al., 1997b) for which the recombinant Phe95→Tyr/Asn194→Asp protein prefers (Table 1). However, the Phe95→Tyr/Asn194→Asp virus does not bind to any of the cultured cells at the same level as the Asn194→Asp virus.

Fig. 5. Binding of recombinant influenza B/Lee/40 viruses to cultured cells.

Fig. 5

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Alexa 488-labeled viruses were bound to chilled cells in 96-well plates at an MOI of 3 and incubated at 4 for 1 hour. The relative binding in each cell line is expressed as a percentage of that of recombinant influenza virus that harbors Asn194→Asp HA.

Agglutination of erythrocytes by recombinant B/Lee/40 viruses

We then examined the ability of recombinant viruses to agglutinate erythrocytes of different animal species, which are known to contain different types, density and distribution of glycans on the cell surface. Consistent with the picomolar dissociation constant of the recombinant Asn194→Asp HA protein for 3′SLN-LN receptors, the Asn194→Asp virus binds to a higher titer for chicken, turkey and guinea pig erythrocytes (containing mixed α(2,3)- and α(2,6)-linked sialic acid receptors) than for sheep, horse and bovine erythrocytes (containing predominantly α(2,6)-linked sialic acid receptors) (Ito et al., 1997a; Medeiros et al., 2001; Takemae et al., 2010) (Table 2). Its binding to horse erythrocytes is particularly low, in agreement with the fact that horse erythrocytes contain 100% Neu5Gc (Suzuki, Matsunaga, and Matsumoto, 1985) that can not be recognized by the Asn194→Asp protein on B/Yamagata/73 HA (Fig. 2b). In sharp contrast, the Phe95→Tyr/Asn194→Asp virus binds at a higher titer to sheep, horse and bovine erythrocytes, consistent with the stronger binding of the Phe95→Tyr/Asn194→Asp HA protein for Neu5Acα(2,6)Gal receptors (in the case of sheep and bovine erythrocytes) (Table 1) and for Neu5Gc-containing sialic acids (in terms of horse erythrocytes) (Fig. 2d). Interestingly, by incubating the virus-erythrocyte mixtures at 37°C for 2 hours to allow the cleavage of sialic acids by viral NA protein, the majority of Asn194→Asp virus is quickly eluted from erythrocytes, while in marked contrast, the binding of the Phe95→Tyr/Asn194→Asp virus hardly changes. After overnight incubation at 37°C, the Phe95→Tyr/Asn194→Asp virus is eluted from most erythrocytes but remains bound to guinea pig and horse erythrocytes at a significant level (Table 2). These data suggest that the activity of NA is not sufficiently high to keep up with the strong binding of the Phe95→Tyr/Asn194→Asp HA, thus a longer working time is needed.

Table 2.

Agglutination of erythrocytes by recombinant B/Lee/40 viruses under different conditionsa

Erythrocytesb Agglutination by virus (HAU/50 mL)
4, 2 hours 4, 2 hours and 37, 2 hours 4, 2 hours and 37, overnight
Asn194→Asp Phe95→Tyr/Asn194→Asp Asn194→Asp Phe95→Tyr/Asn194→Asp Asn194→Asp Phe95→Tyr/Asn194→Asp
cRBC 128 8 <2 8 <2 <2
tRBC 256 16 128 16 <2 2
gpRBC 128 16 <2 16 <2 16
sRBC 64 64 <2 32 <2 <2
hoRBC 4 64 <2 64 <2 32
bRBC 64 16 <2 16 <2 <2
Asialo cRBC <2 <2 <2 <2 <2 <2
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a

HAU were recorded at different time points after erythrocytes were incubated with virus for 2 hours at 4 C, then placed at 37°C for 2 hours, or placed at 37°C overnight.

b

cRBC, chicken red blood cell (RBC); tRBC, turkey RBC; gpRBC, guinea pig RBC; sRBC, sheep RBC; hoRBC, horse RBC; bRBC, bovine RBC. Asialo cRBC: cRBCs were treated with Clostridium perfringens NA to remove sialic acids.

Replication of recombinant B/Lee/40 viruses in MDCK cells and mice

We compared the replication capabilities of the recombinant viruses in MDCK cell line that are known to contain both Neu5Acα(2,3)Gal and Neu5Acα(2,6)Gal receptors (Govorkova et al., 1996; Ito et al., 1997b). Relative to the titer of the Asn194→Asp virus (at 2.8×106 pfu/mL), the Phe95→Tyr/Asn194→Asp virus has a comparable titer of 3.0×106 pfu/ml (Table 3).

Table 3.

In vivo and in vitro replication capabilities of recombinant B/Lee/40 viruses

MDCKa (pfu/mL) Mice lungsb (pfu/mL lung)
Asn194→Asp 2.8±0.2×106 2.7±0.3×105
Phe95→Tyr/Asn194→Asp 3.0±0.1×106 2.2±1.7×102
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a

MDCK cells were infected at an MOI of 0.1 and the titer was determined at 48 hours post-infection.

b

Mice were administered intranasally at 104 PFU, and lungs were harvested at 72 hours post-infection. Titers are expressed as mean ± standard deviation pfu/mL lung lysates that were collected from four mice within each group.

The replication capabilities of the recombinant viruses were also tested in mice. By administering 104 PFU recombinant viruses intranasally, the mice have an average viral titer of 2.2×102 pfu/mL for the Phe95→Tyr/Asn194→Asp virus, in marked contrast to the titer of 2.7×105 pfu/mL for the Asn194→Asp virus, indicating an attenuation of ~1,000 folds for the former (Table 3). This attenuation is in good agreement with the 276 times lower binding affinity of the Phe95→Tyr/Asn194→Asp protein, compared to Asn194→Asp, for Neu5Acα(2,3)Gal receptors (Table 1) that are almost exclusively found in mouse airway epithelial cells (Tang and Chong, 2009). These data suggest that the observed differential replication of these viruses in mice is likely resultant from the different binding affinities of their HA proteins to Neu5Acα(2,3)Gal receptors.

Structural basis for the effects of Phe95→Tyr on binding to receptors

In order to understand the effects of Phe95→Tyr on binding to receptors, we determined the crystal structures of B/Lee/40 HA containing Phe95→Tyr/Asn194→Asp and its complexes with human-like LSTc and avian-like LSTa receptor analogues to 2.53, 2.63 and 2.72 Å, respectively (Table 4). As noted earlier (Ni, Kondrashkina, and Wang, 2013), B/Yamanashi/98 HA differs significantly from B/HK/73 HA in the region of HA1 235~240. It appears that B/Lee/40 HA is also different from B/HK/73 HA (Fig. 6a) but closely resembles B/Yamanashi/98 HA (Fig. 6b). As expected, the extra hydroxyl oxygen atom on the side chain of the introduced Tyr-95 mediates an extra hydrogen bond with His-191, one of the three key residues constituting the receptor-binding site of influenza B virus HA (Fig. 6a, 6b). Moreover, the interaction between the receptor-binding site of HA and the bound receptor analogues, LSTc and LSTa, is strengthened by the two additional hydrogen bonds between the Sia-1 moiety and the hydroxyl oxygen atom of Tyr-95 (Fig. 6c, 6d, Table 5). Thus, the mutation Phe95→Tyr restores all three hydrogen bonds that the hydroxyl oxygen atom of Tyr-98 makes in influenza A/H3 HA (Weis et al., 1988). However, whether and how much this mutation enhances the binding to human or avian receptors depends on the local environment at and around the receptor-binding site (see Discussion).

Table 4.

Data collection and refinement statistics of influenza B/Lee/40 HA Phe95→Tyr/Asn194→Asp and its complexes with LSTc and LSTa

Unliganded Complex with LSTc Complex with LSTa
Data collection statistics
Resolution range (Å) 44.63~2.53 (2.67~2.53) 47.46~2.63 (2.77~2.63) 48.91~2.72 (2.87~2.72)
Space group P21 2 21 P21 2 21 P21 2 21
Unit cell
 a, b, c (Å) 83.7, 128.6, 211.2 83.6, 128.3, 211.8 83.6, 128.4, 211.7
 α, β, γ (°) 90, 90, 90 90, 90, 90 90, 90, 90
Unique reflections 75,220 68,564 59,313
Multiplicity 7.4 (7.0) 5.3 (5.2) 6.6 (6.6)
Completeness (%) 97.9 (93.9) 100.0 (100.0) 95.7 (87.1)
Mean I/sigma(I) 13.1 (4.4) 11.5 (2.4) 13.7 (4.0)
Wilson B-factor (Å2) 33.8 40.9 34.9
Rmerge (%) 11.4 (48.3) 11.1 (49.9) 11.8 (49.1)
Refinement statistics
Rcryst (%) 18.7 (23.6) 20.7 (27.7) 19.4 (24.8)
Rfree (%) 22.2 (27.8) 24.5 (33.3) 22.9 (28.0)
Number of atoms 12,678 12,547 12,583
 Protein 11,653 11,603 11,651
 Ligands 459 594 663
 Water 566 350 269
RMSD
 Bond length (Å)/angles (°) 0.003/0.75 0.007/0.77 0.007/0.84
Ramachandran plot
 Favored, allowed (%) 97.4, 2.6 97.1, 2.9 97.2, 2.8
Average B-factor (Å2) 36.3 44.0 34.1
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Statistics for the highest-resolution shell are shown in parentheses.

Fig. 6. Structural analysis of influenza B/Lee/40 HA containing the Phe95→Tyr/Asn194→Asp mutant and its complexes with avian- and human-like receptor analogues.

Fig. 6

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(a) Superposition of the receptor-binding sites between influenza B/Lee/40 HA Phe95→Tyr/Asn194→Asp mutant (in cyan) and influenza B/HK/73 HA (PDB code: 3BT6) (in yellow). B/HK/73 and B/Yamagata/73 HA differ by one residue, Asn vs. His at HA1 116. The glycosylation at HA1 163 in B/HK/73 HA is also shown. (b) Superposition of the receptor-binding sites between B/Lee/40 HA Phe95→Tyr/Asn194→Asp (in cyan) and B/Yamanashi/98 HA (PDB code: 4M40) (in orange). (c) Complex of B/Lee/40 HA Phe95→Tyr/Asn194→Asp (in yellow) with human-like receptor LSTc (in green). (d) Complex of B/Lee/40 HA Phe95→Tyr/Asn194→Asp (in yellow) with avian-like receptor LSTa (in green). (e) Superposition of the Sia-1 in the complex structures with LSTa by B/Lee/40 HA Phe95→Tyr/Asn194→Asp (in cyan) and B/Yamanashi/98 HA (PDB code: 4M44) (in orange). (f) LSTa in the receptor-binding sites of B/Lee/40 HA Phe95→Tyr/Asn194→Asp and Asn194→Asp. The structure of B/Lee/40 HA Asn194→Asp – LSTa is modeled by using the conformation of LSTa in the B/Lee/40 HA Phe95→Tyr/Asn194→Asp – LSTa structure (in cyan), but with the Sia-1 moiety at the higher position as observed in B/Yamanashi/98 HA (in orange). The glycosylation at HA1 230 in B/Lee/40 HA is also shown. In all panels, the hydrogen bonds are shown in dashed lines according to the color of the corresponding structure. The hydrogen bonds formed by the hydroxyl oxygen atom of Tyr-95 are highlighted in magenta.

Table 5.

Hydrogen bonding interactions of B/Lee/40 HA Phe95→Tyr/Asn194→Asp mutant with bound LSTc and LSTa receptor analogues

Interaction pair B/Lee/HA Phe95→Tyr/Asn194→Asp distance (Å) Interaction pair B/Yamanashi/HA -LSTa (PDB code: 4M44) distance (Å)
LSTc LSTa
Sia-1 O1A……Ser140 OG 3.3 3.2 O1A……Ser140 OG 3.4
O1A……Gly141 N 2.4 2.6 O1A……Gly141 N 2.8
O1B……Ser140 OG 2.9 2.8 O1B……Ser140 OG 2.9
O1B……Gln239 NE2 2.6 2.7 O1B……Gln239 NE2 2.8
O1B……Gln239 OE1 3.2 3.2 O1B……Gln239 OE1 3.3
O4………Thr139 O 3.5 3.6 O4………Thr139 O 3.4
N5………Thr139 O 3.0 3.0 N5………Thr139 O 3.3
O7………Arg200 NH2 2.7 2.5 O7………Asn200 -
O8………Tyr95 OH 3.1 3.3 O8………Phe95 -
O8………Gln239 OE1 3.0 2.9 O8………Gln239 OE1 3.1
O9………Tyr95 OH 3.3 3.3 O9………Phe95 -
O9………Asp193 OD2 2.6 2.5 O9………Asp193 OD2 2.7
O9………Gln197 NE2 3.5 - O9………Gln197 -
O9………Ser240 OG 3.3 3.5 O9………Ser240 OG 3.1
O10Lys136 - - O10Arg136 NH2 2.9
Gal-2 O3………Lys238 O 2.8 - O3………Pro238 -
O4………Lys238 O 3.1 - O4………Pro238 -
O6………Lys238 - 2.7 O6………Pro238 O 2.9
Glc-5 O2………Thr196 OG1 - 3.0 O2………Thr196 -
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In contrast to the relatively higher position of the Sia-1 moiety in the receptor-binding site of B/Yamanashi/98 HA (containing Phe-95), the presence of Tyr-95 pulls the Sia-1 moiety deeper into the receptor-binding site by a rotation of ~18° (Fig. 6e). If everything else is the same, the pull-down of Sia-1 by Tyr-95 could result in substantial shifts in the positions of the following sugar rings. To illustrate this point, we generated a modeled structure of B/Lee/40 HA Asn194→Asp – LSTa by using the conformation of LSTa in the B/Lee/40 HA Phe95→Tyr/Asn194→Asp – LSTa structure, but with the Sia-1 moiety at the higher position as observed in B/Yamanashi/98 HA (Fig. 6f). Clearly, compared to the modeled B/Lee/40 HA Asn194→Asp – LSTa structure, the LSTa in the B/Lee/40 HA Phe95→Tyr/Asn194→Asp extends closer to the glycosylation at HA1 230 in the 240-loop (Fig. 6f). This closer spatial location between the LSTa and the glycosylation at HA1 230 may be responsible for the reduced binding affinity of B/Lee/40 HA Phe95→Tyr/Asn194→Asp for 3′SLN-LN (Table 1).

Discussion

Phe95→Tyr increases the receptor-binding affinity of recombinant influenza B virus HA proteins

Comparing with influenza A virus, naturally occurring influenza B virus has a lower binding affinity for synthetic glycans and natural receptors on cell surface (Matrosovich et al., 1993). Built upon our previous structural studies of influenza B virus HA (Ni, Kondrashkina, and Wang, 2013; Wang, 2010; Wang et al., 2007), we noticed a critical difference between the HA glycoproteins of influenza A and B viruses at the base of the receptor-binding sites, Tyr-98 in influenza A/H1~H15 HA versus Phe-95 in influenza B virus HA. Given the absolutely conserved nature of Tyr-98 among influenza A/H1~H15 HA proteins, we speculated that the presence of HA Phe-95 might be responsible for the lower receptor-binding affinity of influenza B virus.

Here, by using recombinant B/Yamagata/73 HA proteins, we demonstrated that Phe95→Tyr is able to enhance the binding affinity to synthetic glycans by up to 107 times (Fig. 2, Table 1), to a level that is comparable to those of influenza A virus (dissociation constants at picomolar range) (Jayaraman et al., 2011; Pappas et al., 2010; Srinivasan et al., 2008; Viswanathan et al., 2010b). Most significantly, the mutation drastically enhances the binding to three cultured cell lines (Fig. 3) and competitively blocks the binding and infection by influenza A virus (Fig. 4). Similarly, in the background of B/Lee/40 HA Asn194→Asp, the Phe95→Tyr mutation also increases the binding affinity for 6′SLN-LN by ~2,000 times (Table 1).

It is interesting to note that the small-to-large mutation of Phe95→Tyr in our study can be accommodated in the receptor-binding site of influenza B virus HA easily and enhances the receptor-binding affinity. In sharp contrast, the large-to-small mutation of Tyr98→Phe on influenza A/H3 HA almost abolished the binding of the virus to red blood cells, and the recombinant Tyr98→Phe virus can not infect mutant MDCK cells with a reduced level of cell-surface sialic acids (Martin et al., 1998) and replicates poorly in mice (Meisner et al., 2008). Since the hydroxyl oxygen atom on the side chain of Tyr-98 makes one hydrogen bond within the receptor-binding site and two additional ones with the sialic acid receptors (Weis et al., 1988), it is likely that Tyr98→Phe somehow distorts the receptor-binding site and/or substantially weakens the receptor binding in influenza A/H3 HA. On the other hand, the fact that wild-type influenza B virus HA can still bind to sialic acid receptors and red blood cells, albeit at a lower affinity, suggests that the loss of the hydroxyl oxygen atom in Phe-95, compared to a tyrosine residue, has been at least partially compensated, probably by some small local structural adjustments such as those found within the 240-loop and 140-loop (Wang et al., 2007).

The impacts of glycosylations on the receptor binding of influenza virus HA proteins

The different paths that LSTa and LSTc exit from the receptor-binding site provide a plausible explanation for the mutational effects of Asn194→Asp and Phe95→Tyr in this study. Since the Neu5Acα(2,3)Gal receptor (LSTa) exits the receptor-binding site at the N-terminus of the 190-helix and the terminal sugar rings interact with Thr-196 in the absence of the glycosylation at HA1 194 (Fig. 6d), this glycosylation may severely interfere with the binding of Neu5Acα(2,3)Gal receptors to the receptor-binding site. The interference may be responsible for the only modest increase in binding to Neu5Acα(2,3)Gal receptors by the Phe95→Tyr mutation that introduces two strong hydrogen bonds with the Sia-1 moiety, while the corresponding increase for Neu5Acα(2,6)Gal receptors is 107 folds (Fig. 6d, Table 1). Therefore, the removal of the glycosylation at HA1 194 in B/Yamagata/73 HA containing either Asn194→Asp or Phe95→Tyr/Asn194→Asp relieves this interference and yields picomolar dissociation constants for both Neu5Acα(2,3)Gal and Neu5Acα(2,6)Gal receptors (Table 1).

However, in the case B/Lee/40 HA, although the loss of the glycosylation at HA1 194 results in a picomolar dissociation constant for Neu5Acα(2,3)Gal receptors, the introduction of Phe95→Tyr into this background reduces the binding affinity for Neu5Acα(2,3)Gal receptors. It appears that the presence of Tyr-95 pulls down the Sia-1 to a lower position via an 18° rotation, which results in a closer spatial location of the terminal sugar rings of LSTa with the glycosylation at HA1 230 that is unique to B/Lee/40 HA (Fig. 6f). Thus, in addition to their recognized roles in modulating antigenicity of influenza B virus (Nakagawa et al., 2000; Nakagawa et al., 2004; Wang et al., 2008), glycosylations at or near the receptor-binding site of influenza B virus HA also influence the receptor-binding affinity and preference of the virus. This is reminiscent of the glycosylation at HA1 158 of influenza A/H5 HA where its removal contributes to the completely switched binding to human receptors (Imai et al., 2012; Tharakaraman et al., 2013).

The roles of receptor binding of HA in influenza B virus pathogenicity

Recent studies on influenza A virus have demonstrated that manipulating receptor-binding avidity is an important tool that influenza A virus uses in response to neutralization imposed by neutralizing antibodies, which seems to drive antigenic drift (Hensley et al., 2009). Moreover, a single-nucleotide change in the coding sequence of influenza A/H1 HA increased its binding affinity for human receptors, corrected the low receptor affinity of the field isolate and led to efficient airborne transmission in ferrets (Jayaraman et al., 2011). These and other studies (Maines et al., 2009; Pappas et al., 2010; Viswanathan et al., 2010b) suggest that the binding affinity of HA to receptors is an important determinant for influenza A virus infectivity.

The lack of reservoirs of influenza B virus outside of human population is believed to be one of the major reasons that influenza B virus does not undergo antigenic shift, a molecular evolutionary mechanism that is responsible for almost all known human pandemics caused by influenza A virus (Gamblin and Skehel, 2010; Skehel and Wiley, 2000; Steinhauer, 2010; Wang, 2010; Wiley and Skehel, 1987). It remains an open question whether there exists a correlation between the limited host range and the lower receptor binding affinity of HA of naturally occurring influenza B virus. Most recently, Palese and co-workers published an interesting study in which the ectodomain of influenza A/H1 HA was used to replace that of influenza B virus HA in the backbone of influenza B virus. In marked contrast to the wild-type influenza B virus, the chimeric influenza B virus with inserted A/H1 HA resulted in temporary but significant weight loss in infected mice (Hai et al., 2011), suggesting that the stronger receptor binding of influenza A/H1 HA has enhanced the pathogenicity of the recombinant influenza B virus in which it resides.

The enhanced receptor binding affinity of influenza B virus HA upon a single Phe95→Tyr mutation at the receptor-binding site provides a plausible explanation for the very limited host range displayed by influenza B virus. As suggested in previous studies (Martin et al., 1998), a sufficient density of cell-surface sialic acids with a weaker receptor-binding HA or a lower than optimal density of sialic acids with a stronger receptor-binding HA are both viable combinations for effective influenza infection. Thus, the weaker receptor binding of wild-type influenza B virus HA likely imposes a much higher demand for the “perfect” glycan composition on the surface of target cells in a given host, thus the virus may only infect hosts satisfying such a strict requirement. A single Phe95→Tyr mutation was able to promote the binding affinity of influenza B/Yamagata/73 HA to a comparable level as that of influenza A virus HA, regardless of human or avian-like receptors, thus enabling its efficient binding to three cultured cell lines containing a varied range of glycan composition for which the wild-type HA binds very poorly. While it is not known when, how and why influenza B virus HA acquired Tyr95→Phe mutation in the course of evolution, it is likely that some or all of the other viral proteins of influenza B virus have undergone concomitant changes, including a delicate balance between HA and NA (Mitnaul et al., 2000). In addition, influenza viral infection is a complex process involving a myriad of host factors (Brass et al., 2009; Hao et al., 2008; Karlas et al., 2010; Konig et al., 2010; Li et al., 2011; Shapira et al., 2009; Sui et al., 2009; Watanabe, Watanabe, and Kawaoka, 2010). While our data revealed the striking importance of a single-residue mutation at the receptor-binding site to the receptor binding affinity of influenza B virus HA, further studies are needed in order to illustrate its implication to pathogenicity and evolution of influenza B virus and co-evolution of HA with other viral proteins.

Supplementary Material

01

Highlights of our work.

  • The mutation Phe95→Tyr in influenza B virus HA restores all three hydrogen bonds made by Tyr-98 in influenza A/H3 HA

  • The effects of Phe95→Tyr in receptor binding depend on the local environment at and around the receptor-binding site

  • Glycosylations play a key role in receptor binding

  • The binding and replication of recombinant viruses correlate well with the receptor-binding capabilities of HA

Acknowledgments

The authors gratefully thank Dr. Peter Palese for the gene of influenza B/Yamagata/73 HA, Dr. Bernard Moss for the pRB21 and vRB12 vaccinia expression system, Dr. Yoshihiro Kawaoka for the plasmids used to generate recombinant influenza B/Lee/40 viruses, Dr. Robert Couch for insightful discussions, Dr. Pedro Piedra for influenza A/Brisbane/10/2007 (H3N2) - like isolate TX-419 and invaluable discussions, and Drs. David Smith and Jamie Heimburg-Molinaro for performing the glycan microarray analysis. The authors would like to acknowledge Consortium for Functional Glycomics funded by the NIGMS GM62116 and GM98791 for services provided by the Glycan Array Synthesis Core (The Scripps Research Institute, La Jolla, CA) that produced the glycans and mammalian glycan microarray and the Protein-Glycan Interaction Core (Emory University School of Medicine, Atlanta, GA) that assisted with analysis of samples on the array. Q.W. acknowledges support from the National Institutes of Health (R01-AI067839), the Gillson-Longenbaugh Foundation, the Simmons Collaborative Research Fund Award from the Golf Coast Consortium and The Welch Foundation (Q-1826). Use of the Advanced Photon Source was supported by the U. S. Department of Energy, Office of Science, Office of Basic Energy Sciences, under Contract No. DE-AC02-06CH11357. Use of the LS-CAT Sector 21 was supported by the Michigan Economic Development Corporation and the Michigan Technology Tri-Corridor for the support of this research program (Grant 085P1000817).

Footnotes

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