Influenza A penetrates host mucus by cleaving sialic acids with neuraminidase
© Cohen et al.; licensee BioMed Central Ltd. 2013
Received: 17 September 2013
Accepted: 14 October 2013
Published: 22 November 2013
Influenza A virus (IAV) neuraminidase (NA) cleaves sialic acids (Sias) from glycans. Inhibiting NA with oseltamivir suppresses both viral infection, and viral release from cultured human airway epithelial cells. The role of NA in viral exit is well established: it releases budding virions by cleaving Sias from glycoconjugates on infected cells and progeny virions. The role of NA in viral entry remains unclear. Host respiratory epithelia secrete a mucus layer rich in heavily sialylated glycoproteins; these could inhibit viral entry by mimicking sialylated receptors on the cell surface. It has been suggested that NA allows influenza to penetrate the mucus by cleaving these sialylated decoys, but the exact mechanism is not yet established.
We tested IAV interaction with secreted mucus using frozen human trachea/bronchus tissue sections, and bead-bound purified human salivary mucins (HSM) and purified porcine submaxillary mucins (PSM). The protective effect of mucus was analyzed using MDCK cells coated with purified HSM and PSM with known Sia content. Oseltamivir was used to inhibit NA activity, and the fluorescent reporter substrate, 4MU-Neu5Ac, was used to quantify NA activity.
IAV binds to the secreted mucus layer of frozen human trachea/bronchus tissues in a Sia dependent manner. HSM inhibition of IAV infection is Sia dose-dependent, but PSM cannot inhibit infection of underlying cells. HSM competitively inhibits NA cleavage of 4MU-Neu5Ac, reporter substrate. Human IAV effectively cleaves Sias from HSM but not from PSM, and binds to HSM but not to PSM.
IAV interacts with human mucus on frozen tissue sections and mucus-coated beads. Inhibition of IAV infection by sialylated human mucus is dose-dependent, and enhanced when NA is inhibited with oseltamivir. Thus NA cleaves sialylated decoys during initial stages of infection. Understanding IAV interactions with host mucins is a promising new avenue for drug development.
Interactions of Influenza A viruses (IAVs) with mucus were first described in the mid 20th century, and led early researchers to classify influenza as a (ortho)myxovirus – a virus with affinity for mucus [1, 2]. IAVs must penetrate a secreted mucus layer (up to 50 μm thick) to reach target tissues in mammalian airways . Mucus is a defensive layer containing highly glycosylated mucins rich in terminal sialic acids (Sias) . It has been suggested that mucus may protect against IAV infection by presenting sialylated “decoys” that mimic receptors on the cell surface [5, 6]. Influenza viruses bind these unproductive receptors, become trapped in the mucus layer, and can then be removed by the normal process of mucus clearance as part of the innate defense system [7, 8].
Neuraminidase (NA) enzymatic activity cleaves Sias from glycoconjugates on infected cells and progeny virions allowing budding virions to escape from infected cells. NA inhibitors such as oseltamivir inhibit viral release by preventing the cleavage of Sias specifically . It has also been shown that NA inhibition with oseltamivir carboxylate suppresses IAV infection of cultured human airway epithelium (HTBE) . Thus NA activity must have a role during initial infection, but the exact mechanism is not yet known. Since HTBE cells are coated with a secreted mucus layer, it was speculated that NA activity promotes infection by cleaving Sias from mucus . However, other interpretations such as promotion of hemagglutinin-mediated fusion [11, 12] cannot yet be ruled out, and IAV interactions with mucins during initial infection remain poorly characterized.
Mucin glycosylation and sialylation vary significantly between species, and thus could influence influenza host species specificity. Human airway and salivary mucins have been well characterized [13–15] and their constituents and glycosidic linkages differ dramatically from those of other species such as chimpanzees  and pigs . Humans express predominantly N-acetylneuraminic acid (Neu5Ac) . Human airway sialoglycans lack the sialic acid (Sia) N-glycolylneuraminic acid (Neu5Gc), which is the predominant Sia in porcine mucus . In the human upper respiratory tract Sias are predominantly found in α2-6 glycosidic linkage . In contrast, these Sias are mostly α2-3-linked in chimpanzees  and pigs .
Mucus carries a vast array of variable receptors, and mucin sialylation can create sites in the mucus that resemble the target receptors on cells [16, 21]. IAV binding properties are important determinants of host susceptibility and host range, and the type and distribution patterns of sialylated glycans on target tissues seem to be crucial . Notably, binding properties of IAV are traditionally studied in absence of secreted mucins despite the fact that all natural infection sites are abundantly covered with these secreted sialoglycoproteins.
In this study we investigate the interaction of IAV with mucins, and provide experimental evidence for the role of NA during initial infection of mucus-coated cells and tissues. We demonstrate Sia-dependent binding of IAV to secreted mucus on frozen human trachea/bronchus tissues. We present direct in vitro evidence that secreted mucus protects underlying cells from infection by presenting sialylated decoys for hemagglutinin (HA) and competing for NA cleavage activity. We used purified mucus from two different hosts: human and pig, to show direct cleavage of- and direct binding to- sialylated human mucus by human IAV NA and HA, respectively.
Influenza A virus interacts with mucin on human airway tissue
Human mucin protects cells from infection in vitro
High Sia-content PSM (high) is comprised of 10,200 pmol Neu5Gc and 1,800 pmol Neu5Ac (Additional file 1D). However, the presentation of Sia differs between PSM  and HSM [13–15]. Despite having a similar Neu5Ac content, PSM (high) and HSM (low) did not have the same inhibitory effect on IAV infection. Two strains, A/PR/8/34(H1N1) and A/Aichi/2/68(H3N2) were not significantly inhibited by coating monolayers with PSM (high), however coating monolayers with HSM (low) significantly reduced the number of infected cells (Figure 3A and D, P<0.0005, and P<0.0462, respectively). Numbers of cells infected by A/SD/17/2008(H1N1) were reduced in monolayers coated with both PSM (high) and HSM (low), however, fewer infected cells were observed in HSM-coated monolayers compared with PSM-coated monolayers (Figure 3C, P<0.0504). In contrast coating monolayers with either PSM (high) or HSM (low) did not significantly reduce the number of cells infected with A/SD/1/2009(SOIV) (Figure 3B). Thus Sia content, type, and presentation are all important factors for inhibition of IAV infection. Furthermore, mucus inhibition of IAV infection is strain-dependent.
Inhibition of IAV neuraminidase by oseltamivir increases the protective effect of HSM but not PSM
Since the virus NA can potentially cleave sialylated receptors presented on secreted human mucus, inhibition of NA activity may enhance the protective effect of mucus. In order to test this hypothesis, 1 μM oseltamivir was added to IAV prior to challenging the mucus-coated cells. The most prominent reduction of infection rate by oseltamivir was observed for the pandemic A/SD/2009(SOIV) strain. A reduction of ~60% in the number of infected cells was observed in monolayers coated with either buffer or PSM (Figure 3B, PBS and PSM P<0.05). This could be attributed to oseltamivir inhibition of the secondary Sia-binding site found on the virus N1 neuraminidase , rather than to the neuraminidase enzymatic activity. However, the number of infected cells in HSM-coated monolayers was further reduced to <10% upon addition of oseltamivir, even with monolayers that were coated with HSM (low). Thus oseltamivir and HSM have an additive inhibitory effect (HSM + oseltamivir compared with HSM, or with PBS + oseltamivir, P<0.05, see Additional file 2 for complete statistical analysis). The number of infected cells in PSM (high)-coated monolayers and HSM (low)-coated monolayers were reduced to a similar extent by the addition of 1 μM oseltamivir (Figure 3B, 24±8% and 11±7%, respectively, P<0.0853).
Similar results were obtained for the seasonal H1N1 strain (A/PR/8/34(H1N1)). Although the number of infected cells in PSM- or PBS-coated monolayers was not significantly reduced by oseltamivir, 25-30% reduction in the number of infected cells was observed (Figure 3A, gray bars). However, the number of infected cells in HSM-coated monolayers was further reduced to <5% even in monolayers coated with only a low Sia-content HSM (Figure 3A, gray bars, P<0.05).
The clinical isolate A/SD/17/2008(H1N1) contains the oseltamivir-resistant mutation H275Y in the NA gene, as determined by cDNA sequencing (data not shown). An overall reduction of 12-44% in the number of cells infected by this strain was observed in oseltamivir-supplemented samples (Figure 3C, compare gray and black bars for each treatment). However, this effect was mostly not statistically significant, and all of the oseltamivir-supplemented samples had similar number of infected cells regardless of the mucus content (Figure 3C, compare all gray bars). Thus, the oseltamivir effect seen with this strain is likely attributed to inhibition of a secondary Sia binding site of N1 neuraminidase. In contrast, infection with A/Aichi/2/68(H3N2) strain was not affected by addition of 1 μM oseltamivir (Figure 3D, gray bars).
Taken together this suggests that NA sialidase activity is important to release the virus from the HSM layer.
Direct evidence for cleaving of Sias by viral Neuraminidase
Cleaving specificity of viral neuraminidase
HSM directly inhibits viral neuraminidase
Direct binding of IAV to HSM on magnetic beads array
We studied the interactions between IAV and host secreted mucins. Mucins are highly sialylated secretions usually standing (or rather flowing) between the viruses and their target cells on host epithelia. They are part of a “chemical shield”, packed with defensive molecules and innate immune cells , and also form a mechanical clearance mechanism [8, 38, 39]. The small number of existing studies of the human saliva inhibitory activity on IAV infection identify several salivary molecules as potential inhibitors including surfactants, secretory IgA, histatins, defensins and MUC5B mucin [28, 40]. In order to focus on the sialylated mucin aspect of the respiratory tract, we enriched the mucin component of human saliva and porcine submaxillary mucus samples by acid precipitation. Since the Sia content of the mucus samples was controlled throughout the experiments we were able to provide direct experimental evidence for the mechanism of mucus-protection during initial IAV infection.
We measured interactions of several different IAV strains with mucins in vitro. IAV bind to Sias on mucins (Figures 1, 2, and 7), and actively remove Sias from some mucin targets in vitro (Figure 4). We further show that a layer of HSM protects underlying cells from infection in Sia-dose dependent manner (Figure 3) and that this protection is augmented when viral NA (sialidase) is inhibited by oseltamivir in vitro (Figure 3A-B, gray bars). This inhibition was dependent on the source of mucins. Three of the IAV strains that were tested were not inhibited by PSM (Figure 3A-B, and D) but all of the tested strains were inhibited by HSM (Figure 3). Despite having a similar Neu5Ac content (1,800 and 1,500 pmol Neu5Ac, respectively), PSM (high) and HSM (low) do not have the same inhibitory effect on IAV infection. HSM (low) significantly reduced viral infection of three strains (A/PR/8/34(H1N1), A/SD/17/2008(H1N1), and A/Aichi/2/68(H3N2)), but PSM (high) only affected one of the strains (A/SD/17/2008(H1N1)) and was less efficient compared with HSM (low) (Figure 3C). Similarly, HSM competitively inhibited sialidase activity of viral NA as measured by 4MU-Sia cleavage assays, but PSM did not (Figure 6). Inhibition of NA activity with 1 μM oseltamivir reduced the number of infected cells in HSM-coated monolayers down to <10%, depending on the strain (Figure 3A-B), thus exhibiting an additive inhibitory effect. The NA of certain H1N1 strains, including the pandemic A/California/04/2009(H1N1) contains a functional secondary Sia-binding site, which can bind oseltamivir . Indeed oseltamivir reduced the number of cells infected with A/SD/2009(SOIV) by ~60% even in the absence of mucus (Figure 3B); a similar trend was observed for the other two H1N1 strains (Figure 3A and C), but not for the H3N2 strain (Figure 3D). However, an additive inhibitory effect was observed only for the HSM-coated cells (Figure 3A-B, see Additional file 2 for complete statistical analysis). Thus oseltamivir impairs both the escape from sialylated mucin decoy and the binding to target cells, by pandemic IAV.
Incidentally, early studies of human saliva have reported the presence of Neu2en5Ac (N-Acetyl-2,3-dehydro-2-deoxyneuraminic Acid) which is a very potent sialidase inhibitor [41, 42]. Modifications at the C-5 position of Neu2en5Ac were the first improved in vitro NA inhibitors . However, neither Neu2en5Ac nor the modified compound has an in vivo inhibition activity against IAV, possibly due to their rapid clearance . The presence of Neu2enAc in natural mucins might indicate that hosts also secrete their own NA-inhibitors. Alternatively, the presence of Neu2enAc could be due to activity of sialidases in saliva as this molecule also represents an intermediate product of sialidase activity.
Binding specificity of IAV strains is often studied using glycan arrays . These are highly informative but lack the 3D aspect of host cells as well as the overlaying secretions. Mucins can be conceived as the “fluid glycan arrays of nature” thus utilizing mucins for probing viral functions could be immensely informative. Furthermore, due to the constant clearance of airway mucus layer, attenuating the rate of IAV penetration through the mucus layer may be sufficient to prevent infection of the underlying epithelium. Considering the additive inhibitory effect of mucins and NA inhibition, it may be possible to design NA-inhibiting drugs that will have minimal side effects. Humans and other mammals express four NA genes (NEU 1-4), which are present in the brain among other tissues . It is conceivable that some of the reported neuropsychiatric adverse effects of oseltamivir and zanamavir [45, 46] could be caused by cross-reaction with endogenous NA in the brain. Our results warrant further studies to better establish the function of viral NA during in vivo infection and to establish ways by which its role during initial infection could be perturbed. Although most studies to date have focused on the potential ability of NA inhibitors to prevent egress of viral particles from infected cells, our study confirms that these agents might also have a critical impact on viral ingress. Specifically we demonstrate that NA role during initial infection includes cleavage of sialylated mucin decoys to allow virus penetration through the mucus layer. Thus a better understanding of the NA and HA interactions within the complex molecular “ecosystem” of host mucins during initial infection also promises new avenues for drug testing and development.
In this study, we show that mucins protect underlying cells from IAV infection in a sialic acid-dose dependent manner. We demonstrate direct binding to- and cleavage of- sialylated human mucins by multiple IAV strains. Our findings extend previous studies of inhibition by oseltamivir , providing experimental evidence for the specific molecular function of NA during initial infection. We show that NA sialidase activity is required to free virions from sialylated host mucins decoy. Thus sialylated host mucins have an important protective role against IAV infection. Sia composition, presentation and density, are critical for effective inhibition of the virus. Understanding IAV interactions with the complex molecular “ecosystem” of sialylated host mucins promises new avenues for drug testing and development.
Viruses and cultured cells
Three influenza strains A/PR/8/34(H1N1), A/Denver/1/57(H1N1) and A/Aichi/2/68(H3N2) were purchased from ATCC. In addition, three clinical virus isolates A/SD/21/2008(H1N1) and A/SD/17/2008(H1N1) and A/SD/1/2009(SOIV) [47, 48] were also used in this study. MDCK cells were maintained in Dulbeco’s modified Eagle’s medium (DMEM, Cellgro) supplemented with 10% fetal calf serum (FCS). All viruses were propagated in MDCK cells that were transferred to DMEM medium supplemented with 0.2% BSA fraction V (EMD), 25 mM HEPES buffer (Gibco), 2 μg/ml TPCK-trypsin (Worthington Corporation), and 1% penicillin/streptomycin (“DMEM-TPCK”).
Antibodies and lectins
Anti-Influenza A nucleoprotein monoclonal antibody (Anti-NP, MIA-NP-108) was purchased from eEnzyme, TKH2 monoclonal antibody (HB-9654) was purchased from ATCC, biotinylated Sambucus nigra lectin (bSNA) was purchased from Vector laboratories. Biotinylated donkey-anti-mouse IgG antibody and streptavidin conjugated horseradish peroxidase (SA-HRP) were purchased from Jackson Immunoresearch.
Biotinylated probes and mucins
Biotinylated-polyacrylamide (PAA) glycan probes were purchased from GlycoTech. Probes used in this study were: Neu5Ac-PAA-biotin, Neu5Gc-PAA-biotin, Neu5Ac-α2-6GalNAc-PAA-biotin, Neu5Gc-α2-6GalNAc-PAA-biotin, 3’ Sialyllactose-PAA-biotin, 6’ Sialyllactose-PAA-biotin, and Galβ1-3GalNAc-PAA-biotin. Human submaxillary mucins (HSM) and porcine submaxillary mucins (PSM) were biotinylated with EZ-link-NHS-PEG4-biotin (Thermo Scientific) according to the manufacturer’s instructions. Free biotin was removed by dialysis against PBS in a Slide-A-Lyzer dialysis device (Thermo Scientific).
Salivary mucins were produced as previously described with slight modifications . Briefly, human saliva was collected from healthy human donors into tubes and placed on ice, with the approval of the UCSD Human Research Protections Program (protocol #080011). Saliva samples were centrifuged 5 min at 10,000 g, and the pellet was discarded. One percent penicillin/streptomycin was added to the clear supernatant. Mucins were precipitated by adjusting to pH 3.5 with 50 mM hydrochloric acid (HCl), stirring over night at 4°C, and collecting at 800 g for 10 min. The pellet was washed with miliQ water, resuspended in miliQ water, adjusted to pH 7.0 and boiled for 10 min to inhibit protease and glycosidase activity. Mucins were dialyzed against water in a 10,000 MWCO Slide-A-Lyzer dialysis device (Thermo Scientific), and stored in aliquots at -80°C. PSM was prepared following a published protocol . The purity of the mucins was determined by analysis of monosaccharide composition using high performance anion exchange chromatography with Pulsed Amperometric Detection (HPAEC-PAD). In addition, amino acid content was determined by Gas Chromatography Mass Spectrometry (GC-MS) methods (Additional file 1) , both done at the UCSD Glycotechnology Core Resource.
Sialic acid quantification by DMB-HPLC
Sia content was determined by 1,2-diamino-4,5-methylenedioxybenzene dihydrochloride–high performance liquid chromatography analysis (DMB-HPLC) according to a published protocol . Samples were incubated for 1 h with 0.1 M HCl at 80°C to release Sias, and filtered through microcon-10kDa filtration device (Millipore). Free Sias were incubated for 2.5 h at 50°C in the dark with 7 mM DMB (Sigma), 0.75 M 2-mercapto-ethanol, 18 mM Na-hydrosulfite in 1.4 M acetic acid. Sias were separated on 250×4.6 mm Gemini C18 column (Phenomenex) with 7% MeOH, 8% Acetonitrile, 85% H2O solution at 0.9 ml/min using the ELITE Lachrom HPLC system (Hitachi). DMB-labeled Sias were detected at EX=373 EM=488.
Virus tissue-binding assay
Snap frozen human tissues were obtained as part of the UCSD approved IRB protocol #101754. Tissues were embedded in optimal cutting media (OCT) and cut into 5 micrometer-thick sections by Leica CM1800 cryomicrotome. Tissues were air-dried and fixed for 30 min in 10% buffered formalin, blocked with an Avidin/Biotin blocking kit according to the manufacturer’s instructions (Vector Laboratories), followed by a 20 min incubation with 0.3% H2O2 in PBS, and a 10 min incubation with 1% bovine serum albumin in PBS (BSA/PBS). This standard procedure does not affect glycan presentation or virus binding to the tissue. Virus was concentrated using a microcon-10 filtration system (Millipore); approximately 600 hemagglutinating Units (HAU) were mixed 1:1 with 1% BSA/PBS and incubated on the tissues for 1.5 h at room temperature . The virus was fixed after rinsing with 1:1 methanol/acetone for 15 min at -20°C. Tissue sections were then incubated with Anti-NP antibody (1:100) for 1 hour at room temperature, followed by 30 min incubation with biotinylated donkey-anti-mouse IgG antibody (1:500), and a 30 min incubation with SA-HRP (1:500). As a control for sialic acid specific binding, some tissue sections were incubated with 0.25 mM Arthrobacter ureafaciens sialidase in 50 mM sodium Acetate buffer for 2 hours at 37°C, or with 2 mM ice-cold sodium periodate for 30 min at 4°C prior to formalin fixation. Tissues were also stained with TKH2 antibody (1:50) over night at 4°C, followed by 30 min incubation with biotinylated-donkey-anti-mouse-IgG antibody (1:500), and 30 min with SA-HRP (1:500), or with biotinylated SNA (1:1000) 1 h at room temperature, followed by 30 min incubation with SA-HRP (1:500). Color was developed using Chromagen AEC (SK 4200, Vector laboratories) and nuclei were counterstained with Mayer Hematoxylin (Sigma-Aldrich). All antibodies and lectins were diluted in 1% BSA/PBS. Hematoxylin and Eosin (H&E), and Periodic Acid Schiff (PAS, Electron Microscopy Sciences) staining was done as previously described . Microscopy slides were scanned with a NanoZoomer microscope (NanoZoomer 2.0 series, Hamamatsu, Japan).
Infection inhibition assay
MDCK cells in 16-well Lab-Tek chamber slides (Nunc, Thermo Fisher Scientific, NY) were washed with PBS, and layered with 60 μl HSM or PSM diluted in PBS with Ca2+ and Mg2+ (Gibco) to 30, 64, or 240 pmol Sia/μl, or with 60 μl PBS with Ca2+ and Mg2+. Virus was diluted in DMDM-TPCK media to 109 TCID50/ml, and aliquot into two vials, one of the vials contained 2 μM oseltamivir. TCID50 of the virus was calculated according to Spearman-Karber method. The cells were inoculated with 60 μl virus for 1 h at 37°C, washed three times with PBS, and fresh DMEM-TPCK media was added. Following 5.5 h incubation at 37°C, the cells were washed with PBS, fixed for 20 min with 3% paraformaldehyde, permeabilized for 4 min with 0.2% Triton X-100 (Sigma) in PBS, and blocked with 1% BSA/PBS. Media chamber and gasket were detached from the slides and the cells were incubated with Anti-NP antibody (1:100) for 1 hour at room temperature, followed by 30 min incubation with Alexa-488 conjugated donkey-anti-mouse IgG antibody (1:500), Both antibodies were diluted in 1% BSA/PBS. Nuclei were counterstained with Hoechst stain (Life Technologies, NY). Samples were analyzed with DeltaVision Deconvolution Microscope equipped with Coolsnap HQ camera (Applied Precision, WA). Twenty randomly selected images were taken at ×10 magnification from each sample, and the number of cells in each image was determined using Volocity image analysis software (PerkinElmer, MA). The experiment was repeated three times. Data was analyzed by 3-way ANOVA, corrected for multiple comparisons using Tukey’s HSD.
4MU-Sia cleavage assay
NA activity was measured using 2’-(4-methylumbelliferyl)-α-D-N-acetylneuraminic acid (4MU-Neu5Ac)(Sigma-Aldrich), 4MU-N-glycolyl-neuraminic acid (4MU-Neu5Gc) and 4MU-2-keto-3-deoxynononic acid (4MU-Kdn) (both gifts from Ken Kitajima, Nagoya University, Japan) as a substrate as previously described . Briefly, 4MU-Sia compounds were used as substrates in 33 mM 2-(N-Morpholino)ethanesulphonic acid (MES, Sigma-Aldrich), 120 mM NaCl2, 4 mM CaCl2 buffer adjusted to pH 6.5 (MES/CaCl2/NaCl2 buffer). In 96-well plates, 20 μl 4MU-Sia was added to achieve a final concentration of 300-10,000 pmol sia/well. Then 30 μl virus diluted in MES/CaCl2/NaCl2 buffer was added, and incubated for 1 h at 37°C in dark. For some wells oseltamivir was added at a final concentration of 1 μM or 17.5 μM prior to incubation. HSM and PSM inhibition of 4MU-Sia cleavage was done by adding mucus at 4,500-10,000 pmol sia/well to wells containing 25-400 pmol 4MU-Neu5Ac prior to addition of virus. The reaction was stopped by adding 150 μl of 25% ethanol, 0.1M glycine pH 10.7 and measured at excitation 365 nm and emission 450 nm in a SpectraMax M3 spectrophotometer (Molecular Devices).
Magnetic beads sialoglycan array
Sera-Mag SpeedBeads Blocked Streptavidin (Thermo-Fisher cat# 2115-2104-011150) magnetic beads were washed in PBS, and incubated with biotinylated-HSM (bHSM), biotinylated-PSM (bPSM) or biotinylated-polyacrylamide (PAA) glycan probes in 125 mM PBS pH 7.4 for 1 h at room temperature. The glycan-conjugated beads were washed with PBS and incubated with virus diluted in PBS containing 16 μM oseltamivir to 32-64 HAU for 1 h at 37°C with rotation. Beads were washed three times with oseltamivir-containing PBS, and once with PBS. NA activity of bead-bound virus was quantified by adding 100 nmol of 4MU-Neu5Ac and incubating for 30 min at 37°C. Cleavage of 4MU-Neu5Ac, quantified as described above, is directly proportional to the number of IAV bound to the beads.
Mucus cleaving assay
Sera-Mag SpeedBeads Blocked Streptavidin (Thermo-Fisher) magnetic beads were washed in PBS, and incubated with bHSM, bPSM, or Galβ1-3GalNAc-PAA-biotin in 125 mM PBS pH 7.4 for 1 h at room temperature. The glycan-conjugated beads were washed, resuspended in 50 μl MES/CaCl2/NaCl2 buffer, and 50 μl virus (600-2048 HAU) was added. As a control, 50 μl DMEM-TPCK media was added to the beads. Beads were incubated for 1.5 h at room temperature with rotation, washed extensively with PBS, and fixed with 10% buffered formalin for 30 min. Beads were washed, 100 μl 0.1 M HCl was added and Sias were released from the beads by a 30 min incubation at 80°C. Sias were then quantified by DMB-HPLC. Galβ1-3GalNAc-PAA-biotin conjugated beads were used as control for non-specific signal.
The data sets supporting the results of this article are included within the article and its additional files.
We thank Dr. Ken Kitajima (Nagoya University, Japan) for providing the 4MU-N-glycolyl-neuraminic acid and 4MU- 2-keto-3-deoxynononic acid compounds. Dr. Biswa Choudhury at the Glycotechnology Core Facility at UCSD for amino acid and monosaccharide analysis. Dr. Stevan A. Springer (UCSD) for helping with the statistical analysis. Dr. Eillen Tecle (UCSD) for helpful comments on the manuscript. Images were acquired at the Neuroscience Microscopy Shared Facility, UCSD. Human tissues were obtained as part of a UCSD approved IRB protocol. This work was supported by the University of California Laboratory Fees Research Program Award (118645), a Cooperative Agreement from the National Institute of Allergy and Infectious Diseases (1U01AI074521), a grant from the National Institute of Neurological Disorders and Stroke (P30 NS047101), and UCSD Cancer Center Specialized Support grant (P30 CA23100).
- Andrewes CH: Nomenclature of viruses. Nature 1954, 173: 620-621.View ArticleGoogle Scholar
- Burnet FM: Mucoproteins in relation to virus action. Physiol Rev 1951, 31: 131-150.PubMedGoogle Scholar
- Fahy JV, Dickey BF: Airway mucus function and dysfunction. N Engl J Med 2010, 363: 2233-2247. 10.1056/NEJMra0910061PubMedPubMed CentralView ArticleGoogle Scholar
- Cone RA: Barrier properties of mucus. Adv Drug Deliv Rev 2009, 61: 75-85. 10.1016/j.addr.2008.09.008PubMedView ArticleGoogle Scholar
- Duez JM, Sixt N, Pechinot A: Influenza virus infection: don’t forget the role of the mucociliary system! J Antimicrob Chemother 2009, 63: 421-422.PubMedView ArticleGoogle Scholar
- Matrosovich M, Klenk HD: Natural and synthetic sialic acid-containing inhibitors of influenza virus receptor binding. Rev Med Virol 2003, 13: 85-97. 10.1002/rmv.372PubMedView ArticleGoogle Scholar
- Lillehoj ER, Kim KC: Airway mucus: its components and function. Arch Pharm Res 2002, 25: 770-780. 10.1007/BF02976990PubMedView ArticleGoogle Scholar
- Knowles MR, Boucher RC: Mucus clearance as a primary innate defense mechanism for mammalian airways. J Clin Invest 2002, 109: 571-577.PubMedPubMed CentralView ArticleGoogle Scholar
- von Itzstein M, Thomson R: Anti-influenza drugs: the development of sialidase inhibitors. Handb Exp Pharmacol 2009, 189: 111-154. 10.1007/978-3-540-79086-0_5PubMedView ArticleGoogle Scholar
- Matrosovich MN, Matrosovich TY, Gray T, Roberts NA, Klenk HD: Neuraminidase is important for the initiation of influenza virus infection in human airway epithelium. J Virol 2004, 78: 12665-12667. 10.1128/JVI.78.22.12665-12667.2004PubMedPubMed CentralView ArticleGoogle Scholar
- Huang RT, Rott R, Wahn K, Klenk HD, Kohama T: The function of the neuraminidase in membrane fusion induced by myxoviruses. Virology 1980, 107: 313-319. 10.1016/0042-6822(80)90299-8PubMedView ArticleGoogle Scholar
- Huang RT, Dietsch E, Rott R: Further studies on the role of neuraminidase and the mechanism of low pH dependence in influenza virus-induced membrane fusion. J Gen Virol 1985, 66: 295-301. 10.1099/0022-1317-66-2-295PubMedView ArticleGoogle Scholar
- Zalewska A, Zwierz K, Zolkowski K, Gindzienski A: Structure and biosynthesis of human salivary mucins. Acta Biochim Pol 2000, 47: 1067-1079.PubMedGoogle Scholar
- Klein A, Carnoy C, Wieruszeski JM, Strecker G, Strang AM, van Halbeek H, Roussel P, Lamblin G: The broad diversity of neutral and sialylated oligosaccharides derived from human salivary mucins. Biochemistry 1992, 31: 6152-6165. 10.1021/bi00141a028PubMedView ArticleGoogle Scholar
- Thornton DJ, Rousseau K, McGuckin MA: Structure and function of the polymeric mucins in airways mucus. Annu Rev Physiol 2008, 70: 459-486. 10.1146/annurev.physiol.70.113006.100702PubMedView ArticleGoogle Scholar
- Gagneux P, Cheriyan M, Hurtado-Ziola N, van der Linden EC, Anderson D, McClure H, Varki A, Varki NM: Human-specific regulation of alpha 2-6-linked sialic acids. J Biol Chem 2003, 278: 48245-48250. 10.1074/jbc.M309813200PubMedView ArticleGoogle Scholar
- Gerken TA, Jentoft N: Structure and dynamics of porcine submaxillary mucin as determined by natural abundance carbon-13 NMR spectroscopy. Biochemistry 1987, 26: 4689-4699. 10.1021/bi00389a015PubMedView ArticleGoogle Scholar
- Varki NM, Strobert E, Dick EJJ, Benirschke K, Varki A: Biomedical differences between human and nonhuman hominids: potential roles for uniquely human aspects of sialic acid biology. Annu Rev Pathol 2011, 6: 365-393. 10.1146/annurev-pathol-011110-130315PubMedView ArticleGoogle Scholar
- Van Poucke SG, Nicholls JM, Nauwynck HJ, Van Reeth K: Replication of avian, human and swine influenza viruses in porcine respiratory explants and association with sialic acid distribution. Virol J 2010, 7: 38. 10.1186/1743-422X-7-38PubMedPubMed CentralView ArticleGoogle Scholar
- Liu X, Luo M, Guo C, Yan Z, Wang Y, Engelhardt JF: Comparative biology of rAAV transduction in ferret, pig and human airway epithelia. Gene Ther 2007, 14: 1543-1548. 10.1038/sj.gt.3303014PubMedPubMed CentralView ArticleGoogle Scholar
- Matrosovich MN, Matrosovich TY, Gray T, Roberts NA, Klenk HD: Human and avian influenza viruses target different cell types in cultures of human airway epithelium. Proc Natl Acad Sci U S A 2004, 101: 4620-4624. 10.1073/pnas.0308001101PubMedPubMed CentralView ArticleGoogle Scholar
- Ozawa M, Kawaoka Y: Cross talk between animal and human influenza viruses. Annu Rev Anim Biosci 2013, 1: 21-42. 10.1146/annurev-animal-031412-103733PubMedView ArticleGoogle Scholar
- Nicholls JM, Bourne AJ, Chen H, Guan Y, Peiris JS: Sialic acid receptor detection in the human respiratory tract: evidence for widespread distribution of potential binding sites for human and avian influenza viruses. Respir Res 2007, 8: 73. 10.1186/1465-9921-8-73PubMedPubMed CentralView ArticleGoogle Scholar
- Jayaraman A, Chandrasekaran A, Viswanathan K, Raman R, Fox JG, Sasisekharan R: Decoding the distribution of glycan receptors for human-adapted influenza a viruses in ferret respiratory tract. PLoS One 2012, 7: e27517. 10.1371/journal.pone.0027517PubMedPubMed CentralView ArticleGoogle Scholar
- Cohen M, Varki NM, Jankowski MD, Gagneux P: Using unfixed, frozen tissues to study natural mucin distribution. J Vis Exp 2012, 67: e3928.PubMedGoogle Scholar
- Varki A, Kannagi R, Toole BP: Glycosylation Changes in Cancer: Chapter 44. In Essentials of Glycobiology. 2nd edition. Edited by: Varki A, Cummings RD, Esko JD, Freeze HH, Stanley P, Bertozzi CR, Hart GW, Etzler ME. Cold Spring Harbor, New York: Cold Spring Harbor Laboratory Press; 2009:617-632.Google Scholar
- Gahmberg CG, Andersson LC: Selective radioactive labeling of cell surface sialoglycoproteins by periodate-tritiated borohydride. J Biol Chem 1977, 252: 5888-5894.PubMedGoogle Scholar
- White MR, Helmerhorst EJ, Ligtenberg A, Karpel M, Tecle T, Siqueira WL, Oppenheim FG, Hartshorn KL: Multiple components contribute to ability of saliva to inhibit influenza viruses. Oral Microbiol Immunol 2009, 24: 18-24. 10.1111/j.1399-302X.2008.00468.xPubMedPubMed CentralView ArticleGoogle Scholar
- Sung JC, Van Wynsberghe AW, Amaro RE, Li WW, McCammon JA: Role of secondary sialic acid binding sites in influenza N1 neuraminidase. J Am Chem Soc 2010, 132: 2883-2885. 10.1021/ja9073672PubMedPubMed CentralView ArticleGoogle Scholar
- Nayak DP, Reichl U: Neuraminidase activity assays for monitoring MDCK cell culture derived influenza virus. J Virol Methods 2004, 122: 9-15. 10.1016/j.jviromet.2004.07.005PubMedView ArticleGoogle Scholar
- Nadano D, Iwasaki M, Endo S, Kitajima K, Inoue S, Inoue Y: A naturally occurring deaminated neuraminic acid, 3-deoxy-D-glycero-D-galacto-nonulosonic acid (KDN): Its unique occurrence at the nonreducing ends of oligosialyl chains in polysialoglycoprotein of rainbow trout eggs. J Biol Chem 1986, 261: 11550-11557.PubMedGoogle Scholar
- Angata T, Varki A: Chemical diversity in the sialic acids and related alpha-keto acids: an evolutionary perspective. Chem Rev 2002, 102: 439-469. 10.1021/cr000407mPubMedView ArticleGoogle Scholar
- Koerner I, Matrosovich MN, Haller O, Staeheli P, Kochs G: Altered receptor specificity and fusion activity of the haemagglutinin contribute to high virulence of a mouse-adapted influenza a virus. J Gen Virol 2012, 93: 970-979. 10.1099/vir.0.035782-0PubMedView ArticleGoogle Scholar
- Childs RA, Palma AS, Wharton S, Matrosovich T, Liu Y, Chai W, Campanero-Rhodes MA, Zhang Y, Eickmann M, Kiso M, Hay A, Matrosovich M, Feizi T: Receptor-binding specificity of pandemic influenza A (H1N1) 2009 virus determined by carbohydrate microarray. Nat Biotechnol 2009, 27: 797-799. 10.1038/nbt0909-797PubMedPubMed CentralView ArticleGoogle Scholar
- Suzuki Y, Matsunaga M, Matsumoto M: N-Acetylneuraminyllactosylceramide, GM3-NeuAc, a new influenza a virus receptor which mediates the adsorption-fusion process of viral infection: binding specificity of influenza virus A/Aichi/2/68 (H3N2) to membrane-associated GM3 with different molecular species of sialic acid. J Biol Chem 1985, 260: 1362-1365.PubMedGoogle Scholar
- Matrosovich MN, Gambaryan AS, Tuzikov AB, Byramova NE, Mochalova LV, Golbraikh AA, Shenderovich MD, Finne J, Bovin NV: Probing of the receptor-binding sites of the H1 and H3 influenza A and influenza B virus hemagglutinins by synthetic and natural sialosides. Virology 1993, 196: 111-121. 10.1006/viro.1993.1459PubMedView ArticleGoogle Scholar
- Tamura S, Kurata T: Defense mechanisms against influenza virus infection in the respiratory tract mucosa. Jpn J Infect Dis 2004, 57: 236-247.PubMedGoogle Scholar
- Vareille M, Kieninger E, Edwards MR, Regamey N: The airway epithelium: soldier in the fight against respiratory viruses. Clin Microbiol Rev 2011, 24: 210-229. 10.1128/CMR.00014-10PubMedPubMed CentralView ArticleGoogle Scholar
- Lillehoj EP, Kato K, Lu W, Kim KC: Cellular and molecular biology of airway mucins. Int Rev Cell Mol Biol 2013, 303: 139-202.PubMedView ArticleGoogle Scholar
- Hartshorn KL, White MR, Mogues T, Ligtenberg T, Crouch E, Holmskov U: Lung and salivary scavenger receptor glycoprotein-340 contribute to the host defense against influenza A viruses. Am J Physiol Lung Cell Mol Physiol 2003, 285: L1066-L1076.PubMedView ArticleGoogle Scholar
- Meindl P, Tuppy H: 2-Deoxy-2,3-dehydrosialic acids. II. Competitive inhibition of Vibrio cholerae neuraminidase by 2-deoxy-2,3-dehydro-N-acylneuraminic acids. Hoppe Seylers Z Physiol Chem 1969, 350: 1088-1092. 10.1515/bchm2.1969.350.2.1088PubMedView ArticleGoogle Scholar
- Schauer R, Haverkamp J, Wember M, Kamerling JP, Vliegenthart JF: N-acetyl-9-O-L-lactylneuraminic acid, a new acylneuraminic acid from bovine submandibular gland. Eur J Biochem 1976, 62: 237-242. 10.1111/j.1432-1033.1976.tb10153.xPubMedView ArticleGoogle Scholar
- Stevens J, Blixt O, Paulson JC, Wilson IA: Glycan microarray technologies: tools to survey host specificity of influenza viruses. Nat Rev Microbiol 2006, 4: 857-864. 10.1038/nrmicro1530PubMedView ArticleGoogle Scholar
- Monti E, Bonten E, D’Azzo A, Bresciani R, Venerando B, Borsani G, Schauer R, Tettamanti G: Sialidases in vertebrates: a family of enzymes tailored for several cell functions. Adv Carbohydr Chem Biochem 2010, 64: 403-479.PubMedView ArticleGoogle Scholar
- Toovey S, Rayner C, Prinssen E, Chu T, Donner B, Thakrar B, Dutkowski R, Hoffmann G, Breidenbach A, Lindemann L, Carey E, Boak L, Gieschke R, Sacks S, Solsky J, Small I, Reddy D: Assessment of neuropsychiatric adverse events in influenza patients treated with oseltamivir: a comprehensive review. Drug Saf 2008, 31: 1097-1114.PubMedView ArticleGoogle Scholar
- Smith SM, Gums JG: Antivirals for influenza: strategies for use in pediatrics. Paediatr Drugs 2010, 12: 285-299. 10.2165/11532530-000000000-00000PubMedView ArticleGoogle Scholar
- Holmes EC, Ghedin E, Halpin RA, Stockwell TB, Zhang XQ, Fleming R, Davey R, Benson CA, Mehta S, Taplitz R, Liu YT, Brouwer KC, Wentworth DE, Lin X, Schooley RT: Extensive geographical mixing of 2009 human H1N1 influenza A virus in a single university community. J Virol 2011, 85: 6923-6929. 10.1128/JVI.00438-11PubMedPubMed CentralView ArticleGoogle Scholar
- Lu Q, Zhang XQ, Pond SL, Reed S, Schooley RT, Liu YT: Detection in 2009 of the swine origin influenza A (H1N1) virus by a subtyping microarray. J Clin Microbiol 2009, 47: 3060-3061. 10.1128/JCM.01243-09PubMedPubMed CentralView ArticleGoogle Scholar
- Banda K, Gregg CJ, Chow R, Varki NM, Varki A: Metabolism of vertebrate amino sugars with N-glycolyl groups: mechanisms underlying gastrointestinal incorporation of the non-human sialic acid xeno-autoantigen N-glycolylneuraminic acid. J Biol Chem 2012, 287: 28852-28864. 10.1074/jbc.M112.364182PubMedPubMed CentralView ArticleGoogle Scholar
- Faure M, Moennoz D, Montigon F, Fay LB, Breuille D, Finot PA, Ballevre O, Boza J: Development of a rapid and convenient method to purify mucins and determine their in vivo synthesis rate in rats. Anal Biochem 2002, 307: 244-251. 10.1016/S0003-2697(02)00048-9PubMedView ArticleGoogle Scholar
- Hara S, Yamaguchi M, Takemori Y, Furuhata K, Ogura H, Nakamura M: Determination of mono-O-acetylated N-acetylneuraminic acids in human and rat sera by fluorometric high-performance liquid chromatography. Anal Biochem 1989, 179: 162-166. 10.1016/0003-2697(89)90218-2PubMedView ArticleGoogle Scholar
- Kimble B, Nieto GR, Perez DR: Characterization of influenza virus sialic acid receptors in minor poultry species. Virol J 2010, 7: 365. 10.1186/1743-422X-7-365PubMedPubMed CentralView ArticleGoogle Scholar
This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.