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. 2011 Aug 16;22(2):174–180. doi: 10.1093/glycob/cwr112

Receptor-binding specificity of the human parainfluenza virus type 1 hemagglutinin–neuraminidase glycoprotein

Irina V Alymova 2,1, Allen Portner 2,5, Vasiliy P Mishin 3, Jonathan A McCullers 2, Pamela Freiden 2, Garry L Taylor 4
PMCID: PMC3255505  PMID: 21846691

Abstract

The hemagglutinin–neuraminidase (HN) glycoprotein is utilized by human parainfluenza viruses for binding to the host cell. By the use of glycan array assays, we demonstrate that, in addition to the first catalytic-binding site, the HN of human parainfluenza virus type 1 has a second site for binding covered by N-linked glycan. Our data suggest that attachment of the first site to sialic acid (SA)-linked receptors triggers exposure of the second site. We found that both sites bind to α2-3-linked SAs with a preference for a sialyl-Lewisx motif. Binding to α2-3-linked SAs with a sulfated sialyl-Lewis motif as well as to α2-8-linked SAs was unique for the second binding site. Neither site recognizes α2-6-linked oligosaccharides.

Keywords: binding, glycan array, hemagglutinin–neuraminidase, parainfluenza, receptor

Introduction

Human parainfluenza viruses (hPIVs; Paramyxoviridae family) cause a spectrum of respiratory tract infections in young children, the elderly and immunocompromised individuals (Knott et al. 1994; Marx et al. 1997; Reed et al. 1997). They utilize a major surface glycoprotein, hemagglutinin–neuraminidase (HN), to initiate infection [via binding to sialic acid (SA)-containing receptors on the cell surface] and to release progeny viruses [via its neuraminidase (NA) activity] at the end of the replication cycle (Karron and Collins 2007). HN is also involved in the promotion of fusion through an interaction with the fusion (F) glycoprotein (Takimoto et al. 2002; Lamb et al. 2006).

There is a continuing discussion about whether the active sites for receptor binding and NA activities are separated on paramyxovirus HN molecules (Scheid and Choppin 1974; Merz et al. 1981; Morrison and Portner 1991). A crystallographic study of Newcastle disease virus (NDV; an avian paramyxovirus) HN demonstrated the presence of two SA recognition sites on the glycoprotein molecule: a site involved in both receptor binding and NA functionality (hereafter referred to as Site 1; Crennell et al. 2000) and a second receptor-binding site (hereafter referred to as Site 2; Zaitsev et al. 2004). Subsequent studies proposed that both sites participate in NDV binding to cells (Bousse et al. 2004).

The hPIVs were not expected to have an additional Site 2 on the HN molecule. Indeed, the crystal structure of the hPIV-3 HN (the only one among the four types of hPIVs characterized so far) did not reveal the presence of Site 2 on the glycoprotein (Lawrence et al. 2004). However, we have recently shown that Site 2 in hPIV-3 HN might be shielded by an N-linked glycan at residue 523 (Mishin et al. 2010). The masking of Site 2 by an N-linked glycan is not unique to hPIV-3. Other paramyxoviruses were also proposed to shield their binding sites with N-linked glycans. Among them are measles virus (Hashiguchi et al. 2007), Nipah virus (Guillaume et al. 2006) and hPIV-1 (Alymova et al. 2008).

The mutant virus exhibiting an unmasked Site 2 on hPIV-1 HN was isolated during our resistance study with selective parainfluenza virus HN inhibitor BCX 2798 (BioCryst Pharmaceuticals, Inc., Birmingham, AL). BCX 2798 was developed based on binding of the lead compound Neu5Ac2en (which inhibits most NAs or sialidases) to Site 1 of NDV HN (Crennell et al. 2000; Takimoto et al. 2000; Figure 1). The analysis of the Neu5Ac2en-NDV HN binding displayed high conservancy of the amino acid residues forming the Site 1 among all paramyxoviruses, thus, allowing development of hPIVs HN inhibitors (as BCX 2798) based on the NDV HN model. Due to the inhibition of both binding and NA activity, BCX 2798 showed high efficacy against parainfluenza virus infections in vitro and in vivo (Alymova et al. 2004; 2005; 2009).

Fig. 1.

Fig. 1.

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Chemical structures of Neu5Ac2en and BCX 2798 compounds. Neu5Ac2en (2-deoxy-2,3-dehydro-N-acetyl neuraminic acid); BCX 2798 (4-azido-5-isobutyrylamino-2,3-didehydro-2,3,4,5-tetradeoxy-d-glycero-d-galacto-2-nonulopyranosic acid).

Isolated with BCX 2798, this parainfluenza virus with an unmasked Site 2 exhibited the Asn-to-Ser mutation at residue 173 of hPIV-1 HN which resulted in the loss of the N-linked glycan (Alymova et al. 2008). The mutant failed to be efficiently eluted from erythrocytes or released from the surface of infected cells (indicating increased binding) and lost its sensitivity to BCX 2798 (Site 1 inhibitor) in hemagglutination inhibition tests, which measure the ability of the compound to inhibit virus' binding to SA-containing receptors on the red blood cells. The mutant's NA activity and its susceptibly to BCX 2798 in NA inhibition assays did not differ from those of the virus without mutation, indicating that all effects seen were not due to changes in Site 1, but due to the appearance of Site 2. Computer modeling data suggested that Site 2 in hPIV-1 HN occupies a similar location to that identified in NDV HN, namely at the dimer interface with the binding site involving residues from both monomers (Figure 2). The remoteness of hPIV-1 HN residue 173 from Site 1 and its close proximity to Site 2 (only ∼8 Å) was also consistent with the suggestion that the loss of the N-linked glycan at residue 173 exposed Site 2 on hPIV-1 HN.

Fig. 2.

Fig. 2.

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Schematic view of the hPIV-1 HN dimer. The hPIV-1 HN dimer is modeled on the hPIV-3 HN dimer structure as reported previously (Alymova et al. 2008). The two monomers are colored in green and cyan, and show the location of Sites 1 and 2. The approximate location of Asn173 is indicated. The N-linked oligosaccharide, shown as a black oval, occludes Site 2.

In the current study, by the use of glycan array assays and the selective parainfluenza virus HN inhibitor BCX 2798, we confirm the presence of an additional receptor-binding site covered by N-linked glycan on the hPIV-1 HN molecule, characterize receptor-binding specificity of Sites 1 and 2 and report unique binding of Site 2 to α2-8-linked Neu5Ac. Based on the data from the glycan array assays, we suggest that the attachment of Site 1 to SA-linked receptors triggers the exposure of Site 2 and propose the involvement of Site 2 in the hPIV-1 binding, thus, suggesting its biological significance.

Results and discussion

The receptor specificity of hPIV-1 has been studied previously using thin-layer chromatography analysis of ganglioside binding (Suzuki et al. 2001), and more recently, by a similar glycan array analysis (Amonsen et al. 2007) developed by the Consortium for Functional Glycomics (CFG) which facilitates the identification of carbohydrate structures bound by lectins.

To confirm the presence of a second site covered by an N-linked glycan at residue 173 on hPIV-1 HN and to determine its receptor-binding specificity, we utilized the Sendai virus (SeV) reverse genetic system and rescued a recombinant virus in which the SeV HN gene was substituted with that of hPIV-1 (C-39 strain; hereafter referred to as the parent virus). Then, the HN gene in the parent virus was mutated at residue Asn173 to Ser to remove the N-linked glycan (hereafter virus referred to as the N173S mutant). The parent and N173S mutant were treated with a selective inhibitor of Site 1 BCX 2798 at a final concentration of 1, 10, 100 and 1000 µM or phosphate-buffered saline (PBS). Then, receptor-binding specificity expressed in relative fluorescent units (RFUs) was determined by glycan array assays. The assays with 1 and 10 µM of BCX 2798 did not reveal clear differences in binding profiles between the two viruses due to the inability of these doses to inhibit binding activity of Site 1 (data not shown). Application of higher BCX 2798 concentrations (100 and 1000 µM) allowed us to differentiate receptor-binding specificities of Sites 1 and 2 on hPIV-1 HN. The binding profiles of the parent and the N173S mutant in the presence of 1000 µM of BCX 2798 were similar to those determined in the presence of 100 µM of compound (data not shown). Figure 3 shows the results of the glycan array analysis for the parent virus, the parent virus in the presence of 100 μM BCX 2798, the N173S mutant and the mutant in the presence of 100 μM BCX 2798. The binding profile of the parent virus confirmed previous studies by other research groups (Suzuki et al. 2001; Kogure et al. 2006; Amonsen et al. 2007), namely that the hPIV-1 HN recognizes the oligosaccharides of the form Neu5Acα2-3Galβ1-4GlcNAc (glycans 234, 237 and, possibly, 282 since the low signal of 6155 ± 1549 RFU was observed), a 6′-sulfo-Gal derivative of glycan 282 (glycan 44), various sialyl-Lewisx (sLex) epitopes (glycans 228, 229, 230, 231 and 232) and a 6′-sulfo-Gal derivative of sLex (glycan 207), as well as those having a branched GalNAc on the galactose (glycans 208, 209 and 210). In contrast to the study by Amonsen et al. (2007), there was no significant binding detected for N-glycolyl-sLex (glycan 259) with the parent virus. In the presence of the inhibitor that blocks Site 1, there was a much reduced fluorescence signal observed for the parent virus with only two glycans 207 (6′-sulfo derivative of sLex) and 228 (namely an extended sialyl-Lewisx ligand sLex-Lex-Lex) giving relatively high signals (>7000 and 10,000 RFU, respectively). Of note, glycan 228 was the one that exhibited the highest signal in the absence of inhibitor. The N173S mutant retains Site 1, but lacks the N-glycosylation that is proposed to occlude Site 2. Our data indicate that the binding profile for this mutant was similar to that of the parent virus: with a modestly higher or lower intensity, the N173S mutant binds to the same glycans as does the parent virus. Similar to the parent, the highest signals with the mutant virus were observed from binding to glycans 208, 209, 210, 228 and 232. We noted the appearance of a small signal (∼5000 RFU) for ganglioside GD3 that has the structure Neu5Acα2-8Neu5Acα2-3-Galβ1-4Glc (glycan 251) in the N173S binding data that increases substantially (>20,000 RFU) in the presence of 100 μM BCX 2798. Interestingly, other research groups previously reported the ability of the hPIV-1 HN to cleave α2-8-linked SA (Amonsen et al. 2007), as does NDV (Corfield et al. 1982). When screened in the presence of inhibitor, the mutant, in contrast to the parent virus, retained its high binding ability to the majority of SA-linked oligosaccharides. In particular, the N173S recognized sLex (glycans 228–232), its 6′-sulfo derivative (glycan 207), oligosaccharides with GalNAc on the galactose (glycan 209), the Neu5Acα2-3Galβ1-4GlcNAc form of the oligosaccharide (glycans 234 and 237) and its 6′-sulfo-Gal derivative (glycan 44). Similar to the parent virus, the binding of the N173S mutant to glycans 208 and 210 was significantly reduced (more than 10 times) in the presence of inhibitor of Site 1.

Fig. 3.

Fig. 3.

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Glycan array analysis showing binding of the parent and the N173S mutant. The binding of virus to each glycan is displayed in RFU. The average from four replicates for each glycan is plotted with error bars indicating standard error of mean (SEM). The glycan numbers refer to those on version 3.2 of the printed array. Structures for glycan numbers: 44, Neu5Acα2-3(6OSO3)Galβ1-4GlcNAc; 207, Neu5Acα2-3(6OSO3)Galβ1-4(Fucα1-3)GlcNAc; 208, Neu5Acα2-3(GalNAcβ1-4)Galβ1-4GlcNAc; 209, Neu5Acα2-3(GalNAcβ1-4)Galβ1-4GlcNAc; 210, Neu5Acα2-3(GalNAcβ1-4)Galβ1-4Glc; 228, Neu5Acα2-3Galβ1-4(Fucα1-3)GlcNAcb1-3Galβ1-4(Fucα1-3)GlcNAcb1-3Galβ1-4(Fucα1-3)GlcNAc; 229, Neu5Acα2-3Galβ1-4(Fucα1-3)GlcNAc; 230, Neu5Acα2-3Galβ1-4(Fucα1-3)GlcNAc; 231, Neu5Acα2-3Galβ1-4(Fucα1-3)GlcNAcβ1-3Gal; 232, Neu5Acα2-3Galβ1-4(Fucα1-3)GlcNAcβ1-3Galβ1-4GlcNAc; 234, Neu5Acα2-3Galβ1-4GlcNAcβ1-3Galβ1-4GlcNAcβ1-3Galβ1-4GlcNAc; 282, Neu5Aα2-3Galβ1-4GlcNAcβ1-3Galb1-3GlcNAc; 251, Neu5Acα2-8Neu5Acα2-3-Galβ1-4Glc. Details for other glycans can be found at www.functionalglycomics.org.

Analysis of the binding properties of the parent and the N173S mutant viruses in the presence or the absence of selective inhibitor of Site 1 BCX 2798 allowed us to distinguish receptor-binding profiles between the two sites (Table I; Figure 4). The parent and the N173S mutant showed strong binding to glycans 208 and 210 which in both cases was notably decreased (from 12 to 20 times) in the presence of Site 1 inhibitor, indicating that glycans 208 and 210 are bound only by Site 1 (Table I, Group I). Glycans 207 and 251 are uniquely bound by Site 2 (Table I, Group II) because the binding of both viruses to glycan 207 was not significantly reduced upon addition of BCX 2798, and binding to glycan 251 was observed only with the N173S mutant. The binding of the parent virus to glycan 207 in the absence or the presence of BCX 2798 (∼11,231 and 7251 RFU, respectively) but not to glycan 251 (∼156 and 136 RFU, respectively) suggests that during attachment, Site 2 of the parent virus might be partially exposed (possibly through glycan position rearrangement) and interact with glycan 207. This proposition is in agreement with our unpublished data indicating that introduction of the mutations into the region of the Site 2 on hPIV-1 HN (e.g. at residues 516, 523 of hPIV-1 HN) which may prevent its formation, significantly decreases the N173S mutant binding and increases its sensitivity to BCX 2798 when compared with that of the parent virus (manuscript in preparation). The differences in the binding properties of the parent and the N173S mutant (Alymova et al. 2008), as well as glycan array assay data showing inability of the parent virus to bind to glycan 251 (unique for Site 2), suggest that the complete exposure of Site 2 was observed only in the absence of glycosylation at residue 173. Glycan binding (to 209 and 228–234), which was significantly reduced by Site 1 inhibitor BCX 2798 only in the case of the parent virus, was considered to be common for both sites (Table I, Group III). The binding properties of glycans 44 and 237 were difficult to interpret as they did not comply with the principles of glycan distribution into the groups described in the preceding text. These two glycans were separated into Group IV of Table I.

Table I.

Receptor-binding specificities of the parent and the N173S mutant virusesa

Group Glycan number Glycan structure Intensity of binding (RFU ± SEM)
Parent + PBS Parent + 100 µM BCX 2798 N173S + PBS N173S + 100 µM BCX 2798
I 208 Neu5Acα2-3(GalNAcβ1-4)Galβ1-4GlcNAcβ-Sp0 22,046 ± 7405 1272 ± 499 33,825 ± 1311 2593 ± 703
210 Neu5Acα2-3(GalNAcβ1-4)Galβ1-4Glcβ-Sp0 12,092 ± 2929 613 ± 62 19,266 ± 772 1632 ± 416
II 207 Neu5Acα2-3(6-O-Su)Galβ1-4(Fucα1-3)GlcNAcβ-Sp8 11,231 ± 1450 7251 ± 734 8724 ± 1324 30,359 ± 1369
251 Neu5Acα2-8Neu5Acα2-3Galβ1-4Glcβ-Sp0 156 ± 46 136 ± 34 4551 ± 764 22,233 ± 1759
III 209 Neu5Acα2-3(GalNAcβ1-4)Galβ1-4GlcNAcβ-Sp8 27,300 ± 1027 3702 ± 328 23,440 ± 3824 15,630 ± 854
228 Neu5Acα2-3Galβ1-4(Fucα1-3)GlcNAcβ1-3Galb1-4(Fucα1-3) GlcNAcβ1-3Galβ1-4(Fucα1-3)GlcNAcβ-Sp0 31,952 ± 1027 13,807 ± 1319 16,523 ± 683 29,316 ± 994
229 Neu5Acα2-3Galβ1-4(Fucα1-3)GlcNAcβ-Sp0 8179 ± 581 1098 ± 476 2626 ± 145 6792 ± 1340
230 Neu5Acα2-3Galβ1-4(Fucα1-3)GlcNAcβ-Sp8 6489 ± 660 1549 ± 321 4203 ± 217 13,675 ± 154
231 Neu5Acα2-3Galβ1-4(Fuca1-3)GlcNAcβ1-3Galβ-Sp8 9154 ± 1432 84 ± 50 8663 ± 717 6822 ± 1918
232 Neu5Acα2-3Galβ1-4(Fucα1-3)GlcNAcβ1-3Galβ1-4GlcNAcβ-Sp8 12,117 ± 1538 1122 ± 527 16,011 ± 407 15,182 ± 942
234 Neu5Acα2-3Galβ1-4GlcNAcβ1-3Galβ1-4GlcNAcβ1- 3Galβ1-4GlcNAcβ-Sp0 12,831 ± 1033 1929 ± 705 4564 ± 359 7747 ± 1197
IV 44 Neu5Acα2-3[6OSO3]Galβ1-4GlcNAcβ-Sp8 11,567 ± 1980 4916 ± 1105 2846 ± 753 11,637 ± 914
237 Neu5Acα2-3Galβ1-4GlcNAcβ1-3Galβ1-4GlcNAcβ-Sp0 7835 ± 333 4559 ± 593 1783 ± 531 12,509 ± 1330
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RFU, relative fluorescent units; SEM, standard error of mean.

aReceptor binding of viruses treated with BCX 2798 at a final concentration of 100 µM or PBS for 1 h at RT was analyzed on the CFG printed array v3.2 containing 406 glycans (www.functionalglycomics.org).

Fig. 4.

Fig. 4.

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Schematic presentation of hPIV-1 HN receptor-binding specificity.

When analyzing binding properties of the N173S mutant, we noted a significant increase in the intensity of virus binding to glycans 207 and 251 (which are unique for Site 2) in the presence of BCX 2798 (Table I). As was argued previously for NDV HN, changes in Site 1 upon binding and cleavage of the SA-linked receptors (or synthetic substrate) alter the HN dimer interface and create the new SA-binding site (Zaitsev et al. 2004). The new site allows the virus to remain attached to SA-linked cell receptors, while fusion proceeds. We propose that the same is true for the N173S mutant. Our data from glycan array assays indicate that, similar to NDV, Site 2 appears on the N173S mutant only when Site 1 is engaged by inhibitor BCX 2798. The implication of Site 2 in the N173S mutant's fusion is a subject for further studies.

When making conclusions regarding receptor-binding specificities of the parent and the N173S mutant viruses, we took into the consideration the possibility of the influence of SeV background and the technical nature of microarray analysis on interpretation of obtained results. Our previously published study, comparing hPIV-1 and rSeV bearing hPIV-1 HN (instead of SeV HN), did not reveal any differences in biological properties of the HN molecule between the two viruses (Alymova et al. 2004), indicating that its binding profile should not be attributed to the chimeric nature of the N173S mutant. Our data showing binding of hPIV-1 HN to α2-3-linked, but not to α2-6-linked, SA-linked receptors agrees with data obtained by other research groups (Suzuki et al. 2001; Amonsen et al. 2007). Our data from the current and previously published studies (Alymova et al. 2008) both indicate the presence of the Site 2 on the N173S mutant HN. Our current data demonstrate binding of Site 2 to α2-8-linked SA and data by Amonsen et al. (2007) show the ability of hPIV-1 HN to cleave α2-8-linked SA. Taken together, all these facts suggest the validity of our results from glycan array assays on the hPIV-1 HN receptor-binding specificity. However, further crystallographic studies are required to unconditionally confirm the presence of Site 2 on hPIV-1 HN and reveal its biology.

Our new findings support our previously published data proposing that Site 2 in hPIV1-1 HN is occluded by glycosylation on Asn173 (Alymova et al. 2008). Formation of Site 2 is navigated by Site 1 binding to the SA-linked receptors. When exposed, Site 2 is able to bind SA-containing oligosaccharides and is therefore a second receptor-binding site. Site 2, like Site 1, recognizes Neu5Ac that is predominantly linked by an α2-3-linkage to N-acetyllactosamine. Neither site recognizes α2-6-linked Neu5Ac, although Site 2 can uniquely recognize α2-8-linked Neu5Ac (glycan 251).

Recognition of α2-8-linked poly-SAs by viruses was previously reported for bovine rotavirus (Lee et al. 1998), NDV (Corfield et al. 1982), SeV (Holmgren et al. 1980), influenza A/NWS/33 (H1N2; Wu and Air 2004) and representative of the recent H1N1 2009 influenza pandemic, A/California/4/2009 (Childs et al. 2009). This is the first report of binding of hPIV-1 to this type of SA-containing receptor. Besides oligosaccharides of cell surface glycoproteins, the α2-8-linked polysialyl sequences are found on gaingliosides of the brain and platelet membranes and on the neural cell adhesion molecules (N-CAMs). They have been shown to play a role in the development of neural tissue (Bock et al. 1980), in N-CAMs mediated adhesion (Cunningham et al. 1987; Rutishauser et al. 1988), in the metastasis of several human cancers (Seidenfaden et al. 2003), and bacterial pathogenesis. The recent study with Streptococcus mitis demonstrates that direct binding of bacteria to α2-8-linked Neu5Ac on the membrane of human platelets contributes to the pathogenesis of infective endocarditis (Mitchell and Sullam 2009). The role of α2-8-linked SA in the pathogenesis of viral infections and, in particular, infections caused by hPIV-1 is a subject for further investigation.

The biological and clinical significance of the second site on hPIVs is a complex issue. As we demonstrated previously, inoculation of mice with the N173S resulted in accelerated infection (in term of virus lung titers) when compared with that of the parent virus (Alymova et al. 2008). This can be due to both the expanded receptor specificities (additional recognition of α2-8-linked Neu5Ac) and binding abilities as well as due to the loss of glycosylation. Recently published studies indicate that a decrease in the level of glycosylation increases influenza viruses' virulence (Vigerust et al. 2007). The isolation (from children hospitalized due to respiratory complications) of hPIV-1 (Henrickson and Savatski 1992) and hPIV-3 (Mishin et al. 2010) mutants, possessing an open second binding site as a result of the loss of glycosylation, indicates their occurrence in nature. Potentially, infection of humans with the hPIV mutants possessing an open second binding site can lead to a more severe course of disease in comparison to an infection with a variant possessing the masked site.

Together, our results from glycan array assays on receptor-binding specificity of the hPIV-1 HN provide new principal insights on the biology of this important childhood pathogen such as the presence of Site 2 on the HN molecule and its possible involvement in hPIV-1 binding. Confirmation of the proposition that hPIV-1 utilizes its second site for binding might change the strategy for the design of vaccines and HN inhibitors.

Materials and methods

The SeV reverse genetic system was used to rescue parent virus and the N173S mutant as described previously (Alymova et al. 2004, 2008). Rescued viruses were plaque purified and amplified on LLC-MK2 cells. To confirm the presence/absence of the mutations, viral RNAs were extracted from the infected cell supernatants; cDNA synthesis and polymerase chain reaction (PCR) amplification were carried out using the One-step RT–PCR system (Qiagen, Valencia, CA). Sequencing of the HN gene (performed by the Hartwell Center for Bioinformatics and Biotechnology at St Jude) of the parent virus did not reveal any mutations; while sequencing of the HN gene of the N173S confirmed the desired substitution.

For the glycan array assays, viruses were propagated in LLC-MK2 cells in Dulbecco's modified Eagle's medium that contained 0.3% bovine serum albumin and 1 µg/mL acetylated trypsin at 33°C in 5% CO2 for 4–6 days. Then, viruses were concentrated and purified by ultracentrifugation through a gradient of 30–50% sucrose in PBS, as described previously (Thompson et al. 1988). The pellet containing virus particles was resuspended in 200 µL of PBS. The hemagglutination of concentrated purified viruses was determined with 0.5% chicken red blood cells in PBS to evaluate binding activities of the HNs. Normalization of the amount of the virus proteins in each sample preparation was based on the results of the sodium dodecyl sulfate–polyacrylamide gel electrophoresis under reducing conditions and subsequent protein quantification (by Totallab TL100 1D, version 2008.01; Nonlinear USA Inc., Durham, NC). The viruses were diluted in binding buffer at pH 7.0 with the addition of 1% bovine serum albumin and 0.05% Tween 20 to a final total protein concentration of 200 µg/mL. To block Site 1, the parent and N173S viruses were treated with BCX 2798 at a final concentration of 1, 10, 100 and 1000 µM for 1 h at room temperature (RT); for the control, viruses were treated with PBS. Seventy microliters of sample was incubated on the CFGs' printed array (v3.2; containing 406 glycans, www.functionalglycomics.org) for 1 h at RT. The slide was washed and then incubated with primary SeV anti-F mouse monoclonal [IgG2b (Portner et al. 1986)] and secondary Alexa-labeled antibody (provided by CFGs). The fluorescence intensity related to binding was read in a Perkin-Elmer Microarray XL4000 scanner and analyzed using Imagine image analysis software.

Funding

This work was supported by The Wellcome Trust and in part by the National Institute of Allergy and Infectious Diseases (grant PO1 A1054955) and by the American Lebanese Syrian Associated Charities (ALSAC). The glycan analyses were performed by the Protein-Glycan Interaction Core (H) of the CFG, funded by NIGMS (GM62116).

Conflict of interest

None declared.

Abbreviations

CFG, Consortium for Functional Glycomics; F, fusion; HN, hemagglutinin–neuraminidase; hPIVs, human parainfluenza viruses; NA, neuraminidase; N-CAM, neural cell adhesion molecule; NDV, Newcastle disease virus; PBS, phosphate-buffered saline; PCR, polymerase chain reaction; RFU, relative fluorescent unit; SA, sialic acid; SeV, Sendai virus; RT, room temperature.

Acknowledgements

We thank BioCryst Pharmaceuticals, Inc., for providing BCX 2798.

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