The influenza A virus hemagglutinin glycosylation state affects receptor-binding specificity

Vries, Robert P. de; Vries, Erik de; Bosch, Berend Jan; Groot, Raoul J. de; Rottier, Peter J. M.; Haan, Cornelis A. M. de

doi:10.1016/j.virol.2010.03.047

Cited by 121 publications

(179 citation statements)

References 39 publications

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“…Madin-Darby canine kidney (MDCK)-II, HEK293T, and HEK293S GnT1(−) cells were maintained as described previously (3,21). CHO Pro5, CHO lec1, CHO k1, and CHO 15B cells (all from American Type Culture Collection) were maintained at 37°C in α-MEM (Gibco) supplemented with 10% (vol/vol) FCS, 100 U/mL of penicillin, and 100 μg/mL of streptomycin.…”

Section: Methodsmentioning

confidence: 99%

“…Entry assays were performed by replacing WSN-Ren and entry medium after 2 h with full growth medium containing 10 nM BafA1 (3). Luciferase activity was determined after 16 h. When indicated, cells were treated before infection with PNGase F (New England BioLabs) or VCNA (Roche) as described previously (3,21). Cell viability (WST assay; Roche) was not affected.…”

Section: Methodsmentioning

confidence: 99%

“…Soluble trimeric recombinant HA (strain WSN) was produced, purified, and tested for binding efficiency to the four CHO cell lines as described previously (21). In brief, cells (10 4 per well) were fixed in 96-well plates.…”

Section: Methodsmentioning

confidence: 99%

“…Glycosylation patterns of HAs expressed in the four CHO cell lines were examined by SDS/PAGE, followed by Western blot analysis (21). When indicated, HA was incubated with EndoH glycosidase or PNGaseF (New England BioLabs) before electrophoresis in accordance with the manufacturer's instructions.…”

Section: Methodsmentioning

confidence: 99%

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Influenza A virus entry into cells lacking sialylated N-glycans

Vries

Wienholts

et al. 2012

Proc. Natl. Acad. Sci. U.S.A.

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Influenza A virus (IAV) enters host cells after attachment of its hemagglutinin (HA) to surface-exposed sialic acid. Sialylated Nlinked glycans have been reported to be essential for IAV entry [Chu VC, Whittaker GR (2004) Proc Natl Acad Sci USA 102:18153-18158], thereby implicating the requirement for proteinaceous receptors in IAV entry. Here we show, using different N-acetylglucosaminyl transferase 1 (GnT1)-deficient cells, that N-linked sialosides can mediate, but are not required for, entry of IAV. Entry into GnT1-deficient cells was fully dependent on sialic acid. Although macropinocytic entry appeared to be affected by the absence of sialylated N-glycans, dynamin-dependent entry was not affected at all. However, binding of HA to GnT1-deficient cells and subsequent entry of IAV were reduced by the presence of serum, which could be reversed by back-transfection of a GnT1-encoding plasmid. The inhibitory effect of serum was significantly increased by inhibition of the viral receptor-destroying enzyme neuraminidase (NA). Our results indicate that decoy receptors on soluble serum factors compete with cell surface receptors for binding to HA in the absence of sialylated N-glycans at the cell surface. This competition is particularly disturbed by the additional presence of NA inhibitors, resulting in strongly reduced IAV entry. Our results indicate that the balance between HA and NA is important not only for virion release, but also for entry into cells. (IAV) is an enveloped, negative-strand RNA virus that causes respiratory and/or intestinal infections in a variety of animal hosts, including birds and mammals. The IAV envelope contains two glycoproteins: the hemagglutinin (HA) protein, which is responsible for virus cell attachment and fusion, and the neuraminidase (NA) protein, which is the receptordestroying enzyme essential for release of the virus from the host cell after budding. IAV attaches to host cells by binding of HA to sialic acids (SIA) present on the host cell surface. Attachment is followed by entry via endocytic routes delivering the virus to the acidic environment of the late endosome that triggers HA-mediated fusion. The interactions of HA with SIA likely are important determinants not only of attachment, but also of virus uptake, intracellular trafficking, and fusion.Cell surface glycan composition is complex and varies between host and cell type. Sialylated glycans are attached to membrane phospolipids or to membrane proteins via asparagine (N-linked glycans) or serine/threonine (O-linked glycans) residues. How and to what extent binding of IAV to specific sialoglycans determines subsequent endocytosis remains unclear. N-linked glycans have been shown to be required for IAV entry, even though O-linked glycans and/or glycolipids permit efficient sialic acid-dependent binding of IAV to cells devoid of sialylated N-linked glycans (2). Thus, it appears that beyond the well-established requirement for binding to either α2-6 SIAs of human IAVs or α2-3 SIAs of avian IAVs, a specific subset of glyc...

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

See 2 more Smart Citations

Influenza A virus entry into cells lacking sialylated N-glycans

Vries

Wienholts

et al. 2012

Proc. Natl. Acad. Sci. U.S.A.

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“…Molecular modeling of the responsible mutations (T200A and E227A) demonstrated the loss of two potential hydrogen bond interactions with Gln 191 in the 190-helix and with the penultimate galactose, respectively, thereby providing a rationale for the observed differences in H1-receptor binding. (19). The swine origin IAV A/Cal/04/09 replicates efficiently in cell culture and has been shown to replicate in and transmit among guinea pigs with similar efficiency to that of a seasonal H3N2 influenza virus (20).…”

Section: All Influenza a Virus (Iav)mentioning

confidence: 99%

Only Two Residues Are Responsible for the Dramatic Difference in Receptor Binding between Swine and New Pandemic H1 Hemagglutinin

Vries

Moore

et al. 2011

Journal of Biological Chemistry

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In view of its critical role in influenza A virus (IAV) tropism and pathogenesis, we evaluated the receptor binding properties of HA proteins of the closely related swine and new pandemic human IAVs. We generated recombinant soluble trimeric H1 ectodomains of several IAVs and analyzed their sialic acid binding properties using fetuin-binding and glycan array analysis. The results show that closely related swine and new pandemic H1 proteins differ dramatically in their ability to bind these receptors. Although new pandemic H1 protein exhibited hardly any binding, swine H1 bound efficiently to a number of ␣2-6-linked sialyl glycans. The responsible amino acids were identified by analyzing chimeric H1 proteins and by performing systematic site-directed mutagenesis of swine and new pandemic human H1 proteins. The difference was found to map to residues at positions 200 and 227. Although substitution of either residue significantly affected the binding phenotype, substitution of both was found to act synergistically and reverse the phenotype almost completely. Modeling of the T200A and E227A substitutions into the crystal structure of the new pandemic human H1 protein revealed the loss of potential hydrogen bond formation with Gln 191 , which is part of the 190-loop of the receptor binding site, and with the penultimate galactose, respectively. Thus, a residue not belonging to the receptor binding site may affect the interaction of HA with its receptor. Interestingly, whereas alanine at position 200 is found in most new pandemic human viruses, the residue at position 227 in these viruses is invariably a glutamic acid. All influenza A virus (IAV)2 pandemics known so far originated from avian or swine IAV strains that managed to cross the species barrier to humans and acquire the capacity of human-to-human transmission. The most recent example of this was the new H1N1 swine origin IAV that emerged in 2009 and rapidly spread around the world (1, 2). The specificity of the interaction of HA with sialic acid (SIA), the cellular receptor, largely explains the host range of IAVs (3). Thus, viruses that infect humans bind preferentially to SIA linked to the penultimate galactose in an ␣2-6 configuration, whereas avian viruses prefer binding to SIA with ␣2-3 linkages (4). However, the adaptations in HA required for swine IAVs to become infectious for humans and to establish themselves in the human population are much less characterized.The HA receptor binding site (RBS) is formed by three structural elements at the tip of the HA molecule, an ␣-helix composed by residues 190 -198 (the 190-helix) and two loop structures formed by residues 133-138 (the 130-loop) and 220 -229 (the 220-loop). Four conserved residues, comprising Tyr 98 , Trp 153 , His 183 , and Tyr 195 , form the base of the RBS (5). The amino acid residues in the RBS that are critical for the recognition of either avian or human receptors have been well characterized (4, 6, 7). For H1, glutamic acid and glycine residues at positions 190 and 225, respectively, resu...

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Impact of cultivation conditions on N‐glycosylation of influenza virus a hemagglutinin produced in MDCK cell culture

Rödig

Rapp

Bohne

et al. 2013

Biotech & Bioengineering

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Manufacturers worldwide produce influenza vaccines in different host systems. So far, either fertilized chicken eggs or mammalian cell lines are used. In all these vaccines, hemagglutinin (HA) and neuraminidase are the major components. Both are highly abundant glycoproteins in the viral envelope, and particularly HA is able to induce a strong and protective immune response. The quality characteristics of glycoproteins, such as specific activity, antigenicity, immunogenicity, binding avidity, and receptor-binding specificity can strongly depend on changes or differences in their glycosylation pattern (potential N-glycosylation occupancy as well as glycan composition). In this study, capillary gel electrophoresis with laser-induced fluorescence detection (CGE-LIF) based glycoanalysis (N-glycan fingerprinting) was used to determine the impact of cultivation conditions on the HA N-glycosylation pattern of Madin-Darby canine kidney (MDCK) cell-derived influenza virus A PR/8/34 (H1N1). We found that adaptation of adherent cells to serum-free growth has only a minor impact on the HA N-glycosylation pattern. Only relative abundances of N-glycan structures are affected. In contrast, host cell adaptation to serum-free suspension growth resulted in significant changes in the HA N-glycosylation pattern regarding the presence of specific N-glycans as well as their abundance. Further controls such as different suppliers for influenza virus A PR/8/34 (H1N1) seed strains, different cultivation scales and vessels in standard or high cell density mode, different virus production media varying in either composition or trypsin activity, different temperatures during virus replication and finally, the impact of β-propiolactone inactivation resulted-at best-only in minor changes in the relative N-glycan structure abundances of the HA N-glycosylation pattern. Surprisingly, these results demonstrate a rather stable HA N-glycosylation pattern despite various (significant) changes in upstream processing. Only the adaptation of the production host cell line to serum-free suspension growth significantly influenced HA N-glycosylation regarding both, the type of attached glycan structures as well as their abundances.

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The influenza A virus hemagglutinin glycosylation state affects receptor-binding specificity

Cited by 121 publications

References 39 publications

Influenza A virus entry into cells lacking sialylated N-glycans

Influenza A virus entry into cells lacking sialylated N-glycans

Only Two Residues Are Responsible for the Dramatic Difference in Receptor Binding between Swine and New Pandemic H1 Hemagglutinin

Impact of cultivation conditions on N‐glycosylation of influenza virus a hemagglutinin produced in MDCK cell culture

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