Middle East respiratory syndrome coronavirus (MERS-CoV) targets the epithelial cells of the respiratory tract both in humans and in its natural host, the dromedary camel. Virion attachment to host cells is mediated by 20-nm-long homotrimers of spike envelope protein S. The N-terminal subunit of each S protomer, called S1, folds into four distinct domains designated S1 through S1 Binding of MERS-CoV to the cell surface entry receptor dipeptidyl peptidase 4 (DPP4) occurs via S1 We now demonstrate that in addition to DPP4, MERS-CoV binds to sialic acid (Sia). Initially demonstrated by hemagglutination assay with human erythrocytes and intact virus, MERS-CoV Sia-binding activity was assigned to S subdomain S1 When multivalently displayed on nanoparticles, S1 or S1 bound to human erythrocytes and to human mucin in a strictly Sia-dependent fashion. Glycan array analysis revealed a preference for α2,3-linked Sias over α2,6-linked Sias, which correlates with the differential distribution of α2,3-linked Sias and the predominant sites of MERS-CoV replication in the upper and lower respiratory tracts of camels and humans, respectively. Binding is hampered by Sia modifications such as 5--glycolylation and (7,)9--acetylation. Depletion of cell surface Sia by neuraminidase treatment inhibited MERS-CoV entry of Calu-3 human airway cells, thus providing direct evidence that virus-Sia interactions may aid in virion attachment. The combined observations lead us to propose that high-specificity, low-affinity attachment of MERS-CoV to sialoglycans during the preattachment or early attachment phase may form another determinant governing the host range and tissue tropism of this zoonotic pathogen.

Summary All previously characterized broadly neutralizing antibodies to the HIV-1 envelope glycoprotein (Env) target one of four major sites of vulnerability. Here, we define and structurally characterize a unique epitope on Env that is recognized by a recently discovered family of human monoclonal antibodies (PGT151-158). The PGT151 epitope is comprised of residues and glycans at the interface of gp41 and gp120 within a single protomer and glycans from both subunits of a second protomer and represents a neutralizing epitope that is dependent on both gp120 and gp41. As PGT151 binds only to properly formed, cleaved trimers, this distinctive property, and its ability to stabilize Env trimers, has enabled the successful purification of mature, cleaved Env trimers from the cell surface as a complex with PGT151. Here we compare the structural and functional properties of membrane-extracted Env trimers from several clades with those of the soluble, cleaved SOSIP gp140 trimer.

SUMMARY Human and avian influenza viruses recognize different sialic acid-containing receptors, referred to as human-type (NeuAcα2-6Gal) and avian-type (NeuAcα2-3Gal) respectively. This presents a species barrier for aerosol droplet transmission of avian viruses in humans and ferrets. Recent reports have suggested that current human H3N2 viruses no longer have strict specificity towards human-type receptors. Using an influenza receptor glycan microarray with extended airway glycans we find that H3N2 viruses have in fact maintained human-type specificity, but have evolved preference for a subset of receptors comprising branched glycans with extended poly-N-acetyl-lactosamine (poly-LacNAc) chains, a specificity shared with the 2009 pandemic H1N1 (Cal/04) hemagglutinin. Lipid-linked versions of extended sialoside receptors can restore susceptibility of sialidase-treated MDCK cells to infection by both recent (A/Victoria/361/11) and historical (A/Hong Kong/8/1968) H3N2 viruses. Remarkably, these human-type receptors with elongated branches have the potential to increase avidity by simultaneously binding to two subunits of a single hemagglutinin trimer.

A systematic, efficient means of producing diverse libraries of asymmetrically branched N-glycans is needed to investigate the specificities and biology of glycan binding proteins. To that end, we describe a core pentasaccharide that at potential branching positions is modified by orthogonal protecting groups to allow selective attachment of unique saccharide moieties by chemical glycosylation. The appendages were selected in such a way that the antenna of the resulting deprotected compounds could be selectively extended by glycosyltransferases to give libraries of asymmetrical multi-antennary glycans. The power of the methodology was demonstrated by the preparation of a series of complex oligosaccharides that were printed as microarrays and screened for binding to lectins and influenza-virus hemagglutinins, which showed that recognition is modulated by presentation of minimal epitopes in the context of complex N-glycans.

The avian H7N9 influenza outbreak in 2013 resulted from an unprecedented incidence of influenza transmission to humans from infected poultry. The majority of human H7N9 isolates contained a hemagglutinin (HA) mutation (Q226L) that has previously been associated with a switch in receptor specificity from avian-type (NeuAcα2-3Gal) to human-type (NeuAcα2-6Gal), as documented for the avian progenitors of the 1957 (H2N2) and 1968 (H3N2) human influenza pandemic viruses. While this raised concern that the H7N9 virus was adapting to humans, the mutation was not sufficient to switch the receptor specificity of H7N9, and has not resulted in sustained transmission in humans. To determine if the H7 HA was capable of acquiring human-type receptor specificity, we conducted mutation analyses. Remarkably, three amino acid mutations conferred a switch in specificity for human-type receptors that resembled the specificity of the 2009 human H1 pandemic virus, and promoted binding to human trachea epithelial cells.