SummaryInfluenza virus remains a threat because of its ability to evade vaccine-induced immune responses due to antigenic drift. Here, we describe the isolation, evolution, and structure of a broad-spectrum human monoclonal antibody (mAb), MEDI8852, effectively reacting with all influenza A hemagglutinin (HA) subtypes. MEDI8852 uses the heavy-chain VH6-1 gene and has higher potency and breadth when compared to other anti-stem antibodies. MEDI8852 is effective in mice and ferrets with a therapeutic window superior to that of oseltamivir. Crystallographic analysis of Fab alone or in complex with H5 or H7 HA proteins reveals that MEDI8852 binds through a coordinated movement of CDRs to a highly conserved epitope encompassing a hydrophobic groove in the fusion domain and a large portion of the fusion peptide, distinguishing it from other structurally characterized cross-reactive antibodies. The unprecedented breadth and potency of neutralization by MEDI8852 support its development as immunotherapy for influenza virus-infected humans.

SUMMARY The human JC polyomavirus (JCV) causes a fatal demyelinating disease, Progressive Multifocal Leukoencephalopathy (PML), in immunocompromised individuals. Current treatment options for PML are inadequate. Sialylated oligosaccharides and the serotonin receptor are known to be necessary for JCV entry, but the molecular interactions underlying JCV attachment remain unknown. Using glycan array screening and viral infectivity assays, we identify a linear sialylated pentasaccharide with the sequence NeuNAc-α2,6-Gal-β1,4-GlcNAc-β1,3-Gal-β1,4-Glc (LSTc) present on host glycoproteins and glycolipids as a specific JCV recognition motif. The crystal structure of the JCV capsid protein VP1 was solved alone and in complex with LSTc. It reveals extensive interactions with the terminal sialic acid of the LSTc motif and specific recognition of an extended conformation of LSTc. Mutations in the JCV oligosaccharide binding sites abolish cell attachment, viral spread and infectivity, further validating the importance of this interaction. Our findings provide a powerful platform for the development of antiviral compounds.

Simian virus 40 (SV40) has been a paradigm for understanding attachment and entry of nonenveloped viruses, viral DNA replication, and virus assembly, as well as for endocytosis pathways associated with caveolin and cholesterol. We find by glycan array screening that SV40 recognizes its ganglioside receptor GM1 with a quite narrow specificity, but isothermal titration calorimetry shows that individual binding sites have a relatively low affinity, with a millimolar dissociation constant. The high-resolution crystal structure of recombinantly produced SV40 capsid protein, VP1, in complex with the carbohydrate portion of GM1, reveals that the receptor is bound in a shallow solvent-exposed groove at the outer surface of the capsid. Through a complex network of interactions, VP1 recognizes a conformation of GM1 that is the dominant one in solution. Analysis of contacts provides a structural basis for the observed specificity and suggests binding mechanisms for additional physiologically relevant GM1 variants. Comparison with murine Polyomavirus (Polyoma) receptor complexes reveals that SV40 uses a different mechanism of sialic acid binding, which has implications for receptor binding of human polyomaviruses. The SV40 -GM1 complex reveals a parallel to cholera toxin, which uses a similar cell entry pathway and binds GM1 in the same conformation.crystal structure ͉ glycan array ͉ polyomaviruses ͉ viral attachment ͉ protein-carbohydrate complex

Viruses within a family often vary in their cellular tropism and pathogenicity. In many cases, these variations are due to viruses switching their specificity from one cell surface receptor to another. The structural requirements that underlie such receptor switching are not well understood especially for carbohydrate-binding viruses, as methods capable of structure-specificity studies are only relatively recently being developed for carbohydrates. We have characterized the receptor specificity, structure and infectivity of the human polyomavirus BKPyV, the causative agent of polyomavirus-associated nephropathy, and uncover a molecular switch for binding different carbohydrate receptors. We show that the b-series gangliosides GD3, GD2, GD1b and GT1b all can serve as receptors for BKPyV. The crystal structure of the BKPyV capsid protein VP1 in complex with GD3 reveals contacts with two sialic acid moieties in the receptor, providing a basis for the observed specificity. Comparison with the structure of simian virus 40 (SV40) VP1 bound to ganglioside GM1 identifies the amino acid at position 68 as a determinant of specificity. Mutation of this residue from lysine in BKPyV to serine in SV40 switches the receptor specificity of BKPyV from GD3 to GM1 both in vitro and in cell culture. Our findings highlight the plasticity of viral receptor binding sites and form a template to retarget viruses to different receptors and cell types.

SignificanceX-ray crystallography studies of soluble ectodomains of influenza hemagglutinins (HA) have previously revealed details of their two functions in virus infection: receptor binding and membrane fusion. We have now used cryo-EM to determine the structures of full-length hemagglutinin solubilized in detergent, alone, and in complex with the Fab of an infectivity neutralizing antibody. We observe the structure of the region that anchors HA in the virus membrane as a bundle of three α-helices that are joined to the ectodomain by flexible linkages. The Fab binds near the linkages and restricts their flexibility. This may indicate that the flexibility is required for HA-mediated membrane fusion and that interference with it represents a mechanism for neutralizing virus infectivity.

Viral infections are initiated by specific attachment of a virus particle to receptors at the surface of the host cell. For many viruses, these receptors are glycans that are linked to either a protein or a lipid. Glycans terminating in sialic acid and its derivatives serve as receptors for a large number of viruses, including several human pathogens. In combination with glycan array analyses, structural analyses of complexes of viruses with sialylated oligosaccharides have provided insights into the parameters that underlie each interaction. Here, we compare the currently available structural data on viral attachment proteins in complex with sialic acid and its variants. The objective is to define common parameters of recognition and to provide a platform for understanding the determinants of specificity. This information could be of use for the prediction of the location of sialic acid binding sites in viruses for which structural information is still lacking. An improved understanding of the principles that govern the recognition of sialic acid and sialylated oligosaccharides would also advance efforts to develop efficient antiviral agents.

The recently discovered human Merkel cell polyomavirus (MCPyV or MCV) causes the aggressive Merkel cell carcinoma (MCC) in the skin of immunocompromised individuals. Conflicting reports suggest that cellular glycans containing sialic acid (Neu5Ac) may play a role in MCPyV infectious entry. To address this question, we solved X-ray structures of the MCPyV major capsid protein VP1 both alone and in complex with several sialylated oligosaccharides. A shallow binding site on the apical surface of the VP1 capsomer recognizes the disaccharide Neu5Ac-α2,3-Gal through a complex network of interactions. MCPyV engages Neu5Ac in an orientation and with contacts that differ markedly from those observed in other polyomavirus complexes with sialylated receptors. Mutations in the Neu5Ac binding site abolish MCPyV infection, highlighting the relevance of the Neu5Ac interaction for MCPyV entry. Our study thus provides a powerful platform for the development of MCPyV-specific vaccines and antivirals. Interestingly, engagement of sialic acid does not interfere with initial attachment of MCPyV to cells, consistent with a previous proposal that attachment is mediated by a class of non-sialylated carbohydrates called glycosaminoglycans. Our results therefore suggest a model in which sialylated glycans serve as secondary, post-attachment co-receptors during MCPyV infectious entry. Since cell-surface glycans typically serve as primary attachment receptors for many viruses, we identify here a new role for glycans in mediating, and perhaps even modulating, post-attachment entry processes.

The human JC polyomavirus (JCPyV) establishes an asymptomatic, persistent infection in the kidneys of the majority of the population and is the causative agent of the fatal demyelinating disease progressive multifocal leukoencephalopathy (PML) in immunosuppressed individuals. The Mad-1 strain of JCPyV, a brain isolate, was shown earlier to require ␣2,6-linked sialic acid on the lactoseries tetrasaccharide c (LSTc) glycan for attachment to host cells. In contrast, a JCPyV kidney isolate type 3 strain, WT3, has been reported to interact with sialic acid-containing gangliosides, but the role of these glycans in JCPyV infection has remained unclear. To help rationalize these findings and probe the effects of strain-specific differences on receptor binding, we performed a comprehensive analysis of the glycan receptor specificities of these two representative JCPyV strains using high-resolution X-ray crystallography and nuclear magnetic resonance (NMR) spectroscopy, and correlated these data with the results of infectivity assays. We show here that capsid proteins of Mad-1 and WT3 JCPyV can both engage LSTc as well as multiple sialylated gangliosides. However, the binding affinities exhibit subtle differences, with the highest affinity observed for LSTc. Engagement of LSTc is a prerequisite for functional receptor engagement, while the more weakly binding gangliosides are not required for productive infection. Our findings highlight the complexity of virus-carbohydrate interactions and demonstrate that subtle differences in binding affinities, rather than the binding event alone, help determine tissue tropism and viral pathogenesis. IMPORTANCEViral infection is initiated by attachment to receptors on host cells, and this event plays an important role in viral disease. We investigated the receptor-binding properties of human JC polyomavirus (JCPyV), a virus that resides in the kidneys of the majority of the population and can cause the fatal demyelinating disease progressive multifocal leukoencephalopathy (PML) in the brains of immunosuppressed individuals. JCPyV has been reported to interact with multiple carbohydrate receptors, and we sought to clarify how the interactions between JCPyV and cellular carbohydrate receptors influenced infection. Here we demonstrate that JCPyV can engage numerous sialylated carbohydrate receptors. However, the virus displays preferential binding to LSTc, and only LSTc mediates a productive infection. Our findings demonstrate that subtle differences in binding affinity, rather than receptor engagement alone, are a key determinant of viral infection. J C polyomavirus (JCPyV) infects ϳ50% of healthy individuals and causes an asymptomatic, lifelong persistent infection in the kidney (1, 2). The form of the virus that resides in the kidney is nonpathogenic and is excreted in the urine (3, 4). In immunosuppressed individuals, JCPyV can spread from the site of persistence to the central nervous system (CNS) (5, 6) and infect the glial cells astrocytes and oligodendroctyes (7,8). Oligod...