Here we describe a glycan microarray constructed by using standard robotic microarray printing technology to couple amine functionalized glycans to an amino-reactive glass slide. The array comprises 200 synthetic and natural glycan sequences representing major glycan structures of glycoproteins and glycolipids. The array has remarkable utility for profiling the specificity of a diverse range of glycan binding proteins, including C-type lectins, siglecs, galectins, anticarbohydrate antibodies, lectins from plants and microbes, and intact viruses.carbohydrate ͉ lectin ͉ microarray ͉ glycoprotein ͉ glycolipid
The worldwide spread of H5N1 avian influenza has raised concerns that this virus might acquire the ability to pass readily among humans and cause a pandemic. Two anti-influenza drugs currently being used to treat infected patients are oseltamivir (Tamiflu) and zanamivir (Relenza), both of which target the neuraminidase enzyme of the virus. Reports of the emergence of drug resistance make the development of new anti-influenza molecules a priority. Neuraminidases from influenza type A viruses form two genetically distinct groups: group-1 contains the N1 neuraminidase of the H5N1 avian virus and group-2 contains the N2 and N9 enzymes used for the structure-based design of current drugs. Here we show by X-ray crystallography that these two groups are structurally distinct. Group-1 neuraminidases contain a cavity adjacent to their active sites that closes on ligand binding. Our analysis suggests that it may be possible to exploit the size and location of the group-1 cavity to develop new anti-influenza drugs.
The 1918 influenza pandemic resulted in about 20 million deaths. This enormous impact, coupled with renewed interest in emerging infections, makes characterization of the virus involved a priority. Receptor binding, the initial event in virus infection, is a major determinant of virus transmissibility that, for influenza viruses, is mediated by the hemagglutinin (HA) membrane glycoprotein. We have determined the crystal structures of the HA from the 1918 virus and two closely related HAs in complex with receptor analogs. They explain how the 1918 HA, while retaining receptor binding site amino acids characteristic of an avian precursor HA, is able to bind human receptors and how, as a consequence, the virus was able to spread in the human population.
H5N1 influenza A viruses have spread to numerous countries in Asia, Europe and Africa, infecting not only large numbers of poultry, but also an increasing number of humans, often with lethal effects. Human and avian influenza A viruses differ in their recognition of host cell receptors: the former preferentially recognize receptors with saccharides terminating in sialic acid-alpha2,6-galactose (SAalpha2,6Gal), whereas the latter prefer those ending in SAalpha2,3Gal (refs 3-6). A conversion from SAalpha2,3Gal to SAalpha2,6Gal recognition is thought to be one of the changes that must occur before avian influenza viruses can replicate efficiently in humans and acquire the potential to cause a pandemic. By identifying mutations in the receptor-binding haemagglutinin (HA) molecule that would enable avian H5N1 viruses to recognize human-type host cell receptors, it may be possible to predict (and thus to increase preparedness for) the emergence of pandemic viruses. Here we show that some H5N1 viruses isolated from humans can bind to both human and avian receptors, in contrast to those isolated from chickens and ducks, which recognize the avian receptors exclusively. Mutations at positions 182 and 192 independently convert the HAs of H5N1 viruses known to recognize the avian receptor to ones that recognize the human receptor. Analysis of the crystal structure of the HA from an H5N1 virus used in our genetic experiments shows that the locations of these amino acids in the HA molecule are compatible with an effect on receptor binding. The amino acid changes that we identify might serve as molecular markers for assessing the pandemic potential of H5N1 field isolates.
The membrane fusion potential of influenza HA, like many viral membrane-fusion glycoproteins, is generated by proteolytic cleavage of a biosynthetic precursor. The three-dimensional structure of ectodomain of the precursor HA0 has been determined and compared with that of cleaved HA. The cleavage site is a prominent surface loop adjacent to a novel cavity; cleavage results in structural rearrangements in which the nonpolar amino acids near the new amino terminus bury ionizable residues in the cavity that are implicated in the low-pH-induced conformational change. Amino acid insertions at the cleavage site in HAs of virulent avian viruses and those of viruses isolated from the recent severe outbreak of influenza in humans in Hong Kong would extend this surface loop, facilitating intracellular cleavage.
The three-dimensional structures of avian H5 and swine H9 influenza hemagglutinins (HAs) from viruses closely related to those that caused outbreaks of human disease in Hong Kong in 1997 and 1999 were determined bound to avian and human cell receptor analogs. Emerging influenza pandemics have been accompanied by the evolution of receptor-binding specificity from the preference of avian viruses for sialic acid receptors in ␣2,3 linkage to the preference of human viruses for ␣2,6 linkages. The four new structures show that HA binding sites specific for human receptors appear to be wider than those preferring avian receptors and how avian and human receptors are distinguished by atomic contacts at the glycosidic linkage. ␣2,3-Linked sialosides bind the avian HA in a trans conformation to form an ␣2,3 linkage-specific motif, made by the glycosidic oxygen and 4-OH of the penultimate galactose, that is complementary to the hydrogen-bonding capacity of Gln-226, an avian-specific residue. ␣2,6-Linked sialosides bind in a cis conformation, exposing the glycosidic oxygen to solution and nonpolar atoms of the receptor to Leu-226, a human-specific residue. The new structures are compared with previously reported crystal structures of HA͞sialoside complexes of the H3 subtype that caused the 1968 Hong Kong Influenza virus pandemic and analyzed in relation to HA sequences of all 15 subtypes and to receptor affinity data to make clearer how receptor-binding sites of HAs from avian viruses evolve as the virus adapts to humans.
A single amino acid substitution, Asp-63 to was (3,4), the location of regions on the three-dimensional structure of the HA to which antibodies bind have been proposed in the HA of the 1968 influenza virus (5, 6).In these studies, it was noted (5, 6) that in some viruses of both the Hong Kong (H3) subtype and of other subtypes oligosaccharide attachment sites (Asn-X-Ser/Thr) were present in regions of the HA implicated in antibody binding in the 1968 HA, and this was subsequently observed in a study of a HA of the H1 subtype (7,8 (12,13). Hybrid cell culture conditions were based on those described by Fazekas de St. Groth and Scheidegger (14). The antibodies produced from different cloned cells were named hemagglutinin clones (HC) and numbered according to the culture number-e.g., the antibodies used in this study for immunoprecipitation were numbered HC31 and HC100. When antibodies have been characterized by determining the sequence of the HA genes of antigenic variants that they select, they are given another number in parentheses. ThUs, HC31, which selects variants with amino acid substitutions at amino acid residue 198, is finally denoted by HC31(198), and HC100, which selects variants with substitutions at amino acid residue 63, is denoted by HC100(63). The variants selected by these particular antibodies are numbered V31 and V100.Nucleotide Sequence Analyses. Sequences were determined by using the dideoxynucleotide (ddNTP) chain-terminating procedure (15). Each 10-,lI reaction mixture contained Tris HCl, pH 8.3, 0.05 M; magnesium chloride, 0.012 M; dithiothreitol, 0.02 M; dATP, dGTP, dCTP, and dTTP, 0.0004 M each; virus RNA, 7 ,g; human placenta RNase inhibitor (Bethesda Research Laboratories), 3 units; reverse transcriptase, 5 units (Life Sciences, St. Petersburg, FL); and one of ddATP, ddCTP, ddGTP, or ddTTP, 0.00025 M. After 120 min at 42°C, products were analyzed on polyacrylamide gels containing 8% acrylamide. Reactions were primed by using 5'-32P-labeled oligodeoxynucleotides prepared as described by Patel et al. (16) and purified by ion-exchange HPLC (Partisil SAX 10-50). The primers used, numbered according to the sequence of X-31 HA cDNA (17)
The influenza surface glycoprotein hemagglutinin (HA) is a potential target for antiviral drugs because of its key roles in the initial stages of infection: receptor binding and the fusion of virus and cell membranes. The structure of HA in complex with a known inhibitor of membrane fusion and virus infectivity, tert-butyl hydroquinone (TBHQ), shows that the inhibitor binds in a hydrophobic pocket formed at an interface between HA monomers. Occupation of this site by TBHQ stabilizes the neutral pH structure through intersubunit and intrasubunit interactions that presumably inhibit the conformational rearrangements required for membrane fusion. The nature of the binding site suggests routes for the chemical modification of TBHQ that could lead to the development of more potent inhibitors of membrane fusion and potential anti-influenza drugs.crystallography ͉ drug design I nf luenza A virus membranes contain 3 proteins: hemagglutinin (HA), neuraminidase (NA), and the proton channel (M2). HA is responsible during the initial stages of infection for sialic acid-receptor binding and, after virus uptake into endosomes, for fusion of virus and cell membranes (1). M2 transfers protons into the infecting virus in endosomes, and at acidic pH the matrix protein, M1, is dissociated from the genome-transcriptase complex, so that the uncoated complex is transported to the nucleus after membrane fusion (2). At the end of infection, NA cleaves sialic acid from virus and cell glycoconjugates to ensure release of newly-made viruses from infected cells (3). Both M2 and NA are targets of current anti-inf luenza drugs (4 -6). The proton channel, M2, is blocked by the drugs amantadine and rimantadine, and NA is inhibited by the drugs zanamivir and oseltamivir. All 4 drugs are effective upon prompt administration after infection or prophylactically, but concern has been raised by the isolation of viable mutant viruses that are resistant to them (7-11). There is therefore a need to develop new antivirals to act on additional virus targets. Their availability would make possible drug combination therapies to avoid the selection of resistant viruses, a strategy that has been successful in highly active antiretroviral therapy against HIV (12) and was recently reported for the combined use of amantadine and oseltamivir against inf luenza (13). To address the need for new antivirals against inf luenza a number of studies have been made of inhibitors of the receptor binding or membrane fusion activities of HA, particularly the latter (14 -17).The membrane fusion potential of HA is activated in endosomes at acidic pH by the induction of an irreversible reorganization of HA structure (18,19). Comparison of the neutral-pH and fusion-pH structures indicates that at fusion pH the membrane-distal domains of HA dissociate, and extensive structural reorganization occurs that involves extrusion of the ''fusion peptide'' from the interior of the neutral-pH structure, presumably toward the target endosomal membrane with which the virus membrane is to f...
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