Neuraminidase Is Essential for Fowl Plague Virus Hemagglutinin to Show Hemagglutinating Activity

AbstractWhen hemagglutinin (HA) of fowl plague virus (FPV) was expressed in CV-1 cells by a simian virus 40 vector, hemadsorption was barely detectable, although HA was exposed at the cell surface. However, treatment of HA-expressing cells with Vibrio cholerae neuraminidase (VCNA) resulted in extensive hemadsorption. VCNA treatment enhanced the electrophoretic mobility of the HA1 subunit of HA, indicating the removal of sialic acid. When two oligosaccharides in the vicinity of the receptor binding site of FPV HA were deleted by site-specific mutagenesis, VCNA treatment was not required for hemadsorption. Mutants which retained one of these oligosaccharides and mutants in which oligosaccharides not adjacent to the receptor binding site were deleted needed VCNA treatment to show hemadsorption. VCNA treatment also enhanced hemadsorption of vector-expressed HA of the WSN strain, which had a complex-type oligosaccharide in the vicinity of the receptor binding site, but had no effect on hemadsorption of Hong Kong type HA, which has a high-mannose type oligosaccharide adjacent to the receptor binding site. These results indicate that sialic acid on oligosaccharides near the receptor binding site interferes with hemadsorption. Thus, the neuraminidase is essential for FPV HA to show hemagglutinating activity

Structures of Receptor Complexes of a North American H7N2 Influenza Hemagglutinin with a Loop Deletion in the Receptor Binding Site

Human infections with subtype H7 avian influenza viruses have been reported as early as 1979. In 1996, a genetically stable 24-nucleotide deletion emerged in North American H7 influenza virus hemagglutinins, resulting in an eight amino acid deletion in the receptor-binding site. The continuous circulation of these viruses in live bird markets, as well as its documented ability to infect humans, raises the question of how these viruses achieve structural stability and functionality. Here we report a detailed molecular analysis of the receptor binding site of the North American lineage subtype H7N2 virus A/New York/107/2003 (NY107), including complexes with an avian receptor analog (3′-sialyl-N-acetyllactosamine, 3′SLN) and two human receptor analogs (6′-sialyl-N-acetyllactosamine, 6′SLN; sialyllacto-N-tetraose b, LSTb). Structural results suggest a novel mechanism by which residues Arg220 and Arg229 (H3 numbering) are used to compensate for the deletion of the 220-loop and form interactions with the receptor analogs. Glycan microarray results reveal that NY107 maintains an avian-type (α2-3) receptor binding profile, with only moderate binding to human-type (α2-6) receptor. Thus despite its dramatically altered receptor binding site, this HA maintains functionality and confirms a need for continued influenza virus surveillance of avian and other animal reservoirs to define their zoonotic potential

A thyroid hormone receptor-dependent glucocorticoid induction.

Glucocorticoid and thyroid hormones exert their effects in many body tissues by binding to their respective receptors. The search for possible cross-talking mechanisms in overlapping target cells led to the discovery of synergism between a thyroid hormone receptor-binding site and a cryptic glucocorticoid-responsive element. Glucocorticoid responsiveness could only be detected in the presence of thyroid hormone and its receptor. This synergism requires the glucocorticoid receptor (GR) DNA-binding domain and is mediated by the transactivation domains. We found that synergism also occurs when the thyroid hormone receptor is replaced by the retinoic acid receptor or the GR is replaced by the progesterone receptor. Synergism is qualitatively independent of the type of thyroid hormone receptor-binding site and promoter. In several combinations of promoter and response elements, including a retinoic acid response element, T3 induction was only seen in the presence of the cryptic glucocorticoid-responsive element, GR, and glucocorticoids

A Ligand Peptide Motif Selected from a Cancer Patient Is a Receptor-Interacting Site within Human Interleukin-11

Interleukin-11 (IL-11) is a pleiotropic cytokine approved by the FDA against chemotherapy-induced thrombocytopenia. From a combinatorial selection in a cancer patient, we isolated an IL-11-like peptide mapping to domain I of the IL-11 (sequence CGRRAGGSC). Although this motif has ligand attributes, it is not within the previously characterized interacting sites. Here we design and validate in-tandem binding assays, site-directed mutagenesis and NMR spectroscopy to show (i) the peptide mimics a receptor-binding site within IL-11, (ii) the binding of CGRRAGGSC to the IL-11Rα is functionally relevant, (iii) Arg4 and Ser8 are the key residues mediating the interaction, and (iv) the IL-11-like motif induces cell proliferation through STAT3 activation. These structural and functional results uncover an as yet unrecognized receptor-binding site in human IL-11. Given that IL-11Rα has been proposed as a target in human cancer, our results provide clues for the rational design of targeted drugs

B Cell Recognition of the Conserved HIV-1 Co-Receptor Binding Site Is Altered by Endogenous Primate CD4

The surface HIV-1 exterior envelope glycoprotein, gp120, binds to CD4 on the target cell surface to induce the co-receptor binding site on gp120 as the initial step in the entry process. The binding site is comprised of a highly conserved region on the gp120 core, as well as elements of the third variable region (V3). Antibodies against the co-receptor binding site are abundantly elicited during natural infection of humans, but the mechanism of elicitation has remained undefined. In this study, we investigate the requirements for elicitation of co-receptor binding site antibodies by inoculating rabbits, monkeys and human-CD4 transgenic (huCD4) rabbits with envelope glycoprotein (Env) trimers possessing high affinity for primate CD4. A cross-species comparison of the antibody responses showed that similar HIV-1 neutralization breadth was elicited by Env trimers in monkeys relative to wild-type (WT) rabbits. In contrast, antibodies against the co-receptor site on gp120 were elicited only in monkeys and huCD4 rabbits, but not in the WT rabbits. This was supported by the detection of high-titer co-receptor antibodies in all sera from a set derived from human volunteers inoculated with recombinant gp120. These findings strongly suggest that complexes between Env and (high-affinity) primate CD4 formed in vivo are responsible for the elicitation of the co-receptor-site-directed antibodies. They also imply that the naïve B cell receptor repertoire does not recognize the gp120 co-receptor site in the absence of CD4 and illustrate that conformational stabilization, imparted by primary receptor interaction, can alter the immunogenicity of a type 1 viral membrane protein

Substitutions near the hemagglutinin receptor-binding site determine the antigenic evolution of influenza A H3N2 viruses in U.S. swine

Swine influenza A virus is an endemic and economically important pathogen in pigs, with the potential to infect other host species. The hemagglutinin (HA) protein is the primary target of protective immune responses and the major component in swine influenza A vaccines. However, as a result of antigenic drift, vaccine strains must be regularly updated to reflect currently circulating strains. Characterizing the cross-reactivity between strains in pigs and seasonal influenza virus strains in humans is also important in assessing the relative risk of interspecies transmission of viruses from one host population to the other. Hemagglutination inhibition (HI) assay data for swine and human H3N2 viruses were used with antigenic cartography to quantify the antigenic differences among H3N2 viruses isolated from pigs in the United States from 1998 to 2013 and the relative cross-reactivity between these viruses and current human seasonal influenza A virus strains. Two primary antigenic clusters were found circulating in the pig population, but with enough diversity within and between the clusters to suggest updates in vaccine strains are needed. We identified single amino acid substitutions that are likely responsible for antigenic differences between the two primary antigenic clusters and between each antigenic cluster and outliers. The antigenic distance between current seasonal influenza virus H3 strains in humans and those endemic in swine suggests that population immunity may not prevent the introduction of human viruses into pigs, and possibly vice versa, reinforcing the need to monitor and prepare for potential incursions

An Open Receptor-Binding Cavity of Hemagglutinin-Esterase-Fusion Glycoprotein from Newly-Identified Influenza D Virus: Basis for Its Broad Cell Tropism.

Influenza viruses cause seasonal flu each year and pandemics or epidemic sporadically, posing a major threat to public health. Recently, a new influenza D virus (IDV) was isolated from pigs and cattle. Here, we reveal that the IDV utilizes 9-O-acetylated sialic acids as its receptor for virus entry. Then, we determined the crystal structures of hemagglutinin-esterase-fusion glycoprotein (HEF) of IDV both in its free form and in complex with the receptor and enzymatic substrate analogs. The IDV HEF shows an extremely similar structural fold as the human-infecting influenza C virus (ICV) HEF. However, IDV HEF has an open receptor-binding cavity to accommodate diverse extended glycan moieties. This structural difference provides an explanation for the phenomenon that the IDV has a broad cell tropism. As IDV HEF is structurally and functionally similar to ICV HEF, our findings highlight the potential threat of the virus to public health

Second M-3 muscarinic receptor binding site contributes to bronchoprotection by tiotropium

Background and Purpose The bronchodilator tiotropium binds not only to its main binding site on the M-3 muscarinic receptor but also to an allosteric site. Here, we have investigated the functional relevance of this allosteric binding and the potential contribution of this behaviour to interactions with long-acting beta-adrenoceptor agonists, as combination therapy with anticholinergic agents and beta-adrenoceptor agonists improves lung function in chronic obstructive pulmonary disease. Experimental Approach ACh, tiotropium, and atropine binding to M-3 receptors were modelled using molecular dynamics simulations. Contractions of bovine and human tracheal smooth muscle strips were studied. Key Results Molecular dynamics simulation revealed extracellular vestibule binding of tiotropium, and not atropine, to M-3 receptors as a secondary low affinity binding site, preventing ACh entry into the orthosteric binding pocket. This resulted in a low (allosteric binding) and high (orthosteric binding) functional affinity of tiotropium in protecting against methacholine-induced contractions of airway smooth muscle, which was not observed for atropine and glycopyrrolate. Moreover, antagonism by tiotropium was insurmountable in nature. This behaviour facilitated functional interactions of tiotropium with the beta-agonist olodaterol, which synergistically enhanced bronchoprotective effects of tiotropium. This was not seen for glycopyrrolate and olodaterol or indacaterol but was mimicked by the interaction of tiotropium and forskolin, indicating no direct beta-adrenoceptor-M-3 receptor crosstalk in this effect. Conclusions and Implications We propose that tiotropium has two binding sites at the M-3 receptor that prevent ACh action, which, together with slow dissociation kinetics, may contribute to insurmountable antagonism and enhanced functional interactions with beta-adrenoceptor agonists

A Cation-π Interaction in the Binding Site of the Glycine Receptor Is Mediated by a Phenylalanine Residue

Cys-loop receptor binding sites characteristically contain many aromatic amino acids. In nicotinic ACh and 5-HT3 receptors, a Trp residue forms a cation-{pi} interaction with the agonist, whereas in GABAA receptors, a Tyr performs this role. The glycine receptor binding site, however, contains predominantly Phe residues. Homology models suggest that two of these Phe side chains, Phe159 and Phe207, and possibly a third, Phe63, are positioned such that they could contribute to a cation-{pi} interaction with the primary amine of glycine. Here, we test this hypothesis by incorporation of a series of fluorinated Phe derivatives using unnatural amino acid mutagenesis. The data reveal a clear correlation between the glycine EC50 value and the cation-{pi} binding ability of the fluorinated Phe derivatives at position 159, but not at positions 207 or 63, indicating a single cation-{pi} interaction between glycine and Phe159. The data thus provide an anchor point for locating glycine in its binding site, and demonstrate for the first time a cation-{pi} interaction between Phe and a neurotransmitter