Hemagglutinin Receptor Binding Research Articles

Glycan Microarray Analysis of the Hemagglutinins from Modern and Pandemic Influenza Viruses Reveals Different Receptor Specificities

Influenza A virus specificity for the host is mediated by the viral surface glycoprotein hemagglutinin (HA), which binds to receptors containing glycans with terminal sialic acids. Avian viruses preferentially bind to alpha2-3-linked sialic acids on receptors of intestinal epithelial cells, whereas human viruses are specific for the alpha2-6 linkage on epithelial cells of the lungs and upper respiratory tract. To define the receptor preferences of a number of human and avian H1 and H3 viruses, including the 1918 H1N1 pandemic strains, their hemagglutinins were analyzed using a recently described glycan array. The array, which contains 200 carbohydrates and glycoproteins, not only revealed clear differentiation of receptor preferences for alpha2-3 and/or alpha2-6 sialic acid linkage, but could also detect fine differences in HA specificity, such as preferences for fucosylation, sulfation and sialylation at positions 2 (Gal) and 3 (GlcNAc, GalNAc) of the terminal trisaccharide. For the two 1918 HA variants, the South Carolina (SC) HA (with Asp190, Asp225) bound exclusively alpha2-6 receptors, while the New York (NY) variant, which differed only by one residue (Gly225), had mixed alpha2-6/alpha2-3 specificity, especially for sulfated oligosaccharides. Only one mutation of the NY variant (Asp190Glu) was sufficient to revert the HA receptor preference to that of classical avian strains. Thus, the species barrier, as defined by the receptor specificity preferences of 1918 human viruses compared to likely avian virus progenitors, can be circumvented by changes at only two positions in the HA receptor binding site. The glycan array thus provides highly detailed profiles of influenza receptor specificity that can be used to map the evolution of new human pathogenic strains, such as the H5N1 avian influenza.

Infectious salmon anemia virus: causative agent, pathogenesis and immunity.

Infectious salmon anemia (ISA) virus (ISAV), an economically important new pathogen in marine aquaculture, is classified in the family Orthomyxoviridae, genus Isavirus. The main structural properties of this genus include enveloped virions 90-140 nm in diameter with surface projections of a combined receptor-binding hemagglutinin and receptor-destroying enzyme activity demonstrated to be an esterase, hence recently designated HE, and a genome composed of eight segments of linear, single-stranded, negative sense RNA ranging in length from 1.0 to 2.4 kb, with a total size of approximately 14.3 kb. The viral genome encodes at least ten proteins, of which nine are structural and one is non-structural. Examination of more than 160 ISAV isolates has led to the identification of two hemagglutinin subtypes of ISAV, one North American and one European. The immune response against ISAV after infection or vaccination does not provide full protection against the infection. The recent discovery of antibody-mediated uptake and replication of ISAV in macrophage-like fish cell lines suggests that Fc receptor-mediated antibody-dependent enhancement of the ISA virus infection might also occur in vivo, as the virus in Atlantic salmon (Salmo salar) targets endothelial cells lining blood vessels and macrophage-like cells. Cumulative mortalities in Atlantic salmon during natural ISA outbreaks and experimental infections range from 0 to 100%. ISAV causes fatal systemic infections in marine-farmed Atlantic salmon and asymptomatic infections in feral fish. Experimentally induced fatal clinical disease in rainbow trout (Oncorhynchus mykiss) has identified a correlate of virulence of ISAV that may explain its emergence as a fish pathogen.

Effective primary isolation of wild-type Canine distemper virus in MDCK, MV1 Lu and Vero cells without nucleotide sequence changes within the entire haemagglutinin protein gene and in subgenomic sections of the fusion and phospho protein genes

Canine distemper virus (CDV) is an important pathogen of many carnivores. We are developing a field-based model of morbillivirus virulence and pathogenesis through a study of distemper in naturally infected free-ranging raccoons. The isolation of CDV from raccoon tissues is essential for this work. CDV has often been isolated from animals only after co-cultivation of infected tissues with peripheral blood mononuclear cells derived from specific pathogen-free dogs or similar methods. We explored the utility and consequences of a simpler and cheaper alternative: CDV isolation in Vero, MDCK, and MV1 Lu cells. Virus growth was detected first in MDCK cells, whereas viral cytopathic effects were most obvious in Vero cells. CDV growth in MV1 Lu cells was relatively protracted and occurred without the formation of cytopathic effects. In primary CDV isolates, the entire nucleotide sequence of the receptorbinding haemagglutinin ( H) gene, and subgenomic fusion ( F) and phospho ( P) protein gene sequences corresponding to nt 5399–5733 and 2132–2563 of CDV reference strain Onderstepoort, respectively, were identical to those in matched infected tissues. Virus isolation confirmed the presence of CDV in instances where RT-PCR failed to detect CDV in infected tissues. Different viral phenotypes and genotypes were detected. The conservation of H gene sequences in primary CDV isolates suggests that MDCK, MV1 Lu, and Vero cells express proper receptors for wild-type CDV.

Differences between HA receptor-binding sites of avian influenza viruses isolated from Laridae and Anatidae.

A comparative study of the hemagglutinin (HA) receptor binding site (RBS) of a number of H13 influenza viruses isolated from Laridae family of birds (gulls) and other influenza viruses obtained from the Anatidae family (ducks) was conducted. The affinity of all viruses to alpha N-acetylneuraminic acid (Neu5Ac alpha), 3'sialyllactose (3'SL), and sialylglycopolymers bearing 3'-sialyl(N-acetyllactosamine) (3'SLN-PAA), [Neu5Ac alpha(2-3)Gal beta(1-4)][-Fuc alpha(1-3)]GlcNAc beta (SLe(x)-PAA), and [Neu5Ac alpha(2-3)Gal beta(1-3)][-Fuc alpha(1-4)]GlcNAc beta (SLe(a)-PAA), was determined. The last three polymer glycoconjugates were synthesized for determining the contribution of carbohydrate chains after the galactose link to the binding with the receptor. The difference in affinity between 3'SL and Neu5Ac alpha in all studied H13 viruses is small, which indicates a less significant role of the galactose moiety in the binding to the receptor. The results of virus binding with polymer sialylglycoconjugates indicates that the method of linking, the third monosaccharide moiety, and the presence of an extra fucose substitute in this moiety may influence the binding considerably. For viruses isolated from ducks, the suitable polymer is SLe(a)-PAA (i.e., a 1-3 linkage between galactose and glucosamine is optimal). This finding is in accord with the data that H13 viruses isolated from the gulls differ based on their ability to interact with polymer sialylglycoconjugates. The affinity to all three polymers is uniform, and the presence of GlcNAc-linked fucose does not prevent the binding. A comparative analysis of six sequenced HA H13 viruses and other subtype viruses showed presence of substantial differences in the composition of amino acids of this region in H13 viruses.

Changes in the receptorbinding haemagglutinin protein of wild-type morbilliviruses are not required for adaptation to Vero cells.

We examined the consequences of isolation and adaptation to Vero cells for the receptorbinding haemagglutinin (H) gene of four syncytia-forming isolates of canine distemper virus (CDV) and of a dolphin morbillivirus isolate. A Vero-adapted CDV isolate exhibited biased hypermutation, since 11 out of 12 nucleotide differences to other isolates from the same epidemic were U-C transitions. Most of these transitions appeared to have taken place during in vitro cultivation. Previously, biased hypermutation in morbilliviruses has almost exclusively been described for subacute sclerosing panencephalitis and measles inclusion body encephalitis, which are rare measles virus brain infections. Amino acid changes in the H proteins were not required for Vero cell adaptation, suggesting that Vero cells express receptors for wild-type morbilliviruses. This strongly indicate the existence of other morbillivirus receptors than CD46 and CDw150.

Dendritic cells and measles virus infection.

Measles is a major cause of childhood mortality in developing countries which is mainly attributed to the ability of measles virus (MV) to suppress general immune responses. Paradoxically, virus-specific immunity is efficiently induced, which leads to viral clearance from the host and confers long-lasting protection against reinfection. As sensitisers of pathogen encounter and instructors of the adaptive immune response, dendritic cells (DCs) may play a decisive role in the induction and quality of the MV-specific immune activation. The ability of MV wild-type strains in particular to infect DCs in vitro is dearly established, and the receptor binding haemagglutinin protein of these viruses essentially determines this particular tropism. DC maturation as induced early after MV infection is likely to be of crucial importance for the induction of MV-specific immunity. DCs may, however, be instrumental in MV-induced immunosuppression. (1) T cell depletion could be brought about by DC-T cell fusion or TRAIL-mediated induction of apoptosis. (2) Inhibition of stimulated IL-12 production from MV-infected DCs might affect T cell responses in qualitative terms in favouring Th2 and suppressing Th1 responses. (3) The viral glycoprotein complex expressed at high levels on infected DCs late in infection is able to directly inhibit T cell proliferation by surface contact-dependent negative signalling. This most likely accounts for the failure of infected DC cultures to stimulate allogeneic and inhibit mitogen-stimulated T cell proliferation in vitro and the pronounced proliferative unresponsiveness of T cell ex vivo to polyclonal and antigen-specific stimulation which is a central finding of MV-induced immunosuppression.

Correlation between levels of apoptosis, levels of infection and haemagglutinin receptor binding interaction of various subtypes of influenza virus: does the viral neuraminidase have a role in these associations

Previously, we have shown that an H3N2 influenza virus (clone 7a) induced more apoptosis in MDCK cells than an H1N1 (A/Fiji) influenza virus and that the virion neuraminidase (NA) played a role in the induction of apoptosis. In this study we have examined a further 6 N2 (H3/H2) and 3 N1 (Hsw/H1) viruses and confirmed that the N2 viruses induce more apoptosis in MDCK cells than the N1 viruses. Furthermore, the level of apoptosis, the level of cell infection and the NA activity of the virus preparations paralleled each other for all the viruses. The levels of infection depended upon the degree of interaction of the viral haemagglutinin (HA) with its receptors: while all the viruses utilised NeuAcα-2,6 Gal containing receptors, the H3/H2 viruses showed a greater interaction than the Hsw/H1 viruses. Removal of sialic acid from virions by treatment with bacterial NA enhanced infection and apoptosis but the effect was much greater for the A/Fiji virus than for the clone 7a virus. Thus, while the relative interaction of the HAs for their receptors is the major factor influencing infectivity and apoptosis, the viral NA possibly plays an indirect role by removing sialic acid from the HA, thereby increasing its receptor binding.

Mechanisms of differential induction of apoptosis by H3N2 and H1N1 influenza viruses

Background: The abilities of seven H3N2 and four H1N1 influenza viruses to infect MDCK cells and produce apoptosis were measured and related to their neuraminidase (NA) activity and the role of individual influenza virus proteins in induction of apoptosis was also investigated by transient transfection assays in HeLa cells with expression vectors containing individual virus genes. Methods: MDCK cells were inoculated at a high multiplicity of infection (5 EID 50/cell) and apoptosis was measured by cytotoxicity and morphology while infection was quantified by fluorescence staining with anti-nucleoprotein (NP) antibody. The Invitrogen Voyager™ vector expressing influenza virus genes as VP22 fusion proteins was transfected into HeLa cells using lipofectamine. Results and discussion: H3N2 viruses with high NA activities (1.4–1.8 μmol/l/min) induced high levels of apoptosis (83–94%) and infected 91–98% of cells, while H1N1 viruses with low NA activities (0.22–0.33 μmol/l/min) were poor apoptosis inducers (11–19%) and infected few (15–21%) cells. The differences in % infected cells reflected differences in haemagglutinin (HA) receptor binding affinity. Treatment of viruses with bacterial NA to remove sialyl groups from oligosaccharides on virus HA increased their ability to infect MDCK cells and the increase correlated inversely with the number of potential glycosylation sites around the receptor binding site. Transfection experiments showed that NA (from clone 7a but not A/Fiji) and NS1 (from clone 7a and A/Fiji) induced apoptosis, while NP (clone 7a and A/Fiji) did not.

Virus–receptor interactions of human parainfluenza viruses types 1, 2 and 3

Human parainfluenza viruses types 1, 2 and 3 (HPF 1, 2 and 3) are important pathogens in children. While these viruses share common structures and replication strategies, they target different parts of the respiratory tract; the most common outcomes of infection with HPF3 are bronchiolitis and pneumonia, while HPF 1 and 2 are associated with croup. While the HPF3 fusion protein (F) is critical for membrane fusion, our previous work revealed that the receptor binding hemagglutinin–neuraminidase (HN) is also essential to the fusion process; interaction between HN and its sialic acid-containing receptor on cell surfaces is required for HPF3 mediated cell fusion. Using our understanding of HPF3 HN's functions in the cell-binding and viral entry process, we are investigating the ways in which these processes differ in HPF 1 and 2, in part by manipulating receptor availability. Three experimental treatments were used to compare the HN–receptor interaction of HPF 1, 2 and 3: infection at high multiplicity of infection (m.o.i.); bacterial neuraminidase treatment of cells infected at low m.o.i.; and viral neuraminidase treatment of cells infected at low m.o.i. (using Newcastle disease virus [NDV] neuraminidase or UV irradiated HPF3 as sources of neuraminidase). In cells infected with HPF3, we have shown that infection with high m.o.i. blocks fusion, by removing sialic acid receptors for the viral HN. However, in cells infected with HPF 1 and 2, infection with high m.o.i. did not block fusion; the fusion increases with increasing m.o.i.. In cells infected with HPF 1 and 2, neither bacterial nor NDV neuraminidase blocked cell fusion, using amounts of neuraminidase that completely block fusion of HPF3 infected cells. However, when inactivated HPF3 was used as a source of viral neuraminidase, the treatment inhibited fusion of cells infected with HPF 1 and 2 as well as 3. The differences found between these viruses in terms of their interaction with the cell, ability to modulate cell–cell fusion and reponse to exogenous neuraminidases of various specificities, may reflect salient differences in biological properties of the three viruses.

Studies of the Binding Properties of Influenza Hemagglutinin Receptor-Site Mutants

Site-specific mutations have been made in the influenza hemagglutinin (HA) receptor binding site to assess the contribution of individual amino acid residues to receptor recognition. Screening of mutant HAs, expressed using recombinant vaccinia virus-infected cells, for their abilities to bind human erythrocytes indicated that substitutions involving conserved residues Y98F, H183F, and L194A severely restricted binding and that the substitution W153A prevented cell surface expression of HA. Mutation of residues E190 and S228 that are in positions to form hydrogen bonds with the 9-OH of sialic acid appeared to increase erythrocyte binding slightly, as did the substitution G225R. Substitutions of other residues that are directly or indirectly involved in receptor binding, S136T, S136A, Y195F, G225D, and L226P, had intermediate effects on binding between these two extremes. Estimates of changes in receptor binding specificity based on inhibition of binding to erythrocytes by nonimmune horse sera indicated that mutants G225R and L226P, unlike wild-type HA, were not inhibited; Y195F and G225D mutants were, like wild type, inhibited; and erythrocyte binding by mutants S136A, S136T, E190A, and S228G was only partially inhibited. Viruses containing mutant HAs Y98F, S136T, G225D, and S228G that cover the range of erythrocyte binding properties observed were also constructed by transfection. All four transfectant viruses replicated in MDCK cells and embryonated hens' eggs as efficiently as wild-type X-31 virus, although the Y98F mutant virus was unable to agglutinate erythrocytes. Mutant MDCK cells that have reduced levels of cell surface sialic acids were susceptible to infection by S136T, G225D, and S228G transfectant viruses and by wild type but not by the Y98F transfectant virus.

Evolution of H5 subtype avian influenza A viruses in North America

The phylogenetic relationships of the hemagglutinin (HA) and non-structural (NS) genes from avian influenza (AI) H5 subtype viruses of North American origin are presented. Analysis of the HA genes of several previously uncharacterized isolates from waterfowl and turkeys provided clear evidence of significant sequence variation and existence of multiple virus clades or sub-lineages, maintained in migratory waterfowl. Phylogenetic analysis of NS gene sequences further demonstrated multiple sub-lineages and also demonstrated re-assortment of two NS alleles in wild duck populations. Based on currently available HA 1 gene sequences, at least four clades exist with waterfowl isolates included in three of the four groups. The most genetically unstable of these sub-lineages is composed of recent poultry isolates from the outbreak of AI in Central Mexico. This group of viruses, which replicated unabated in chickens for at least 16 months, exhibited an increased rate of mutation in both the HA and NS gene. Comparison of the HA 1 sequence data for all available North American H5 subtype viruses demonstrated minimal variation both in and around the amino acids predicted to be involved in the HA receptor binding site. The sequences also revealed that migratory waterfowl, live poultry market chicken, and turkey isolates uniformly lack a glycosylation site at amino acid 236 in the HA protein which is present in commercial chicken isolates.

Differences in sialic acid-galactose linkages in the chicken egg amnion and allantois influence human influenza virus receptor specificity and variant selection.

Human influenza viruses are more efficiently isolated by inoculating patient samples into the amniotic rather than the allantoic cavity of embryonated chicken eggs. This type of cultivation selects virus variants with mutations around the hemagglutinin (HA) receptor binding site. To understand the molecular basis of these phenomena, we investigated the abundances of sialic acid (SA) linked to galactose (Gal) by the alpha-2,3 linkage (SA alpha2,3Gal) and SA alpha2,6Gal in egg amniotic and allantoic cells and in Madin-Darby canine kidney (MDCK) cells. Using SA-Gal linkage-specific lectins (Maackia amurensis agglutinin specific for SA alpha2,6Gal and Sambucus nigra agglutinin specific for SA alpha2,3Gal), we found SA alpha2,3Gal in both allantoic and amniotic cells and SA alpha2,6Gal in only the amniotic cells. MDCK cells contained both linkages. To investigate how this difference in abundances of SA alpha2,3Gal and SA alpha2,6Gal in allantoic and amniotic cells affects the appearance of host cell variants in eggs, we determined the receptor specificities and HA amino acid sequences of two different patient viruses which were isolated and passaged in the amnion or in the allantois and which were compared with MDCK cell-grown viruses. We found that the viruses maintained high SA alpha2,6Gal specificities when grown in MDCK cells or following up to two amniotic passages; however, further passages in either the amnion or allantois resulted in the acquisition of, or a complete shift to, SA alpha2,3Gal specificity, depending on the virus strain. This change in receptor specificity was accompanied by the appearance of variants in the population with Leu-to-Gln mutations at position 226 in their HA. These findings suggest that lack of SA alpha2,6Gal linkages in the allantois of chicken eggs is a selective pressure for the appearance of host cell variants with altered receptor specificities and amino acid changes at position 226.

Generation and characterization of variants of NWS/G70C influenza virus after in vitro passage in 4-amino-Neu5Ac2en and 4-guanidino-Neu5Ac2en

The compounds 4-amino-Neu5Ac2en (5-acetylamino-2,6-anhydro-4-amino-3,4,5- trideoxy-D-glycerol-D-galacto-non-2-enoic acid) and 4-guanidino-Neu5Ac2en (5-acetylamino-2,6-anhydro-4-guanidino-3,4,5- trideoxy-D-glycerol-D-galacto-non-2-enoic acid), which selectively inhibit the influenza virus neuraminidase, have been tested in vitro for their ability to generate drug-resistant variants. NWS/G70C virus (H1N9) was cultured in each drug by limiting-dilution passaging. After five or six passages in either compound, there emerged viruses which had a reduced sensitivity to the inhibitors in cell culture. Variant viruses were up to 1,000-fold less sensitive in plaque assays, liquid culture, and a hemagglutination-elution assay. In addition, cross-resistance to both compounds was seen in all three assays. Some isolates demonstrated drug dependence with an increase in both size and number of plaques in a plaque assay and an increase in virus yield in liquid culture in the presence of inhibitors. No significant difference in neuraminidase enzyme activity was detected in vitro, and no sequence changes in the conserved sites of the neuraminidase were found. However, changes in conserved amino acids in the hemagglutinin were detected. These amino acids were associated with either the hemagglutinin receptor binding site, Thr-155, or the left edge of the receptor binding pocket, Val-223 and Arg-229. Hence, mutations at these sites could be expected to affect the affinity or specificity of the hemagglutinin binding. Compensating mutations resulting in a weakly binding hemagglutinin thus seem to be circumventing the inhibition of the neuraminidase by allowing the virus to be released from cells with less dependence on the neuraminidase.

Effects of body temperature on respiratory rate in young children.

<h3>Abstract</h3> Influenza A viruses (IAV) initiate infection <i>via</i> binding of the viral hemagglutinin (HA) to sialylated glycan receptors on host cells. HAs receptor specificity towards sialic acid (SA) is well studied and clearly critical for virus infection, but the contribution of the highly complex cellular plasma membrane to the cellular specificity remains elusive. In addition, some studies indicated that other host cell factors such as the epidermal growth factor receptor might contribute to the initial virus-cell contact and further downstream signaling¹. Here we use two complementary methods, glycan arrays and single-virus force spectroscopy (SVFS) to compare influenza virus receptor specificity with actual host cell binding. Unexpectedly, our study reveals that HAs receptor binding preference does not necessarily reflect virus-cell specificity. We propose SVFS as a tool to elucidate the cell binding preference of IAV thereby including the complex environment of sialylated receptors within the plasma membrane of living cells.