DNA aptamers against the receptor binding region of hemagglutinin prevent avian influenza viral infection

The entrance of influenza virus into host cells is facilitated by the attachment of the globular region of viral hemagglutinin to the sialic acid receptors on host cell surfaces. In this study, we have cloned the cDNA fragment encoding the entire globular region (residues 101–257) of hemagglutinin of the H9N2 type avian influenza virus (A/ck/Korea/ms96/96). The protein segment (denoted as the H9 peptide), which was expressed and purified in E. coli, was used for the immunization of BALB/c mice to obtain the anti-H9 antiserum. To identify specific DNA aptamers with high affinity to H9 peptide, we conducted the SELEX method; 19 aptamers were newly isolated. A random mixture of these aptamers showed an increased level of binding affinity to the H9 peptide. The sequence alignment analysis of these aptamers revealed that 6 aptamers have highly conserved consensus sequences. Among these, aptamer C7 showed the highest similarity to the consensus sequences. Therefore, based on the C7 aptamer, we synthesized a new modified aptamer designated as C7-35M. This new aptamer showed strong binding capability to the viral particles. Furthermore, it could prevent MDCK cells from viral infection by strong binding to the viral particles. These results suggest that our aptamers can recognize the hemagglutinin protein of avian influenza virus and inhibit the binding of the virus to target receptors required for the penetration of host cells.     

Essential sequence of influenza B virus hemagglutinin DNA to provide protection against lethal homologous viral infection

Hemagglutinin (HA) is the main surface glycoprotein of influenza B virus. The B/Ibaraki/2/85 virus HA gene is 1758 bp in length, including signal peptide sequence, HA1 sequence, and HA2 sequence. We previously proved that B/Ibaraki/2/85 HA DNA induced immune response and provided effective protection in mice against challenge with homologous virus. In this study, a series of recombinant plasmids encoding truncated HA gene were constructed by PCR. BALB/c mice were immunized with the plasmids and challenged with a lethal dose of homologous virus. The essential sequence of HA DNA against influenza virus was explored by evaluation of survival rate, lung virus titer, bodyweight change, and serum anti-HA antibody titer of mice. The result showed that serial deletion did not deprive HA DNA of its protective ability until 885 nucleotides (295 amino acids) at 3'-terminal or 9 nucleotides of the signal peptide sequence at 5'-terminal were deleted. When the signal peptide sequence was kept intact and the 5'-terminal deletion started at the beginning of the HA1 sequence, deletion of 51 nucleotides (17 amino acids) made HA DNA lose its protective ability. This suggests that the sequence nt94-876 of B/Ibaraki/2/85 virus HA DNA played an important role in protection against infection.     

Regulation of receptor binding affinity of influenza virus hemagglutinin by its carbohydrate moiety.

The hemagglutinin (HA) of the fowl plague virus (FPV) strain of influenza A virus has two N-linked oligosaccharides attached to Asn123 and Asn149 in the vicinity of the receptor binding site. The effect of these carbohydrate side chains on the binding of HA to neuraminic acid-containing receptors has been analyzed. When the oligosaccharides were deleted by site-specific mutagenesis, HA expressed from a simian virus 40 vector showed enhanced hemadsorbing activity. Binding was so strong under these conditions that erythrocytes were no longer released by viral neuraminidase and that release was significantly reduced when neuraminidase from Vibrio cholerae was used. Similarly, when these oligosaccharides were removed selectively from purified viruses by N-glycosidase F, such virions were unable to elute from receptors, although they retained neuraminidase activity. Thus, release of FPV from cell receptors depends on the presence of the HA glycans at Asn123 and Asn149. On the other hand, receptor binding was abolished when these oligosaccharides were sialylated after expression in the absence of neuraminidase (M. Ohuchi, A. Feldmann, R. Ohuchi, and H.-D. Klenk, Virology 212:77-83, 1995). These observations indicate that the receptor affinity of FPV HA is controlled by oligosaccharides adjacent to the receptor binding site.     

A single amino acid substitution in 1918 influenza virus hemagglutinin changes receptor binding specificity

The receptor binding specificity of influenza viruses may be important for host restriction of human and avian viruses. Here, we show that the hemagglutinin (HA) of the virus that caused the 1918 influenza pandemic has strain-specific differences in its receptor binding specificity. The A/South Carolina/1/18 HA preferentially binds the alpha2,6 sialic acid (human) cellular receptor, whereas the A/New York/1/18 HA, which differs by only one amino acid, binds both the alpha2,6 and the alpha2,3 sialic acid (avian) cellular receptors. Compared to the conserved consensus sequence in the receptor binding site of avian HAs, only a single amino acid at position 190 was changed in the A/New York/1/18 HA. Mutation of this single amino acid back to the avian consensus resulted in a preference for the avian receptor.     

Infectivity studies of influenza virus hemagglutinin receptor binding site mutants in mice

The replicative properties of influenza virus hemagglutinin (HA) mutants with altered receptor binding characteristics were analyzed following intranasal inoculation of mice. Among the mutants examined was a virus containing a Y98F substitution at a conserved position in the receptor binding site that leads to a 20-fold reduction in binding. This mutant can replicate as well as wild-type (WT) virus in MDCK cells and in embryonated chicken eggs but is highly attenuated in mice, exhibiting titers in lungs more than 1,000-fold lower than those of the WT. The capacity of the Y98F mutant to induce antibody responses and the structural locations of HA reversion mutations are examined.     

Delay of influenza hemagglutinin refolding into a fusion-competent conformation by receptor binding: a hypothesis

Two subunits of influenza hemagglutinin (HA), HA1 and HA2, represent one of the best-characterized membrane fusion machines. While a low pH conformation of HA2 mediates the actual fusion, HA1 establishes a specific connection between the viral and cell membranes via binding to the sialic acid-containing receptors. Here we propose that HA1 may also be involved in modulating the kinetics of HA refolding. We hypothesized that binding of the HA1 subunit to its receptor restricts the major refolding of the low pH-activated HA to a fusion-competent conformation and, in the absence of fusion, to an HA-inactivated state. Dissociation of the HA1-receptor connection was considered to be a slow kinetic step. To verify this hypothesis, we first analyzed a simple kinetic scheme accounting for the stages of dissociation of the HA1/receptor bonds, inactivation and fusion, and formulated experimentally testable predictions. Second, we verified these predictions by measuring the extent of fusion between HA-expressing cells and red blood cells. Three experimental approaches based on 1) the temporal inhibition of fusion by lysophosphatidylcholine, 2) rapid dissociation of the HA1-receptor connections by neuraminidase treatment, and 3) substitution of membrane-anchored receptors by a water-soluble sialyllactose all provided support for the proposed role of the release of HA1-receptor connections. Possible biological implications of this stage in HA refolding and membrane fusion are being discussed.     

Characterization of influenza hemagglutinin interactions with receptor by NMR

In influenza, the envelope protein hemagglutinin (HA) plays a critical role in viral entry by first binding to sialic acid receptors on the cell surface and subsequently mediating fusion of the viral and target membranes. In this work, the receptor binding properties of influenza A HA from different subtypes (H1 A/California/04/09, H5 A/Vietnam/1205/04, H5 A/bar-headed goose/Qinghai/1A/05, and H9 A/Hong Kong/1073/99) have been characterized by NMR spectroscopy. Using saturation transfer difference (STD) NMR, we find that all HAs bind to the receptor analogs 2,3-sialyllactose and 2,6-sialyllactose, with subtle differences in the binding mode. Using competition STD NMR, we determine the receptor preferences for the HA subtypes. We find that H5-Qinghai and H9-Hong Kong HA bind to both receptor analogs with similar affinity. On the other hand, H1 exhibits a clear preference for 2,6-sialyllactose while H5-Vietnam exhibits a clear preference for 2,3-sialyllactose. Together, these results are interpreted within the context of differences in both the amino acid sequence and structures of HA from the different subtypes in determining receptor preference.     

GPI- and transmembrane-anchored influenza hemagglutinin differ in structure and receptor binding activity

We investigated the influence of a glycosylphosphatidylinositol (GPI) anchor on the ectodomain of the influenza hemagglutinin (HA) by replacing the wild type (wt) transmembrane and cytoplasmic domains with a GPI lipid anchor. GPI-anchored HA (GPI-HA) was transported to the cell surface with equal efficiency and at the same rate as wt-HA. Like wt-HA, cell surface GPI-HA, and its ectodomain released with the enzyme PI-phospholipase C (PI-PLC), were 9S trimers. Compared to wt-HA, the GPI-HA ectodomain underwent additional terminal oligosaccharide modifications; some of these occurred near the receptor binding pocket and completely inhibited the ability of GPI-HA to bind erythrocytes. Growth of GPI-HA-expressing cells in the presence of the mannosidase I inhibitor deoxymannojirimycin (dMM) abrogated the differences in carbohydrate modification and restored the ability of GPI-HA to bind erythrocytes. The ectodomain of GPI-HA produced from cells grown in the presence or absence of dMM underwent characteristic low pH-induced conformational changes (it released its fusion peptides and became hydrophobic and proteinase sensitive) but at 0.2 and 0.4 pH units higher than wt-HA, respectively. These results demonstrate that although GPI-HA forms a stable trimer with characteristics of the wt, its structure is altered such that its receptor binding activity is abolished. Our results show that transmembrane and GPI-anchored forms of the same ectodomain can exhibit functionally important differences in structure at a great distance from the bilayer.     

Direct role of viral hemagglutinin in B-cell mitogenesis by influenza viruses

The mitogenic activity of influenza virus is a function of the hemagglutinin (HA) molecule. Purified HA is mitogenic for murine B lymphocytes but not T lymphocytes. Furthermore, like the intact virus, HA of the H2 (but not H3) subtype is mitogenic only for B cells expressing the class II major histocompatibility complex glycoprotein I-E. Since virus bearing uncleaved HA is as mitogenic as virus bearing cleaved HA, the membrane fusion activity of the HA molecule is not involved.     

Exosome-driven antigen transfer for MHC class II presentation facilitated by the receptor binding activity of influenza hemagglutinin

The mechanisms underlying MHC class I-restricted cross-presentation, the transfer of Ag from an infected cell to a professional APC, have been studied in great detail. Much less is known about the equivalent process for MHC class II-restricted presentation. After infection or transfection of class II-negative donor cells, we observed minimal transfer of a proteasome-dependent "class I-like" epitope within the influenza neuraminidase glycoprotein but potent transfer of a classical, H-2M-dependent epitope within the hemagglutinin (HA) glycoprotein. Additional experiments determined transfer to be exosome-mediated and substantially enhanced by the receptor binding activity of incorporated HA. Furthermore, a carrier effect was observed in that incorporated HA improved exosome-mediated transfer of a second membrane protein. This route of Ag presentation should be relevant to other enveloped viruses, may skew CD4(+) responses toward exosome-incorporated glycoproteins, and points toward novel vaccine strategies.     

Receptor binding and pH stability — How influenza A virus hemagglutinin affects host-specific virus infection

Influenza A virus strains adopt different host specificities mainly depending on their hemagglutinin (HA) protein. Via HA, the virus binds sialic acid receptors of the host cell and, upon endocytic uptake, HA triggers fusion between the viral envelope bilayer and the endosomal membrane by a low pH-induced conformational change leading to the release of the viral genome into the host cell cytoplasm. Both functions are crucial for viral infection enabling the genesis of new progeny virus.     

A complex of influenza hemagglutinin with a neutralizing antibody that binds outside the virus receptor binding site.

The structure of a complex of influenza hemagglutinin (HA) with a neutralizing antibody shows that the antibody binds to HA at a distance from the virus receptor binding site. Comparison of the properties of this antibody and its Fab with those of an antibody that recognizes an epitope overlapping the receptor binding site leads to two main conclusions. First, inhibition of receptor binding is an important component of neutralization. Second, the efficiency of neutralization by the antibodies ranks in the same order as their avidities for HA, and their large size makes these antibodies highly efficient at neutralization, regardless of the location of their epitope in relation to the virus receptor binding site. These observations provide rationales for the range of antibody specificities that are detected in immune sera and for the distribution of sequence changes on the membrane-distal surface of influenza HAs that occur during 'antigenic drift.'     

Tight binding of influenza virus hemagglutinin to its receptor interferes with fusion pore dilation

Deletion of oligosaccharide side chains near the receptor binding site of influenza virus A/USSR/90/77 (H1N1) hemagglutinin (HA) enhanced the binding of HA to erythrocyte receptors, as was also observed with A/FPV/Rostock/34 (H7N1). Correlated with the enhancement of binding activity, the cell fusion activity of HA was reduced. A mutant HA in which three oligosaccharide side chains were deleted showed the highest level of binding and the lowest level of fusion among the HAs tested. The cell fusion activity of the oligosaccharide deletion mutant of HA, however, was drastically elevated when the binding activity was reduced by deletion of four amino acids adjacent to the receptor binding site. Thus, a reciprocal relationship was observed between the receptor binding and the cell fusion activities of H1/USSR HA. No difference was observed, however, in lipid mixing activity, so-called hemifusion, between wild-type (WT) and oligosaccharide deletion mutant HAs. Soluble dye transfer testing showed that even the HA with the lowest cell fusion activity was able to form fusion pores through which a small molecule such as calcein could pass. However, electron microscopic studies revealed that a large molecule such as hemoglobin hardly passed through the fusion pores formed by the mutant HA, whereas hemoglobin did efficiently pass through those formed by the WT HA. These results suggested that interference in the process of dilation of fusion pores occurs when the binding of HA to the receptor is too tight. Since the viral nucleocapsid is far larger than hemoglobin, appropriate receptor binding affinity is important for virus entry.     

Analysis of influenza virus hemagglutinin receptor binding mutants with limited receptor recognition properties and conditional replication characteristics

To examine the range of selective processes that potentially operate when poorly binding influenza viruses adapt to replicate more efficiently in alternative environments, we passaged a virus containing an attenuating mutation in the hemagglutinin (HA) receptor binding site in mice and characterized the resulting mutants with respect to the structural locations of mutations selected, the replication phenotypes of the viruses, and their binding properties on glycan microarrays. The initial attenuated virus had a tyrosine-to-phenylalanine mutation at HA1 position 98 (Y98F), located in the receptor binding pocket, but viruses that were selected contained second-site pseudoreversion mutations in various structural locations that revealed a range of molecular mechanisms for modulating receptor binding that go beyond the scope that is generally mapped using receptor specificity mutants. A comparison of virus titers in the mouse respiratory tract versus MDCK cells in culture showed that the mutants displayed distinctive replication properties depending on the system, but all were less attenuated in mice than the Y98F virus. An analysis of receptor binding properties confirmed that the initial Y98F virus bound poorly to several different species of erythrocytes, while all mutants reacquired various degrees of hemagglutination activity. Interestingly, both the Y98F virus and pseudoreversion mutants were shown to bind very inefficiently to standard glycan microarrays containing an abundance of binding substrates for most influenza viruses that have been characterized to date, provided by the Consortium for Functional Glycomics. The viruses were also examined on a recently developed microarray containing glycans terminating in sialic acid derivatives, and limited binding to a potentially interesting subset of glycans was revealed. The results are discussed with respect to mechanisms for HA-mediated receptor binding, as well as regarding the species of molecules that may act as receptors for influenza virus on host cell surfaces.     

Influenza viral hemagglutinin complicated shape is advantageous to its binding affinity for sialosaccharide receptor

Do the complexity and the bulkiness of a protein affect the affinity between protein and ligand? We attempted to investigate this problem by using ab initio fragment molecular orbital (FMO) method to calculate the binding energy between human influenza viral hemagglutinin (HA) and human oligo-saccharide receptor. We compared the binding energies of 4 different sizes of human A virus HA H3 subtype complexed with human receptor Neu5Ac(alpha2-6)Gal as a model. The full shape receptor binding domain complexed with Neu5Ac(alpha2-6)Gal had the highest binding energy 170.3kcal/mol at the FMO-HF/STO-3G level, which was 52.3kcal/mol higher than that of the smallest domain-receptor complex. These data provide the consideration of the backyard bulkiness beyond the binding site of protein to the protein-ligand stability.     

Selection of an antiviral RNA aptamer against hemagglutinin of the subtype H5 avian influenza virus

Avian influenza is an acute viral respiratory disease caused by RNA viruses of the family Orthomyxoviridae. The influenza A virus subtype H5 can cause severe illness and results in almost 100% mortality rate among livestock. Hemagglutinin (HA) present in the virus envelope plays an essential role in the initiation of viral infection. In this study, we investigated the efficacy of using HA as a target for antiviral therapy through nucleic acid aptamers. After purification of the receptor binding domain (HA1) of HA protein, activity of recombinant HA1 was confirmed by using hemagglutination assay. We selected RNA aptamer candidates after 15 rounds of iterative Systematic Evolution of Ligands by EXponential enrichment (SELEX) targeting the biologically active HA protein. The selected RNA aptamer HAS15-5, which specifically binds to HA1, exhibited significant antiviral efficacy according to the results of a hemagglutination inhibition assay using egg allantoic fluids harboring the virus. Thus, the RNA aptamer HAS15-5, which acts by blocking and inhibiting the receptor-binding domain of viral HA, can be developed as a novel antiviral agent against type H5 avian influenza virus.     

Formation of influenza virus particles lacking hemagglutinin on the viral envelope.

We investigated the intracellular block in the transport of hemagglutinin (HA) and the role of HA in virus particle formation by using temperature-sensitive (ts) mutants (ts134 and ts61S) of influenza virus A/WSN/33. We found that at the nonpermissive temperature (39.5 degrees C), the exit of ts HA from the rough endoplasmic reticulum to the Golgi complex was blocked and that no additional block was apparent in either the exit from the Golgi complex or post-Golgi complex transport. When MDBK cells were infected with these mutant viruses, they produced noninfectious virus particles at 39.5 degrees C. The efficiency of particle formation at 39.5 degrees C was essentially the same for both wild-type (wt) and ts virus-infected cells. When compared with the wt virus produced at either 33 or 39.5 degrees C or the ts virus formed at 33 degrees C, these noninfectious virus particles were lighter in density and lacked spikes on the envelope. However, they contained the full complement of genomic RNA as well as all of the structural polypeptides of influenza virus with the exception of HA. In these spikeless particles, HA could not be detected at the limit of 0.2% of the HA present in wt virions. In contrast, neuraminidase appeared to be present in a twofold excess over the amount present in ts virus formed at 33 degrees C. These observations suggest that the presence of HA is not an obligatory requirement for the assembly and budding of influenza virus particles from infected cells. The implications of these results and the possible role of other viral proteins in influenza virus morphogenesis are discussed.     

Fluorescent ligands for the hemagglutinin of influenza A: synthesis and ligand binding assays

Two fluorescent conjugates of sialic acid have been prepared, with a convenient synthetic route that involves preparation of an unsaturated benzyl ester by cross-metathesis, followed by combined hydrogenation/ hydrogenolysis to provide a sialoside bearing a delta-carboxybutyl group, suitable for coupling with the chosen fluorophores. The fluorescent conjugates bound to bromelain-cleaved hemagglutinin (BHA) with affinities in the low microM range. Binding was accompanied by approximately 4.5-fold fluorescence enhancement for the dansyl conjugate 1 and approximately 3-fold fluorescence quenching for the pyrene conjugate 3.     

On the origin of influenza A hemagglutinin

Recent advances in phylogenetic methods have produced some reassessments of the ages of the most recent common ancestor of hemagglutinin proteins in known strains of influenza A. This paper applies Bayesian phylogenetic analysis implemented in BEAST to date the nodes on the influenza A hemagglutinin tree. The most recent common ancestor (MRCA) of influenza A hemagglutinin proteins is located with 95% confidence between 517 and 1497 of the Common Era (AD), with the center of the probability distribution at 1056 AD. The implications of this revised dating for both historical and current epidemiology are discussed. Influenza A can be seen as an emerging disease of mediaeval and early modern times.