Quantitative Characterization of Glycan-Receptor Binding of H9N2 Influenza A Virus Hemagglutinin

Avian influenza subtypes such as H5, H7 and H9 are yet to adapt to the human host so as to establish airborne transmission between humans. However, lab-generated reassorted viruses possessing hemagglutinin (HA) and neuraminidase (NA) genes from an avian H9 isolate and other genes from a human-adapted (H3 or H1) subtype acquired two amino acid changes in HA and a single amino acid change in NA that confer respiratory droplet transmission in ferrets. We previously demonstrated for human-adapted H1, H2 and H3 subtypes that quantitative binding affinity of their HA to α2→6 sialylated glycan receptors correlates with respiratory droplet transmissibility of the virus in ferrets. Such a relationship remains to be established for H9 HA. In this study, we performed a quantitative biochemical characterization of glycan receptor binding properties of wild-type and mutant forms of representative H9 HAs that were previously used in context of reassorted viruses in ferret transmission studies. We demonstrate here that distinct molecular interactions in the glycan receptor-binding site of different H9 HAs affect the glycan-binding specificity and affinity. Further we show that α2→6 glycan receptor-binding affinity of a mutant H9 HA carrying Thr-189→Ala amino acid change correlates with the respiratory droplet transmission in ferrets conferred by this change. Our findings contribute to a framework for monitoring the evolution of H9 HA by understanding effects of molecular changes in HA on glycan receptor-binding properties.     

When comparative information leads us astray: the receptor-binding region of colicin E9

In an effort to develop derivatives of the Escherichia coli antimicrobial protein colicin E9 that exhibit novel interactions with a target cell, we mutagenized a 10-amino acid region located at the C terminus of the colicin receptor-binding domain. We subsequently selected for those colicin molecules that retain the antimicrobial phenotype and found that, despite a mutagenic strategy that alters every amino acid in the targeted domain, more than 70% of the engineered colicins retained antimicrobial activity. This result is all the more surprising given the extensive phylogenetic conservation of this receptor-binding domain, which originally suggested the operation of strong selective constraints on the amino acid sequence of this region. This apparent contradiction between our experimental results and the comparative data is resolved by exploring the fitness consequences of the experimentally induced amino acid substitutions. In 17 of 52 cases we examined, the fitness of cells harboring the functional engineered colicins was lower than that of our control line (containing wild-type colicin E9), and in 33 of 52 cases, equal to it. Paradoxically, two of the engineered colicins appear to confer a higher fitness to the producer cell lines. While the mechanism linking changes in the amino acid sequence of the colicin receptor-binding domain and the growth rate of the cells remains unclear, these results illustrate the surprising versatility of the colicin/receptor interaction and underscore the importance of distinguishing molecular function from organismal fitness.     

Subtype- and antigenic site-specific differences in biophysical influences on evolution of influenza virus hemagglutinin

ABSTRACT: BACKGROUND: Influenza virus undergoes rapid evolution by both antigenic shift and antigenic drift. Antibodies, particularly those binding near the receptor-binding site of hemagglutinin (HA) or the neuraminidase (NA) active site, are thought to be the primary defense against influenza infection, and mutations in antibody binding sites can reduce or eliminate antibody binding. The binding of antibodies to their cognate antigens is governed by such biophysical properties of the interacting surfaces as shape, non-polar and polar surface area, and charge. Methods: To understand forces shaping evolution of influenza virus, we have examined HA sequences of human influenza A and B viruses, assigning each amino acid values reflecting total accessible surface area, non-polar and polar surface area, and net charge due to the side chain. Changes in each of these values between neighboring sequences were calculated for each residue and mapped onto the crystal structures. Results: Areas of HA showing the highest frequency of changes agreed well with previously identified antigenic sites in H3 and H1 HAs, and allowed us to propose more detailed antigenic maps and novel antigenic sites for H1 and influenza B HA. Changes in biophysical properties differed between HAs of different subtypes, and between different antigenic sites of the same HA. For H1, statistically significant differences in several biophysical quantities compared to residues lying outside antigenic sites were seen for some antigenic sites but not others. Influenza B antigenic sites all show statistically significant differences in biophysical quantities for all antigenic sites, whereas no statistically significant differences in biophysical quantities were seen for any antigenic site is seen for H3. In many cases, residues previously shown to be under positive selection at the genetic level also undergo rapid change in biophysical properties. Conclusions: The biophysical consequences of amino acid changes introduced by antigenic drift vary from subtype to subtype, and between different antigenic sites. This suggests that the significance of antibody binding in selecting new variants may also be variable for different antigenic sites and influenza subtypes.     

Evolutionary Patterning of Hemagglutinin Gene Sequence of 2009 H1N1 Pandemic

The 2009 H1N1 swine flu is the first pandemic in decades. Infectivity of the influenza virus for human host depends largely on its ability to evade antibodies specific for viral protein called hemagglutinin (HA) that mediates attachment to the host. In the present study we analysed large number of HA gene sequences available in Flu Database maintained at NCBI. Our sequence based analysis clearly demonstrates that the amino acid usage pattern may dramatically change during the course of evolution, and there exists a clear link between a particular pattern of amino acid usage of HA genes and its potential to become infectious. Structural studies revealed how binding efficiency between the HA and sialic acid may alter the pandemic potential of infection. Our work highlights the evolutionary significance and biochemical basis of the selective advantage of certain amino acids of HA in 2009 and provides a link between the characteristics changes in HA protein and their potential to pronounce a global menace to public health.     

Antigenic structure of influenza A virus subtype H5 hemagglutinin: mechanism of acquiring stability to monoclonal antibodies in escape-mutants

The analysis of escape mutants of the avian influenza virus of H5 subtype (strain A/Mallard/Pennsylvania/10218/84) revealed the location and structure of two antigenic sites in the hemagglutinin (HA) molecule. Several escape mutants exhibited unusual features in the reactions with monoclonal antibodies (Mabs), being completely resistant in the infectivity neutralization test to the Mabs used for their selection, and retaining the ability to bind the Mabs as revealed by enzyme-linked immunosorbent assay. An enhancement of the binding by an amino acid change in a different antigenic site was demonstrated, as well as a complete abolishment of the binding by a mutation selected by passage in the presence of an excess of the non-neutralizing Mab of high binding ability. The observed effects did not result from the changes in the affinity of the mutant HA toward sialic receptors. The data suggest that one amino acid change in HA may prevent the virus neutralization by different mechanisms for different Mabs: either the binding of the Mab to HA is prevented, or the bound Mab is unable to block the receptor-binding pocket of HA. Different mechanisms of the acquisition of resistance to Mabs in the course of the selection of escape mutants are discussed.     

The receptor-binding and membrane-fusion properties of influenza virus variants selected using anti-haemagglutinin monoclonal antibodies.

A monoclonal antibody raised against X-31 influenza virus reacted with the majority of natural H3N2 viruses isolated between 1968 and 1982. A number of variants of X-31 and of a receptor-binding mutant of X-31 were selected by the antibody during virus replication in eggs and MDCK cells. Antibody-binding assays indicated that the viruses selected were not antigenic variants and analyses using derivatized erythrocytes showed that their receptor-binding properties differed from those of the parent viruses. The amino acid substitutions in the variants were all located in the vicinity of the receptor-binding site and the structural consequences are discussed in relation to the three-dimensional structure of X-31 HA. In addition all of the variants fused membranes at higher pH than wild-type virus indicating that structural modifications in the distal globular region of HA influence the low pH-induced conformational change required for membrane fusion.     

Monoclonal antibodies to CD44 and their influence on hyaluronan recognition

Antibodies to CD44 have been used to inhibit a variety of processes which include lymphohemopoiesis, lymphocyte migration, and tumor metastasis. Some, but not all, CD44-mediated functions derive from its ability to serve as a receptor for hyaluronan (HA). However, sites on CD44 that interact with either ligands or antibodies are poorly understood. Interspecies rat/mouse CD44 chimeras were used to analyze the specificity of 25 mAbs and to determine that they recognize at least seven epitopes. Amino acid substitutions that resulted in loss of antibody recognition were all located in the region of homology to other cartilage link family proteins. While at least five epitopes were eliminated by single amino acid replacements, multiple residues had to be changed to destroy binding by other antibodies. One antibody was sensitive to changes in any of three separate parts of the molecule and some antibodies to distinct epitopes cross-blocked each other. Certain antibodies had the ability to increase HA binding by lymphocytes but this did not correlate absolutely with antibody specificity and was only partially attributable to CD44 cross-linking. Antibodies that consistently blocked HA recognition were all sensitive to amino acid changes within a short stretch of CD44. Such blocking antibodies interacted with CD44 more strongly than ligand in competition experiments. One large group of antibodies blocked ligand binding, but only with a particular cell line. This detailed analysis adds to our understanding of functional domains within CD44 and requirements for antibodies to influence recognition of one ligand.     

Accumulation of amino acid substitutions promotes irreversible structural changes in the hemagglutinin of human influenza AH3 virus during evolution

During protein evolution the amino acid substitutions accumulate with time. However, the effect of accumulation of the amino acid substitutions to structural changes has not been estimated well. We will propose that the discordance of amino acid substitution on the HA protein of influenza A virus is useful for the assessment of structural changes during evolution. Discordance value can be obtained from the experimental data of tolerance or intolerance by introducing site directed mutagenesis at the homologous positions of two HA proteins holding the same amino acid residues. The value of discordance correlated to the number of amino acid differences among proteins. In the H3HA discordance rate was calculated to be 0.45% per one amino acid change. Furthermore, discordance of amino acid substitutions suggests that tolerable amino acid substitutions in different order have a probability of promoting irreversible divergence of the HA protein to different subtypes.     

Evolution of the Hemagglutinin Protein of the New Pandemic H1N1 Influenza Virus: Maintaining Optimal Receptor Binding by Compensatory Substitutions

Pandemic influenza A H1N1 (pH1N1) virus emerged in 2009. In the subsequent 4 years, it acquired several genetic changes in its hemagglutinin (HA). Mutations may be expected while virus is adapting to the human host or upon evasion from adaptive immune responses. However, pH1N1 has not displayed any major antigenic changes so far. We examined the effect of the amino acid substitutions found to be most frequently occurring in the pH1N1 HA protein before 1 April 2012 on the receptor-binding properties of the virus by using recombinant soluble HA trimers. Two changes (S186P and S188T) were shown to increase the receptor-binding avidity of HA, whereas two others (A137T and A200T) decreased binding avidity. Construction of an HA protein tree revealed the worldwide emergence of several HA variants during the past few influenza seasons. Strikingly, two major variants harbor combinations of substitutions (S186P/A137T and S188T/A200T, respectively) with opposite individual effects on binding. Stepwise reconstruction of the HA proteins of these variants demonstrated that the mutations that increase receptor-binding avidity are compensated for by the acquisition of subsequent mutations. The combination of these substitutions restored the receptor-binding properties (avidity and specificity) of these HA variants to those of the parental virus. The results strongly suggest that the HA of pH1N1 was already optimally adapted to the human host upon its emergence in April 2009. Moreover, these results are in agreement with a recent model for antigenic drift, in which influenza A virus mutants with high and low receptor-binding avidity alternate.     

不同血清型肉毒毒素受体结合域研究进展

肉毒毒素(botulinum neurotoxin, BoNT)是人类已知毒性最强的蛋白质之一,可以引起肌肉松弛麻痹,严重时可导致死亡。肉毒毒素共分为7种血清型(BoNT/A-BoNT/G),根据氨基酸序列差异可进一步分为40多种亚型。肉毒毒素分子结构由3个基本结构域组成：重链羧基端细胞受体结合域、氨基端的易位域和轻链催化域。在运动神经元表面,受体结合域首先与聚唾液酸神经节苷脂结合,随后与突触囊泡蛋白2或突触囊泡结合蛋白结合形成双受体复合物。每种血清型的受体结合域都必须与其相应受体结合才能发挥作用。肉毒毒素的结构功能及其对宿主的作用一直都是研究热点。近年来,因受体结合域可以促进肉毒毒素与运动神经元膜特异性结合,而成为新的研究方向。本综述将概述不同血清型肉毒毒素与受体结合过程中受体结合域结构变化和结合位点差异。通过分析不同血清型及亚型的序列以及受体结合域结构特征,可以更好地了解细胞受体结合域的序列差异和功能,并为肉毒毒素的治疗策略提供新思路。     

Functional dissection of the Moloney murine leukemia virus envelope protein gp70.

The envelope protein of Moloney murine leukemia virus (Mo-MLV) is a complex glycoprotein that mediates receptor binding and entry via fusion with cell membranes. By using a series of substitution mutations and truncations in the Mo-MLV external envelope surface protein gp70, we have identified regions important for these processes. Firstly, truncations of gp70 revealed that the minimal continuous receptor-binding region is amino acids 9 to 230, in broad agreement with other studies. Secondly, within this region there are two key basic amino acids, Arg-83 and Arg-95, that are essential for receptor binding and may interact with a negatively charged residue(s) or with the pi electrons of the aromatic ring on a hydrophobic residue(s) in the basic amino acid transporter protein that is the Mo-MLV ecotropic receptor. Finally, we showed that outside the minimal receptor-binding region at amino acids 2 to 8, there is a region that is essential for postbinding fusion events.     

Characterization of the diphtheria toxin receptor-binding domain

The carboxyl-terminal region of diphtheria toxin (DT) has been analysed in order to determine regions of receptor recognition. Biochemical cleavage of the toxin with hydroxylamine (HA) was used to generate the peptides HA9DT (residues 454–535), HA6DT (residues 482–535), and HA3DT (residues 454–461). Characterization of HA6DT demonstrated that the final 54 amino acids of DT are sufficient to constitute the receptor-binding domain of the toxin. Within HA9DT, the region encompassing HA3DT and containing the highly cationic polyphosphate-binding site did not contribute to the binding ability of HA6DT. Consistent with this observation, HA3DT itself did not compete for binding of radiolabelled DT to Vero cells. A 30-amino acid synthetic peptide composed of residues 506–535 did not block receptor binding of DT, indicating that residues toward the amino-terminus of HA6DT, or the entire HA6DT region, are required for receptor recognition.     

Generating and characterizing monoclonal and polyclonal antibodies against avian H5N1 hemagglutinin protein

Accurate and timely diagnoses are central to H5N1 infection control. Here we describe the cloning and expression of the HA1 protein of the A/Vietnam/1203/04 strain in a bacterial system to generate mono-/polyclonal antibodies. All of the eight generated monoclonal antibodies recognized the same linear epitope on the top globular region of the HA structure—a highly conserved epitope among all circulating H5N1 clades identified by amino acid alignment. Results from immunofluorescence staining and Western blotting indicate that all monoclonal antibodies interacted with a denatured form of HA proteins, while the resultant polyclonal antibodies recognized both denatured and native HA proteins on H5N1 reverse-genetics (RG) viruses. Results from flow cytometry and microneutralization assays indicate that the polyclonal antibodies blocked viral binding and neutralized H5N1-RG viruses. Our results may prove useful to establishing future H5N1 mono-and polyclonal antibodies, and perhaps contribute to the development of an alternative H5N1 vaccine.     

Analysis of the receptor-binding site of murine coronavirus spike protein.

It has been found that a domain composed of 330 amino acids of the N terminus of murine coronavirus spike protein [S1N(330)] is involved in receptor-binding activity (H. Kubo, Y.K. Yamada, and F. Taguchi, J. Virol. 68:5403-5410, 1994). To delineate the amino acid sequences involved in receptor-binding activity, we have compared the S1N(330) proteins of seven different mouse hepatitis virus MHV strains that are able to utilize the MHV receptor protein. Three conserved regions (sites I, II, and III) were found to consist of more than 10 identical amino acids, and they were analyzed for receptor-binding activity by site-directed mutagenesis. S1N(330) with a substitution at position 62 from the N terminus of S1 in region I and that with substitutions at positions 212, 214, and 216 in region II showed no receptor-binding activity. The S1N(330) mutants without receptor-binding activity were not able to prevent virus binding to the receptor. These results suggest that the receptor-binding site on S1N(330) is composed of regions located apart from each other in the protein's primary structure, in which Thr at position 62 as well as amino acids located at positions 212, 214, and 216 are particularly important.     

Identification, cloning and characterization of a novel nuclear protein, HA95, homologous to A-kinase anchoring protein 95

Previously, we have identified and characterized nuclear AKAP95 from man which targets cyclic AMP (cAMP)-dependent protein kinase (PKA)-type II to the condensed chromatin/spindle region at mitosis. Here we report the cloning of a novel nuclear protein with an apparent molecular mass of 95 kDa that is similar to AKAP95 and is designated HA95 (homologous to AKAP95). HA95 cDNA sequence encodes a protein of 646 amino acids that shows 61% homology to the deduced amino acid sequence of AKAP95. The HA95 gene is located on chromosome 19p13.1 immediately upstream of the AKAP95 gene. Both HA95 and AKAP95 genes contain 14 exons encoding similar regions of the respective proteins, indicating a previous gene duplication event as the origin of the two tandem genes. Despite their apparent similarity, HA95 does not bind RII in vitro. HA95 contains a putative nuclear localization signal in its N-terminal domain. It is localized exclusively into the nucleus as demonstrated in cells transfected with HA95 fused to either green fluorescence protein or the c-myc epitope. In the nucleus, the HA95 protein is found as complexes directly associated with each other or indirectly associated via other nuclear proteins. In interphase, HA95 is co-localized with AKAP95, but the two proteins are not biochemically associated. At metaphase, both proteins co-localize with condensed chromosomes. The similarity in sequence and localization of HA95 and AKAP95 suggests that the two molecules constitute a novel family of nuclear proteins that may exhibit related functions.     

Receptor-binding profiles of H7 subtype influenza viruses in different host species

Influenza viruses of gallinaceous poultry and wild aquatic birds usually have distinguishable receptor-binding properties. Here we used a panel of synthetic sialylglycopolymers and solid-phase receptor-binding assays to characterize receptor-binding profiles of about 70 H7 influenza viruses isolated from aquatic birds, land-based poultry, and horses in Eurasia and America. Unlike typical duck influenza viruses with non-H7 hemagglutinin (HA), all avian H7 influenza viruses, irrespective of the host species, displayed a poultry-virus-like binding specificity, i.e., preferential binding to sulfated oligosaccharides Neu5Acα2-3Galβ1-4(6-O-HSO(3))GlcNAc and Neu5Acα2-3Galβ1-4(Fucα1-3)(6-O-HSO(3))GlcNAc. This phenotype correlated with the unique amino acid sequence of the amino acid 185 to 189 loop of H7 HA and seemed to be dependent on ionic interactions between the sulfate group of the receptor and Lys193 and on the lack of sterical clashes between the fucose residue and Gln222. Many North American and Eurasian H7 influenza viruses displayed weak but detectable binding to the human-type receptor moiety Neu5Acα2-6Galβ1-4GlcNAc, highlighting the potential of H7 influenza viruses for avian-to-human transmission. Equine H7 influenza viruses differed from other viruses by preferential binding to the N-glycolyl form of sialic acid. Our data suggest that the receptor-binding site of contemporary H7 influenza viruses in aquatic and terrestrial birds was formed after the introduction of their common precursor from ducks to a new host, presumably, gallinaceous poultry. The uniformity of the receptor-binding profile of H7 influenza viruses in various wild and domestic birds indicates that there is no strong receptor-mediated host range restriction in birds on viruses with this HA subtype. This notion agrees with repeated interspecies transmission of H7 influenza viruses from aquatic birds to poultry.     

Identification of spike protein residues of murine coronavirus responsible for receptor-binding activity by use of soluble receptor-resistant mutants.

We previously demonstrated by site-directed mutagenesis analysis that the amino acid residues at positions 62 and 214 to 216 in the N-terminal region of mouse hepatitis virus (MHV) spike (S) protein are important for receptor-binding activity (H. Suzuki and F. Taguchi, J. Virol. 70:2632-2636, 1996). To further identify the residues responsible for the activity, we isolated the mutant viruses that were not neutralized with the soluble form of MHV receptor proteins, since such mutants were expected to have mutations in amino acids responsible for receptor-binding activity. Five soluble-receptor-resistant (srr) mutants isolated had mutations in a single amino acid at three different positions: one was at position 65 (Leu to His) (srr11) in the S1 subunit and three were at position 1114 (Leu to Phe) (srr3, srr4, and srr7) and one was at position 1163 (Cys to Phe) (srr18) in the S2 subunit. The receptor-binding activity examined by a virus overlay protein blot assay and by a coimmunoprecipitation assay showed that srr11 S protein had extremely reduced binding activity, while the srr7 and srr18 proteins had binding activity similar to that of wild-type cl-2 protein. However, when cell surface receptors were used for the binding assay, all srr mutants showed activity similar to that of the wild type or only slightly reduced activity. These results, together with our previous observations, suggest that amino acids located at positions 62 to 65 of S1, a region conserved among the MHV strains examined, are important for receptor-binding activity. We also discuss the mechanism by which srr mutants with a mutation in S2 showed high resistance to neutralization by a soluble receptor, despite their sufficient level of binding to soluble receptors.     

Accumulation of amino acid substitutions promotes irreversible structural changes in the hemagglutinin of human influenza AH3 virus during evolution

In order to clarify the effect of an accumulation of amino acid substitutions on the hemadsorption character of the influenza AH3 virus hemagglutinin (HA) protein, we introduced single-point amino acid changes into the HA1 domain of the HA proteins of influenza viruses isolated in 1968 (A/Aichi/2/68) and 1997 (A/Sydney/5/97) by using PCR-based random mutation or site-directed mutagenesis. These substitutions were classified as positive or negative according to their effects on the hemadsorption activity. The rate of positive substitutions was about 50% for both strains. Of 44 amino acid changes that were identical in the two strains with regard to both the substituted amino acids and their positions in the HA1 domain, 22% of the changes that were positive in A/Aichi/2/68 were negative in A/Sydney/5/97 and 27% of the changes that were negative in A/Aichi/2/68 were positive in A/Sydney/5/97. A similar discordance rate was also seen for the antigenic sites. These results suggest that the accumulation of amino acid substitutions in the HA protein during evolution promoted irreversible structural changes and therefore that antigenic changes in the H3HA protein may not be limited.     

禽流感病毒血凝素HA的可变性解析

以重组的蒙古鸭H5N2禽流感病毒A/Duck/Mongolia/54/01的血凝素HA蛋白的cDNA为模板,进行PCR随机突变,表达只有单个氨基酸突变的H5HA基因共计38个.根据红细胞吸附反应,分析这些突变HA的功能,仍然具有红细胞吸附活性的单个氨基酸突变的HA约占89%,说明H5HA单个氨基酸突变的容许率是相当高的.HA1区突变数目大约是HA2区的两倍.对失去红细胞吸附功能和某些仍然拥有红细胞吸附功能的HA及单个氨基酸突变的位置与结构的关系进行探讨.有两个位点氨基酸突变了两次,但都不影响红细胞吸附功能,对红细胞吸附功能的影响,似乎主要由位置决定,而不是取决于取代的氨基酸的种类.位点179位和122位的突变是不允许的;位点179位于H5N1的受体结合区域RBD内,122位位于A抗原决定簇区附近,推测在H5HA三维结构上,这两个位点位于HA分子的内部,维持着H5HA的结构.HA1Cys位点4和HA2Cys位点148的突变是不允许的.这两个Cys正好形成HA1和HA2连接的桥梁,对维持H5HA结构也是相当重要的.本实验中HA先后失去了三个糖基化位点,但并不影响吸附红细胞的功能.总之,通过实验分析以研究某些氨基酸改变的效果,寻找关键位点是否突变,可以作为评估H5N1野毒株大流行潜力的分子标志.     

Structure-function analysis of synthetic and recombinant derivatives of transforming growth factor alpha.

Transforming growth factor alpha (TGF-alpha) is a 50-amino-acid peptide that stimulates cell proliferation via binding to cell surface receptors. To identify the structural features of TGF-alpha that govern receptor-ligand interactions, we prepared synthetic peptide fragments and recombinant mutant proteins of TGF-alpha. These TGF-alpha derivatives were tested in receptor binding and mitogenesis assays. Synthetic peptides representing the N terminus, the C terminus, or the individual disulfide constrained rings of TGF-alpha did not exhibit receptor-binding or mitogenic activity. Replacement of the cysteines with alanines at positions 8 and 21, 16 and 32, and 34 and 43 or at positions 8 and 21 and 34 and 43 yielded inactive mutant proteins. However, mutant proteins containing substitutions or deletions in the N-terminal region retained significant biologic activity. Conservative amino acid changes at residue 29 or 38 or both and a nonconservative amino acid change at residue 12 had little effect on binding or mitogenesis. However, nonconservative amino acid changes at residues 15, 38, and 47 produced dramatic decreases in receptor binding (23- to 71-fold) and mitogenic activity (38- to 125-fold). These studies indicate that at least three distinct regions of TGF-alpha contribute to biologic activity.