Subtyping of influenza virus A hemagglutinine with hybridization microarray

An oligonucleotide microarray for influenza A hemagglutinine subtyping was presented. The number of probes for determination of each subtype hemagglutinine (H1-H13, H15, H16, pandemic flu H1N1)varied from 13 to 28. When testing of the microarray using 40 type A influenza virus isolates the hemagglutinin subtypes were unambiguously determined for 36 specimens.     

Chimeric influenza A viruses with a functional influenza B virus neuraminidase or hemagglutinin

Reassortment of influenza A and B viruses has never been observed in vivo or in vitro. Using reverse genetics techniques, we generated recombinant influenza A/WSN/33 (WSN) viruses carrying the neuraminidase (NA) of influenza B virus. Chimeric viruses expressing the full-length influenza B/Yamagata/16/88 virus NA grew to titers similar to that of wild-type influenza WSN virus. Recombinant viruses in which the cytoplasmic tail or the cytoplasmic tail and the transmembrane domain of the type B NA were replaced with those of the type A NA were impaired in tissue culture. This finding correlates with reduced NA content in virions. We also generated a recombinant influenza A virus expressing a chimeric hemagglutinin (HA) protein in which the ectodomain is derived from type B/Yamagata/16/88 virus HA, whereas both the cytoplasmic and the transmembrane domains are derived from type A/WSN virus HA. This A/B chimeric HA virus did not grow efficiently in MDCK cells. However, after serial passage we obtained a virus population that grew to titers as high as wild-type influenza A virus in MDCK cells. One amino acid change in position 545 (H545Y) was found to be responsible for the enhanced growth characteristics of the passaged virus. Taken together, we show here that the absence of reassortment between influenza viruses belonging to different A and B types is not due to spike glycoprotein incompatibility at the level of the full-length NA or of the HA ectodomain.     

Binding kinetics of sulfatide with influenza A virus hemagglutinin

Association of a sulfated galactosyl ceramide, sulfatide, with the viral envelope glycoprotein hemagglutinin (HA) delivered to the cell surface is required for influenza A virus (IAV) replication through efficient translocation of the newly synthesized viral nucleoprotein from the nucleus to the cytoplasm. To determine whether the ectodomain of HA can bind to sulfatide, a secreted-type HA (sHA), in which the transmembrane region and cytoplasmic tail were deleted, was generated by using a baculovirus expression system. The receptor binding ability and antigenic structure of sHA were evaluated by a hemagglutination assay, solid-phase binding assay and hemagglutination inhibition assay. sHA showed subtype-specific antigenicity and binding ability to both sulfatide and gangliosides. Kinetics of sHA binding to sulfatide and GD1a was demonstrated by quartz crystal microbalance (QCM) analysis. QCM analysis showed that the sHA bound with the association rate constant (k _on) of 1.41?×?10⁴ M^?1 sec^?1, dissociation rate constant (k _off) of 2.03?×?10^?4 sec^?1 and K _d of 1.44?×?10^?8 M to sulfatide immobilized on a sensor chip. The k _off values of sHA were similar for sulfatide and GD1a, whereas the k _on value of sHA binding to sulfatide was 2.56-times lower than that of sHA binding to GD1a. The results indicate that sulfatide directly binds to the ectodomain of HA with high affinity.     

A compact microarray for subtyping of influenza a virus

A version of the universal oligonucleotide hybridization microchip with the size of 6 × 5 spots (4 × 4 mm) has been proposed, which operates on the principle of “one spot-one subtype.” This microchip may be the prototype of a biosensor for fixation of influenza A virus and typing of 15 subtypes of hemagglutinin and 9 subtypes of neuraminidase.     

Gnarled-trunk evolutionary model of influenza A virus hemagglutinin

Human influenza A viruses undergo antigenic changes with gradual accumulation of amino acid substitutions on the hemagglutinin (HA) molecule. A strong antigenic mismatch between vaccine and epidemic strains often requires the replacement of influenza vaccines worldwide. To establish a practical model enabling us to predict the future direction of the influenza virus evolution, relative distances of amino acid sequences among past epidemic strains were analyzed by multidimensional scaling (MDS). We found that human influenza viruses have evolved along a gnarled evolutionary pathway with an approximately constant curvature in the MDS-constructed 3D space. The gnarled pathway indicated that evolution on the trunk favored multiple substitutions at the same amino acid positions on HA. The constant curvature was reasonably explained by assuming that the rate of amino acid substitutions varied from one position to another according to a gamma distribution. Furthermore, we utilized the estimated parameters of the gamma distribution to predict the amino acid substitutions on HA in subsequent years. Retrospective prediction tests for 12 years from 1997 to 2009 showed that 70% of actual amino acid substitutions were correctly predicted, and that 45% of predicted amino acid substitutions have been actually observed. Although it remains unsolved how to predict the exact timing of antigenic changes, the present results suggest that our model may have the potential to recognize emerging epidemic strains.     

Influenza C virus hemagglutinin: comparison with influenza A and B virus hemagglutinins

The complete nucleotide sequence of the influenza C/California/78 virus RNA 4 was obtained by using cloned cDNA derived from the RNA segment. This gene is 2,071 nucleotides long and can code for a polypeptide of 654 amino acids. Although there are no convincing sequence homologies between RNA 4 and the hemagglutinin genes of influenza A and B viruses, we suggest, on the basis of structural features, that RNA 4 of the influenza C virus codes for the hemagglutinin. The structural features which are common to the hemagglutinins of influenza A, B, and C viruses include (i) a hydrophobic signal peptide, (ii) an arginine cleavage site between the hemagglutinin 1 and 2 subunits, (iii) hydrophobic regions at the amino and carboxyl termini of the hemagglutinin 2 subunit, and (iv) several conserved cysteine residues. Additional evidence that RNA 4 of influenza C virus codes for the hemagglutinin is that the tripeptide Ile-Phe-Gly, known to be present at the amino terminus of the hemagglutinin 2 subunit of influenza C virus, is encoded by RNA 4 at a point immediately adjacent to the presumptive arginine cleavage site. The lack of primary sequence homology between the influenza C virus hemagglutinin and the influenza A or B virus hemagglutinins, which all have similar functions, might be attributed to convergent rather than divergent evolution. However, the structural similarities among the influenza A, B, and C virus hemagglutinins strongly suggest that the three hemagglutinin genes have diverged from a common precursor.     

Compensatory evolution of net-charge in influenza A virus hemagglutinin

The propagation of influenza A virus depends on the balance between the activities of hemagglutinin (HA) for binding to host cells and neuraminidase (NA) for releasing from infected cells (HA-NA balance). Since the host cell membrane and the sialic acid receptor are negatively charged, the amino acid substitutions increasing (charge+) and decreasing (charge-) the positive charge of HA subunit 1 (HA1) enhance and reduce, respectively, the binding avidity and affinity. The positive charge of HA1 in human influenza A virus bearing subtype H3N2 (A/H3N2 virus) was observed to have increased during evolution, but the evolutionary mechanism for this observation was unclear because this may disrupt the HA-NA balance. Here we show, from the phylogenetic analysis of HA for human A/H3N2 and A/H1N1 viruses, that the relative frequencies of charge+ and charge- substitutions were elevated on the branches where the number of N-glycosylation sites (NGS) increased and decreased, respectively, compared to those where the number of NGS did not change. On the latter branches, the net-charge of HA1 appeared to have been largely maintained to preserve its structure and function. Since the charge+ and charge- substitutions in HA1 have opposite effects to the gain and loss of NGS on the binding and release of the virus, the net-charge of HA1 may have evolved to compensate for the effect of the gain and loss of NGS, probably through changing the avidity. Apparently, the relative frequency of charge- substitutions in HA1 of A/H3N2 virus was elevated after the introduction of oseltamivir, and that of charge+ substitutions in HA1 of A/H1N1 virus was elevated after the spread of oseltamivir resistance. These observations may also be explained by the compensatory effect of the net-charge in HA1 on the NA activity for keeping the HA-NA balance.     

A second generation universal microarray for subtyping influenza a virus

The microchip for influenza A subtyping working on the ??one spot-one subtype?? principle was developed. Each spot contains a set of oligonucleotide probes specific for particular subtypes of hemagglutinin, neuraminidase and matrix protein (influenza A marker). Reliability of the proposed chip is the same as for the full-size microchip for separate hemagglutinin and neuraminidase typing which was created in our group earlier. The image was visualized by labeling the analyzed nucleic acid by either fluorescent dye or biotin with the following fixation in streptavidin-gold nanoparticles and development by silver precipitation. In the second case, the image was analyzed using an ordinary scanner that essentially simplifies influenza A subtyping.     

Protection of mice against a lethal influenza virus challenge after immunization with yeast-derived secreted influenza virus hemagglutinin.

The A/Victoria/3/75 (H3N2-subtype) hemagglutinin (HA) gene was engineered for expression in Pichia pastoris as a soluble secreted molecule. The HA cDNA lacking the C-terminal transmembrane anchor-coding sequence was fused to the Saccharomyces cerevisiae alpha-mating factor secretion signal and placed under control of the methanol-inducible P. pastoris alcohol oxidase 1 (AOX1) promoter. Growth of transformants on methanol-containing medium resulted in the secretion of recombinant non-cleaved soluble hemagglutinin (HA0s). Remarkably, the pH of the induction medium had an important effect on the expression level, the highest level being obtained at pH 8.0. The gel filtration profile and the reactivity against a panel of different HA-conformation specific monoclonal antibodies indicated that HA0s was monomeric. Analysis of the N-linked glycans revealed a typical P. pastoris type of glycosylation, consisting of glycans with 10-12 glycosyl residues. Mice immunized with purified soluble hemagglutinin (HA0s) showed complete protection against a challenge with 10 LD50 of mouse-adapted homologous virus (X47), whereas all control mice succumbed. Heterologous challenge with X31 virus [A/Aichi/2/68 (H3N2-subtype)], resulted in significantly higher survival rates in the immunized group compared with the control group. These results, together with the safety, reliability and economic potential of P. pastoris, as well as the flexibility and fast adaptation of the expression system may allow development of an effective recombinant influenza vaccine.     

Attenuated influenza virus construct with enhanced hemagglutinin protein expression

Influenza A viruses encoding an altered viral NS1 protein have emerged as promising live attenuated vaccine platforms. A carboxy-terminal truncation in the NS1 protein compromises its interferon antagonism activity, making these viruses attenuated in the host yet still able to induce protection from challenge with wild-type viruses. However, specific viral protein expression by NS1-truncated viruses is known to be decreased in infected cells. In this report, we show that recombinant H5N1 and H1N1 influenza viruses encoding a truncated NS1 protein expressed lower levels of hemagglutinin (HA) protein in infected cells than did wild-type viruses. This reduction in HA protein expression correlated with a reduction in HA mRNA levels in infected cells. NS1 truncation affected the expression of HA protein but not that of the nucleoprotein (NP). This segment specificity was mapped to the terminal sequences of their specific viral RNAs. Since the HA protein is the major immunogenic component in influenza virus vaccines, we sought to restore its expression levels in NS1-truncated viruses in order to improve their vaccine efficacy. For this purpose, we generated an NS1-truncated recombinant influenza A/Puerto Rico/8/34 (rPR8) virus carrying the G3A C8U "superpromoter" mutations in the HA genomic RNA segment. This strategy retained the attenuation properties of the recombinant virus but enhanced the expression level of HA protein in infected cells. Finally, mice immunized with rPR8 viruses encoding a truncated NS1 protein and carrying the G3A C8U mutations in the HA segment demonstrated enhanced protection from wild-type virus challenge over that for mice vaccinated with an rPR8 virus encoding the truncated NS1 protein alone.     

Immunogenic structure of the influenza virus hemagglutinin

We chemically synthesized 20 peptides corresponding to 75% of the HA1 molecule of the influenza virus. Antibodies to the majority (18) of these peptides were capable of reacting with the hemagglutinin molecule. These 18 peptides are not confined to the known antigenic determinants of the hemagglutinin molecule, but rather are scattered throughout its three-dimensional structure. In contrast, antibody raised to intact hemagglutinin did not react with any of the 20 peptides. Taken together these results suggest that the immunogenicity of an intact protein molecule is not the sum of the immunogenicity of its pieces.     

Isolation of influenza virus A hemagglutinin C-terminal domain by hemagglutinin proteolysis in octylglucoside micelles

A method of isolation of hydrophobic membrane-bound C-terminal domain of influenza virus A hemagglutinin (HA) is suggested. The method is based on the insertion of HA into octylglucoside micelles followed by pepsin or thermolysin hydrolysis. Subsequent treatment of proteolytic digests with chloroform-hexafluoroisopropanol mixture resulted in the extraction of a few hydrophobic peptides into organic phase. Mass-spectrometry (MALDI-TOF) analysis revealed that the peptides with ion masses corresponding to the anchoring C-terminal domain with or without modifications predominated in the organic solution. The data obtained confirmed our speculation on the possibility of the suggested isolation scheme following from the strong interactions of anchoring domains in compact trimeric structure of HA spikes combined with micelle protection effect. Several appropriate peptides presence in the organic phase apparently arises from the presence of a few accessible proteolytic sites in HA transmembrane region.     

Structural features influencing hemagglutinin cleavability in a human influenza A virus.

The cleavability of the hemagglutinin (HA) molecule is related to the virulence of avian influenza A viruses, but its influence on human influenza virus strains is unknown. Two structural features are involved in the cleavage of avian influenza A virus HAs: a series of basic amino acids at the cleavage site and an oligosaccharide side chain in the near vicinity. The importance of these properties in the cleavability of a human influenza A virus (A/Aichi/2/68) HA was investigated by using mutants that contained or lacked an oligosaccharide side chain and had either four or six basic amino acids. All mutants except the one that contains a single mutation at the glycosylation site were cleaved, although not completely, demonstrating that a series of basic amino acids confers susceptibility to cellular cleavage enzymes among human influenza virus HAs. The mutants containing six basic amino acids at the cleavage site showed limited polykaryon formation upon exposure to low pH, indicating that cleavage was adequate to impart fusion activity to the HA. Deletion of the potential glycosylation site had no effect on the cleavability of these mutants; hence, the oligosaccharide side chain appears to have no role in human influenza virus HA cleavage. The inability to induce high cleavability in a human influenza A virus HA by insertion of a series of basic amino acids at the cleavage site indicates that other, as yet unidentified structural features are needed to enhance the susceptibility of these HAs to cellular proteases.     

Apical budding of a recombinant influenza A virus expressing a hemagglutinin protein with a basolateral localization signal

Influenza virions bud preferentially from the apical plasma membrane of infected epithelial cells, by enveloping viral nucleocapsids located in the cytosol with its viral integral membrane proteins, i.e., hemagglutinin (HA), neuraminidase (NA), and M2 proteins, located at the plasma membrane. Because individually expressed HA, NA, and M2 proteins are targeted to the apical surface of the cell, guided by apical sorting signals in their transmembrane or cytoplasmic domains, it has been proposed that the polarized budding of influenza virions depends on the interaction of nucleocapsids and matrix proteins with the cytoplasmic domains of HA, NA, and/or M2 proteins. Since HA is the major protein component of the viral envelope, its polarized surface delivery may be a major force that drives polarized viral budding. We investigated this hypothesis by infecting MDCK cells with a transfectant influenza virus carrying a mutant form of HA (C560Y) with a basolateral sorting signal in its cytoplasmic domain. C560Y HA was expressed nonpolarly on the surface of infected MDCK cells. Interestingly, viral budding remained apical in C560Y virus-infected cells, and so did the location of NP and M1 proteins at late times of infection. These results are consistent with a model in which apical viral budding is a shared function of various viral components rather than a role of the major viral envelope glycoprotein HA.     

流感病毒血凝素蛋白裂解机制的研究进展

血凝素(Hemagglutinin,HA)是流感病毒的主要表面抗原之一,诱导机体产生中和抗体,介导病毒囊膜与靶细胞膜融合,从而启动病毒对宿主细胞的感染过程。HA蛋白以前体形式合成,需经宿主蛋白酶水解为HA1、HA2两个亚单位,并以二硫键连接,病毒才获得感染性。研究表明宿主蛋白酶的分布与流感病毒感染后的致病力和组织嗜性有直接关系。潜在的裂解酶及其抑制因子的发现为流感的防治提供了新的思路,成为干预治疗的新潜在靶点。就当前国内外关于流感病毒血凝素的结构与功能、裂解机制及其应用的研究进展进行综述。     

Oligonucleotide microarray for subtyping of influenza virus A neuraminidase

Microarray for influenza A neuraminidase subtyping was presented. Selection of oligoprobes proceeded in two steps. First step included selection of peptides specific for each subtype of neuraminidase. At the second step oligoprobes were calculated using found peptides structures with the subsequent additional selection of the most specific and representative probes. From 19 to 24 probes were used for determination of each subtype of neuraminidase. Microchip testing for 19 samples with the most widespread types (N1 and N2) specifies in unequivocal definition 18 of them and only one isolate has not been identified.     

Integrity of a recombinant hemagglutinin protein of an avian influenza virus

An open reading frame representing cDNA from a hemagglutinin (HA) encoding gene of a low pathogenic avian influenza virus (AIV) subtype H10N7 was cloned in the pNMT1-TOPO vector under the control of thiamine response promoter. This construct was designated as pNMT1-HA. The pNMT1-HA construct was transformed into Schizosaccharomyces pombe for expression of HA antigen. The correct expression of recombinant HA protein was confirmed by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot. The level of expression of recombinant HA protein was approximately 0.2% of total soluble protein. Purified yeast-derived recombinant HA protein showed hemagglutination activity. The 2-D and 3-D scanning images of recombinant HA protein were observed with an atomic force microscope (AFM). The structural integrity of the HA protein under AFM and hemagglutination activity provided support that the recombinant HA protein may be suitable for development of AIV subunit vaccine for mass administration to poultry.     

Capturing a fusion intermediate of influenza hemagglutinin with a cholesterol-conjugated peptide, a new antiviral strategy for influenza virus

We previously described fusion-inhibitory peptides that are targeted to the cell membrane by cholesterol conjugation and potently inhibit enveloped viruses that fuse at the cell surface, including HIV, parainfluenza, and henipaviruses. However, for viruses that fuse inside of intracellular compartments, fusion-inhibitory peptides have exhibited very low antiviral activity. We propose that for these viruses, too, membrane targeting via cholesterol conjugation may yield potent compounds. Here we compare the activity of fusion-inhibitory peptides derived from the influenza hemagglutinin (HA) and show that although the unconjugated peptides are inactive, the cholesterol-conjugated compounds are effective inhibitors of infectivity and membrane fusion. We hypothesize that the cholesterol moiety, by localizing the peptides to the target cell membrane, allows the peptides to follow the virus to the intracellular site of fusion. The cholesterol-conjugated peptides trap HA in a transient intermediate state after fusion is triggered but before completion of the refolding steps that drive the merging of the viral and cellular membranes. These results provide proof of concept for an antiviral strategy that is applicable to intracellularly fusing viruses, including known and emerging viral pathogens.