A Single Amino Acid in the Stalk Region of the H1N1pdm Influenza Virus HA Protein Affects Viral Fusion,Stability and Infectivity

The 2009 H1N1 pandemic (H1N1pdm) viruses have evolved to contain an E47K substitution in the HA2 subunit of the stalk region of the hemagglutinin (HA) protein. The biological significance of this single amino acid change was investigated by comparing A/California/7/2009 (HA2-E47) with a later strain, A/Brisbane/10/2010 (HA2-K47). The E47K change was found to reduce the threshold pH for membrane fusion from 5.4 to 5.0. An inter-monomer salt bridge between K47 in HA2 and E21 in HA1, a neighboring highly conserved residue, which stabilized the trimer structure, was found to be responsible for the reduced threshold pH for fusion. The higher structural and acid stability of the HA trimer caused by the E47K change also conferred higher viral thermal stability and infectivity in ferrets, suggesting a fitness advantage for the E47K evolutionary change in humans. Our study indicated that the pH of HA fusion activation is an important factor for influenza virus replication and host adaptation. The identification of this genetic signature in the HA stalk region that influences vaccine virus thermal stability also has significant implications for influenza vaccine production.     

Antigenic structure of the influenza B virus hemagglutinin: nucleotide sequence analysis of antigenic variants selected with monoclonal antibodies.

We report here the complete nucleotide sequence of the hemagglutinin (HA) gene of influenza B virus B/Oregon/5/80 and, through comparative sequence analysis, identify amino acid substitutions in the HA1 polypeptide responsible for the antigenic alterations in laboratory-selected antigenic variants of this virus. The complete nucleotide sequence of the B/Oregon/5/80 HA gene was established by a combination of chemical sequencing of a full-length cDNA clone and dideoxy sequencing of the virion RNA. The nucleotide sequence is very similar to previously reported influenza B virus HA gene sequences and differs at only nine nucleotide positions from the B/Singapore/222/79 HA gene (Verhoeyen et al., Nucleic Acids Res. 11:4703-4712, 1983). The nucleotide sequences of the HA1 portions of the HA genes of 18 laboratory-selected antigenic variants were determined by the dideoxy method. Comparison of the deduced amino acid sequences of the parental and variant HA1 polypeptides revealed 16 different amino acid substitutions at nine positions. All amino acid substitutions resulted from single-point mutations, and no double mutants were detected, demonstrating that as in the influenza A viruses, single amino acid substitutions are sufficient to alter the antigenicity of the HA molecule. Many of the amino acid substitutions in the variants occurred at positions also observed to change in natural drift strains. The substitutions appear to identify at least two immunodominant regions which correspond to proposed antigenic sites A and B on the influenza A virus H3 HA.     

Identification of amino acids of influenza virus HA responsible for resistance to a fusion inhibitor, Stachyflin

We have recently described a novel hemagglutinin (HA) conformational change inhibitor of human influenza virus, Stachyflin (Yoshimoto et al, Arch. Virol., 144, 1-14, 1999). Stachyflin-resistant variants of human influenza A/WSN/33 (H1N1) virus were isolated in vitro and the nucleotide sequences of their HA genes were determined. The relation of amino acid substitutions and Stachyflin resistance was analyzed with in vitro membrane fusion between HA-expressing cells and octadecylrhodamine (R18)-labelled chick erythrocytes (RBC). The amino acid substitutions, lysine to arginine at position 51 or lysine to glutamic acid at position 121 of the HA2 subunit of the HA protein was enough to confer a Stachyflin-resistant phenotype of HA protein. The molecular mechanism of anti-HA conformational change activity of Stachyflin is discussed.     

Acid-induced changes in thermal stability and fusion activity of influenza hemagglutinin

The conformational and thermal stability of full-length hemagglutinin (HA) of influenza virus (strain X31) has been investigated using a combination of differential scanning calorimetry (DSC), analytical ultracentrifugation, fluorescence, and circular dichroism (CD) spectroscopy as a function of pH. HA sediments as a rosette comprised of 5-6 trimers (31-35 S) over the pH range of 7.4-5.4. The DSC profile of HA in the native state at pH 7.4 is characterized by a single cooperative endotherm with a transition temperature (Tm) of 66 degrees C and unfolding enthalpy (DeltaH(cal)) of 800 kcal x (mol of trimer)(-1). Upon acidification to pH 5.4, there is a significant decrease in the transition temperature (from 66 to 45 degrees C), unfolding enthalpy [from 800 to 260 kcal x (mol of trimer)(-1)], and DeltaH(cal)/DeltaH(vH) ratio (from 3.0 to approximately 1.3). Whereas the far- and near-UV ellipticities are maintained over this pH range, there is an acid-induced increase in surface hydrophobicity and decrease in intrinsic tryptophanyl fluorescence. The major contribution to the DSC endotherm arises from unfolding HA1 domains. The relationship between acid-induced changes in thermal stability and the fusion activity of HA has been examined by evaluating the kinetics and extent of fusion of influenza virus with erythrocytes over the temperature and pH range of the DSC measurements. Surprisingly, X31 influenza virus retains its fusion activity at acidic pH and temperatures significantly below the unfolding transition of HA. This finding is consistent with the notion that the fusion activity of influenza virus may involve structural changes of only a small fraction of HA molecules.     

Quaternary structure of influenza virus hemagglutinin after acid treatment.

Hemagglutinin (HA), a trimeric spike glycoprotein of influenza virus, mediates fusion between the viral envelope and the membrane of an endosome during virus entry. Fusion is triggered by low pH, which induces an irreversible conformational change in the protein. Several studies have indicated that intersubunit contacts along the trimer interfaces may be broken during this alteration. To determine whether HA dissociates into individual subunits as a consequence of the conformational change, we used velocity gradient sedimentation in the presence of Triton X-100. We also determined the resistance of acid-treated HA to dissociation by sodium dodecyl sulfate, a property of the HA trimer. At pH 7.0, isolated HA sedimented as a 9S trimer and gave the characteristic trimer pattern after sodium dodecyl sulfate-polyacrylamide gel electrophoresis. After acidification the HA remained trimeric irrespective of whether it was exposed to acid in intact virus particles or in solubilized form. Only when very low concentrations of HA were acidified did a fraction dissociate to dimers and monomers. In contrast, the water-soluble ectodomain fragment of HA (BHA) readily dissociated under a variety of conditions. Negative-stain electron microscopy supported the notion that HA molecules in virus particles do not dissociate upon acidification and may form larger oligomeric structures in the plane of the viral membrane. Taken together, the results suggested that it is the trimeric HA, or higher-order structures thereof, that are active in the acid-induced fusion reaction. Further, the results emphasized the role of the transmembrane anchors of HA in preventing dissociation of the trimer.     

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.     

Studies of the membrane fusion activities of fusion peptide mutants of influenza virus hemagglutinin.

Influenza virus hemagglutinin (HA) fuses membranes at endosomal pH by a process which involves extrusion of the NH2-terminal region of HA2, the fusion peptide, from its buried location in the native trimer. We have examined the amino acid sequence requirements for a functional fusion peptide by determining the fusion capacities of site-specific mutant HAs expressed by using vaccinia virus recombinants and of synthetic peptide analogs of the mutant fusion peptides. The results indicate that for efficient fusion, alanine can to some extent substitute for the NH2-terminal glycine of the wild-type fusion peptide but that serine, histidine, leucine, isoleucine, or phenylalanine cannot. In addition, mutants containing shorter fusion peptides as a result of single amino acid deletions are inactive, as is a mutant containing an alanine instead of a glycine at HA2 residue 8. Substitution of the glycine at HA2 residue 4 with an alanine increases the pH of fusion, and valine-for-glutamate substitutions at HA2 residues 11 and 15 are without effect. We confirm previous reports on the need for specific HAo cleavage to generate functional HAs, and we show that both inappropriately cleaved HA and mutant HAs, irrespective of their fusion capacities, upon incubation at low pH undergo the structural transition required for fusion.     

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.     

Structural characterization of an early fusion intermediate of influenza virus hemagglutinin

The hemagglutinin (HA) envelope protein of influenza virus mediates viral entry through membrane fusion in the acidic environment of the endosome. Crystal structures of HA in pre- and postfusion states have laid the foundation for proposals for a general fusion mechanism for viral envelope proteins. The large-scale conformational rearrangement of HA at low pH is triggered by a loop-to-helix transition of an interhelical loop (B loop) within the fusion domain and is often referred to as the "spring-loaded" mechanism. Although the receptor-binding HA1 subunit is believed to act as a "clamp" to keep the B loop in its metastable prefusion state at neutral pH, the "pH sensors" that are responsible for the clamp release and the ensuing structural transitions have remained elusive. Here we identify a mutation in the HA2 fusion domain from the influenza virus H2 subtype that stabilizes the HA trimer in a prefusion-like state at and below fusogenic pH. Crystal structures of this putative early intermediate state reveal reorganization of ionic interactions at the HA1-HA2 interface at acidic pH and deformation of the HA1 membrane-distal domain. Along with neutralization of glutamate residues on the B loop, these changes cause a rotation of the B loop and solvent exposure of conserved phenylalanines, which are key residues at the trimer interface of the postfusion structure. Thus, our study reveals the possible initial structural event that leads to release of the B loop from its prefusion conformation, which is aided by unexpected structural changes within the membrane-distal HA1 domain at low pH.     

Mutations in Hemagglutinin and Polymerase Alter the Virulence of Pandemic A(H1N1) Influenza Virus

To study the pathogenicity factors of the pandemic A(H1N1) influenza virus, a number of mutant variants of the A/Hamburg/5/2009 (H1N1)pdm09 strain were obtained through passage in chicken embryos, mouse lungs, and MDCK cell culture. After 17 lung-to-lung passages of the A/Hamburg/5/2009 in mice, the minimum lethal dose of the derived variant decreased by five orders of magnitude compared to that of the parental virus. This variant differed from the original virus by nine amino acid residues in the following viral proteins: hemagglutinin (HA), neuraminidase (NA), and components of the polymerase complex. Additional passaging of the intermediate variants and cloning made it possible to obtain pairs of strains that differed by a single amino acid substitution. Comparative analysis of replicative activity, receptor specificity, and virulence of these variants revealed two mechanisms responsible for increased pathogenicity of the virus for mice. Thus, (1) substitutions in HA (Asp225Gly or Gln226Arg) and compensatory mutation decreasing the charge of HA (Lys123Asn, Lys157Asn, Gly158Glu, Asn159Asp, or Lys212Met) altered viral receptor-binding specificity and restored the functional balance between HA and NA; (2) Phe35Leu substitution in the PA protein increased viral polymerase activity.     

Role of hemagglutinin surface density in the initial stages of influenza virus fusion: lack of evidence for cooperativity

Membrane fusion mediated by influenza virus hemagglutinin (HA) is believed to proceed via the cooperative action of multiple HA trimers. To determine the minimal number of HA trimers required to trigger fusion, and to assess the importance of cooperativity between these HA trimers, we have generated virosomes containing coreconstituted HAs derived from two strains of virus with different pH dependencies for fusion, X-47 (optimal fusion at pH 5.1; threshold at pH 5.6) and A/Shangdong (optimal fusion at pH 5.6; threshold at pH 6.0), and measured fusion of these virosomes with erythrocyte ghosts by a fluorescence lipid mixing assay. Virosomes with different X-47-to-A/Shangdong HA ratios, at a constant HA-to-lipid ratio, showed comparable ghost-binding activities, and the low-pH-induced conformational change of A/Shangdong HA did not affect the fusion activity of X-47 HA. The initial rate of fusion of these virosomes at pH 5.7 increased directly proportional to the surface density of A/Shangdong HA, and a single A/Shangdong trimer per virosome appeared to suffice to induce fusion. The reciprocal of the lag time before the onset of fusion was directly proportional to the surface density of fusion-competent HA. These results support the notion that there is no cooperativity between HA trimers during influenza virus fusion.     

Studies on the adaptation of influenza viruses to MDCK cells

The amino acid sequences and biological properties of the haemagglutinin of three variants of the influenza virus X-31 (H3N2) selected for their capacity to grow in MDCK cells are reported. In two variants, amino acid substitutions at HA1 residues 8 and 144 correlated with the loss of a site for glycosylation and specific changes in antigenicity, respectively. In all three variants substitution of an arginine residue for histidine at HA1 position 17 was correlated with increased pH optima of haemolysis. The importance of this substitution for cleavage of the haemagglutinin precursor required to produce infectious virus is discussed in relation to the three-dimensional structure of X-31 haemagglutinin.     

Morphological changes and fusogenic activity of influenza virus hemagglutinin.

The kinetics of low-pH induced fusion of influenza virus with liposomes have been compared to changes in the morphology of influenza hemagglutinin (HA). At pH 4.9 and 30 degrees C, the fusion of influenza A/PR/8/34 virus with ganglioside-bearing liposomes was complete within 6 min. Virus preincubated at pH 4.9 and 30 degrees C in the absence of liposomes for 2 or 10 min retained most of its fusion activity. However, fusion activity was dramatically reduced after 30 min, and virtually abolished after a 60-min preincubation. Cryo-electron microscopy showed that the hemagglutinin spikes of virions exposed to pH 4.9 at 30 degrees C for 10 min underwent no major morphological changes. After 30 min, however, the spike morphology changed dramatically, and further changes occurred for up to 60 min after exposure to low pH. Because the morphological changes occur at a rate corresponding to the loss of fusion activity, and because these changes are much slower than the rate at which fusion occurs, we conclude that the morphologically altered HA is inactive with respect to fusion-promoting activity. Molecular modeling studies indicate that the formation of an extended coiled coil within the HA trimer, as proposed for HA at low pH, requires a major conformational change in HA, and that the morphological changes we observe are consistent with the formation of an extended coiled coil. These results imply that the crystallographically determined low-pH form of HA does occur in the intact virus, but that this form is not a precursor of viral fusion. It is speculated that the motion to the low-pH form may be responsible for the membrane destabilization leading to fusion.     

The structure of a membrane fusion mutant of the influenza virus haemagglutinin.

The haemagglutinin glycoprotein (HA) of influenza virus specifically mediates fusion of the viral and host cell endosomal membranes at the acidic pH of endosomes. The HAs from mutant viruses with raised fusion pH optima contain amino acid substitutions in regions of the HA structure thought to be involved in the fusion process [Daniels et al. (1985b) Cell, 40, 431-439]. We have determined the neutral pH crystal structure of one such mutant, HA2 112 Asp----Gly. A water molecule appears to partially replace the aspartate side chain, and no changes are observed in the surrounding structure. It appears that four intra-chain hydrogen bonds that stabilize the location of the N-terminus of HA2 are lost in the mutant, resulting in a local destabilization that facilitates the extrusion of the N-terminus at higher pH.     

Intermediates in influenza induced membrane fusion.

Our results show that the mechanism by which influenza virus fuses with target membranes involves sequential complex changes in the hemagglutinin (HA, the viral fusion protein) and in the contact site between virus and target membrane. To render individual steps amenable to study, we worked at 0 degree C which decreased the rate of fusion and increased the efficiency. The mechanism of fusion at 0 degree C and 37 degrees C was similar. The process began with a conformational change in HA which exposed the fusion peptides but did not lead to dissociation of the tops of the ectodomain of the trimer. The change in the protein led to immediate hydrophobic attachment of the virus to the target liposomes. Attachment was followed by a lag period (4-8 min at 0 degree C, 0.6-2 s at 37 degrees C) during which rearrangements occurred in the site of membrane contact between the virus and liposome. After a further series of changes the final bilayer merger took place. This final fusion event was not pH dependent. At 0 degree C efficient fusion occurred without dissociation of the top domains of the HA trimer, suggesting that a transient conformation of HA is responsible for fusion at physiological temperatures. The observations lead to a revised model for HA mediated fusion.     

A common neutralizing epitope conserved between the hemagglutinins of influenza A virus H1 and H2 strains.

When mice were immunized with the A/Okuda/57 (H2N2) strain of influenza virus, a unique monoclonal antibody designated C179 was obtained. Although C179 was confirmed to recognize the hemagglutinin (HA) glycoprotein by immunoprecipitation assays, it did not show hemagglutination inhibition activity to any of the strains of the three subtypes of influenza A virus. However, it neutralized all of the H1 and H2 strains but not the H3 strains. Moreover, it inhibited polykaryon formation induced by the H1 and H2 strains but not by the H3 strains. Two antigenic variants against C179 were obtained, and nucleotide sequence analysis revealed that amino acid sequences, from 318 to 322 of HA1 and from 47 to 58 of HA2, conserved among H1 and H2 strains were responsible for the recognition of C179. Since the two sites were located close to each other at the middle of the stem region of the HA molecule, C179 seemed to recognize these sites conformationally. These data indicated that binding of C179 to the stem region of HA inhibits the fusion activity of HA and thus results in virus neutralization and inhibition of cell-cell fusion. This is the first report which describes the presence of conserved antigenic sites on HA not only in a specific subtype but also in two subtypes of influenza A virus.     

Crystal structure of unliganded influenza B virus hemagglutinin

Here we report the crystal structure of hemagglutinin (HA) from influenza B/Hong Kong/8/73 (B/HK) virus determined to 2.8 Å. At a sequence identity of ~25% to influenza A virus HAs, B/HK HA shares a similar overall structure and domain organization. More than two dozen amino acid substitutions on influenza B virus HAs have been identified to cause antigenicity alteration in site-specific mutants, monoclonal antibody escape mutants, or field isolates. Mapping these substitutions on the structure of B/HK HA reveals four major epitopes, the 120 loop, the 150 loop, the 160 loop, and the 190 helix, that are located close in space to form a large, continuous antigenic site. Moreover, a systematic comparison of known HA structures across the entire influenza virus family reveals evolutionarily conserved ionizable residues at all regions along the chain and subunit interfaces. These ionizable residues are likely the structural basis for the pH dependence and sensitivity to ionic strength of influenza HA and hemagglutinin-esterase fusion proteins.     

Specific Single or Double Proline Substitutions in the “Spring-loaded” Coiled-Coil Region of the Influenza Hemagglutinin Impair or Abolish Membrane Fusion Activity

We tested the role of the “spring-loaded” conformational change in the fusion mechanism of the influenza hemagglutinin (HA) by assessing the effects of 10 point mutants in the region of high coiled-coil propensity, HA2 54–81. The mutants included proline substitutions at HA2 55, 71, and 80, as well as a double proline substitution at residues 55 and 71. Mutants were expressed in COS or 293T cells and assayed for cell surface expression and structural features as well as for their ability to change conformation and induce fusion at low pH. We found the following: Specific mutations affected the precise carbohydrate structure and folding of the HA trimer. All of the mutants, however, formed trimers that could be expressed at the cell surface in a form that could be proteolytically cleaved from the precursor, HA0, to the fusion-permissive form, HA1-S-S-HA2. All mutants reacted with an antibody against the major antigenic site and bound red blood cells. Seven out of ten mutants displayed a wild-type (wt) or moderately elevated pH dependence for the conformational change. V55P displayed a substantial reduction (~60– 80%) in the initial rate of lipid mixing. The other single mutants displayed efficient fusion with the same pH dependence as wt-HA. The double proline mutant V55P/ S71P displayed no fusion activity despite being well expressed at the cell surface as a proteolytically cleaved trimer that could bind red blood cells and change conformation at low pH. The impairment in fusion for both V55P and V55P/S71P was at the level of outer leaflet lipid mixing. We interpret our results in support of the hypothesis that the spring-loaded conformational change is required for fusion. An alternate model is discussed.     

Changes in the phenotypic properties of highly pathogenic influenza A virus of H5N1 subtype induced by N186I and N186T point mutations in hemagglutinin

The change in the phenotypic properties resulting from amino acid substitutions in the hemagglutinin (HA) molecule is an important link in the evolutionary process of influenza viruses. It is believed to be one of the mechanisms of the emergence of highly pathogenic strains of influenza A viruses, including subtype H5N1. Using the site-directed mutagenesis, we introduced mutations in the HA gene of the H5N1 subtype of influenza A virus. The obtained virus variants were analyzed and compared using the following parameters: optimal pH of conformational transition (according to the results of the hemolysis test), specificity of receptor binding (using a set of synthetic analogues of cell surface sialooligosaccharides), thermoresistance (heat-dependent reduction of hemagglutinin activity), virulence in mice, and the kinetics of replication in chicken embryos, and reproductive activity at different temperatures (RCT-based). N186I and N186T mutations in the HA protein increased the virulence of the original virus in mice. These mutations accelerated virus replication in the early stages of infection in chicken embryos and increased the level of replication at late stages. In addition, compared to the original virus, the mutant variants replicated more efficiently at lower temperatures. The obtained data clearly prove the effect of amino acid substitutions at the 186 position of HA on phenotypic properties of the H5N1 subtype of influenza A.