Inhibition of fusion activity of influenza A haemagglutinin mediated by HA2-specific monoclonal antibodies.

The effect of seven monoclonal antibodies (MAbs) specific to the light chain (HA2) of influenza A haemagglutinin (HA) on its fusion activity was investigated. These MAbs, which are non-virus neutralizing, defined four distinct antigenic sites on HA2 glycopolypeptide and the corresponding epitopes were attributed to the sequence stretches on HA2. The accessibility of all seven HA2 epitopes significantly increased after trypsin cleavage and pH 5 treatment of the HA (X-31). The influence of anti-HA2 MAbs on the fusion process was followed by cell-cell fusion of CHO cells expressing precursor HA, virus-liposome fusion assay, and haemolysis mediated by virus. MAb CF2, which bound directly to the fusion peptide 1-35 of HA2, was positive in all three fusion-inhibition assays and was the only one inhibiting the polykaryon formation of CHO-X-31 cells. Two other MAbs belonging to the same antigenic site but not binding directly to the fusion peptide inhibited virus to liposome fusion (EB12) or inhibited haemolysis (BB8). Moreover, MAb IIF4 binding to distinct antigenic site within 125-175 HA2 inhibited haemolysis, too. Thus, fusion activity of HA may be inhibited by anti-HA2 MAbs, mainly those binding to or near the fusion peptide. These antibodies represent useful probes for studies of influenza virus to cell membrane fusion.

A monoclonal antibody specific to the HA2 glycoprotein of influenza A virus hemagglutinin that inhibits its fusion activity reduces replication of the virus.

Monoclonal antibody (MAb) CF2, which binds to the fusion peptide of influenza A virus hemagglutinin (HA) (amino acids (aa) 1-35 of the N-terminus of the light chain of HA), inhibited the fusion activity of HA. This MAb preferentially bound to pH 5-treated virus (with conformationally altered HA) and bound only weakly to the native wild type (wt) virus. However, a significant binding of MAb CF2 to the amantadine resistant virus mutant Ab4 (with a mutation at aa 17 of HA1 leading to a destabilization of HA trimer) was obtained without pH 5 treatment. Exploiting the fusion-inhibition activity of MAb CF2 the effect of this antibody on the virus replication in vitro was followed using both the wt virus and the amantadine resistant mutant Ab4. No reduction of replication of wt virus and a low reduction of replication of Ab4 mutant (by about 20%) was detected by radioimmunoassay after preincubation of the virus with a high concentration of MAb CF2 at room temperature. An increased reduction of replication of Ab4 mutant (by about 40%) was observed in cell radioimmunoassay (RIA) and in plaque assay when the virus was preincubated with MAb at 37 degrees C. Under these conditions a reduction of the wt virus replication also occurred by about 40%. This is the first report on the capacity of a MAb specific to HA2 gp, the light chain of influenza A virus HA, to reduce replication of the virus. This capacity in relation to (i) the affinity of the antibody to the virus, and (ii) the accessibility of corresponding epitopes on the virus surface as well as the proposed mechanism of inhibition of replication of the virus are discussed.

An antibody that prevents the hemagglutinin low pH fusogenic transition.

We have determined the structure of a complex of influenza hemagglutinin (HA) with an antibody that binds simultaneously to the membrane-distal domains of two HA monomers, effectively cross-linking them. The antibody prevents the low pH structural transition of HA that is required for its membrane fusion activity, providing evidence that a rearrangement of HA membrane-distal domains is an essential component of the transition.

Studies on influenza haemagglutinin fusion peptide mutants generated by reverse genetics.

Influenza haemagglutinin (HA) is responsible for fusing viral and endosomal membranes during virus entry. In this process, conformational changes in the HA relocate the HA(2) N-terminal 'fusion peptide' to interact with the target membrane. The highly conserved HA fusion peptide shares composition and sequence features with functionally analogous regions of other viral fusion proteins, including the presence and distribution of glycines and large side-chain hydrophobic residues. HAs with mutations in the fusion peptide were expressed using vaccinia virus recombinants to examine the requirement for fusion of specific hydrophobic residues and the significance of glycine spacing. Mutant HAs were also incorporated into infectious influenza viruses for analysis of their effects on infectivity and replication. In most cases alanine, but not glycine substitutions for the large hydrophobic residues, yielded fusion-competent HAs and infectious viruses, suggesting that the conserved spacing of glycines may be structurally significant. When viruses containing alanine substitutions for large hydrophobic residues were passaged, pseudoreversion to valine was observed, indicating a preference for large hydrophobic residues at specific positions. Viruses were also obtained with serine, leucine or phenylalanine as the N-terminal residue, but these replicated to significantly lower levels than wild-type virus with glycine at this position.

The strong positive correlation between effective affinity and infectivity neutralization of highly cross-reactive monoclonal antibody IIB4, which recognizes antigenic site B on influenza A virus haemagglutinin.

Monoclonal antibody (MAb) IIB4 displays a rare combination of virus neutralization (VN) activity and broad cross-reactivity with influenza A virus strains of the H3 subtype isolated in a period from 1973 to 1988. The epitope of this antibody has been identified as around HA1 residues 198, 199 and 201. Here we report that residues 155, 159, 188, 189 and 193 also influence the binding of this antibody. We have used this antibody to study the relationship between antibody affinity and VN activity. Using one MAb and a single epitope on the haemagglutinin (HA) of different influenza viruses we found a strong positive correlation between effective affinity and VN activity of MAb IIB4. A 10-fold increase in effective affinity corresponded to the 2000-fold increase in VN titre. It follows from the law of mass action that for an effective affinity K=9x10(8) l/mol, 50% VN was achieved at approx. 10% occupation of HA spikes with antibody. In contrast, for an effective affinity K=6x10(7) l/mol, to achieve 50% VN, occupation of up to 98% of HA spikes was required. An effective affinity about K=6x10(7) l/mol thus represents the limiting value for VN because a further decrease in the affinity cannot be compensated by a higher concentration of antibody.

Temperature dependence of fusion by sendai virus.

Studies of the temperature dependence of liposome fusion by Sendai virus indicate that fusion occurs maximally at 55 degrees C. The fusion capacity of the virus is also inactivated maximally by preincubation at this temperature and, under the same conditions, the F glycoprotein becomes resistant to proteolysis. By analogy with the activation at elevated temperatures of fusion by influenza virus our results suggest that temperature is also a variable in the activation of fusion by paramyxoviruses and possibly in the activation of other members of the group of viruses that includes myxo-, paramyxo-, retro-, and filoviruses, which all contain cleaved, trimeric fusion glycoproteins.

Electron microscopy of the human respiratory syncytial virus fusion protein and complexes that it forms with monoclonal antibodies.

Full-length fusion (F) glycoprotein of human respiratory syncytial virus (HRSV) and a truncated anchorless mutant lacking the C-terminal 50 amino acids were expressed from vaccinia recombinants and purified by immunoaffinity chromatography and sucrose gradient centrifugation. Electron microscopy of full-length F protein in the absence of detergents revealed micelles, (i.e., rosettes) containing two distinct types of protein rods, one cone-shaped and the other lollipop-shaped. Analysis of membrane anchorless F molecules indicated that they were similar to the cone-shaped rods and that rosettes, which they formed on storage, were made up of lollipop-shaped rods. The two forms of F protein may represent different structures that the molecule may adopt before and after activation for its role in membrane fusion. Studies of complexes of these structures with monoclonal antibodies of known specificity provide information on the three-dimensional organization of antigenic sites on the F protein and confirm the oligomeric structure, possibly trimeric, of both full-length F and membrane anchorless F.

Interaction of mutant influenza virus hemagglutinin fusion peptides with lipid bilayers: probing the role of hydrophobic residue size in the central region of the fusion peptide.

The amino-terminal region of the membrane-anchored subunit of influenza virus hemagglutinin, the fusion peptide, is crucial for membrane fusion of this virus. The peptide is extruded from the interior of the protein and inserted into the lipid bilayer of the target membrane upon induction of a conformational change in the protein by low pH. Although the effects of several mutations in this region on the fusion behavior and the biophysical properties of the corresponding peptides have been studied, the structural requirements for an active fusion peptide have still not been defined. To probe the sensitivity of the fusion peptide structure and function to small hydrophobic perturbations in the middle of the hydrophobic region, we have individually replaced the alanine residues in positions 5 and 7 with smaller (glycine) or bulkier (valine) hydrophobic residues and measured the extent of fusion mediated by these hemagglutinin constructs as well as some biophysical properties of the corresponding synthetic peptides in lipid bilayers. We find that position 5 tolerates a smaller and position 7 a larger hydrophobic side chain. All peptides contained segments of alpha-helical (33-45%) and beta-strand (13-16%) conformation as determined by CD and ATR-FTIR spectroscopy. The order parameters of the peptide helices and the lipid hydrocarbon chains were determined from measurements of the dichroism of the respective infrared absorption bands. Order parameters in the range of 0.0-0.6 were found for the helices of these peptides, which indicate that these peptides are most likely aligned with their alpha-helices at oblique angles to the membrane normal. Some (mostly fusogenic) peptides induced significant increases of the order parameter of the lipid hydrocarbon chains, suggesting that the lipid bilayer becomes more ordered in the presence of these peptides, possibly as a result of dehydration at the membrane surface.

The central structural feature of the membrane fusion protein subunit from the Ebola virus glycoprotein is a long triple-stranded coiled coil.

The ectodomain of the Ebola virus Gp2 glycoprotein was solubilized with a trimeric, isoleucine zipper derived from GCN4 (pIIGCN4) in place of the hydrophobic fusion peptide at the N terminus. This chimeric molecule forms a trimeric, highly alpha-helical, and very thermostable molecule, as determined by chemical crosslinking and circular dichroism. Electron microscopy indicates that Gp2 folds into a rod-like structure like influenza HA2 and HIV-1 gp41, providing further evidence that viral fusion proteins from diverse families such as Orthomyxoviridae (Influenza), Retroviridae (HIV-1), and Filoviridae (Ebola) share common structural features, and suggesting a common membrane fusion mechanism.

Antigen distortion allows influenza virus to escape neutralization.

The structure of the hemagglutinin (HA) of a mutant influenza virus that escapes neutralization by a monoclonal antibody shows that the mutation causes changes in HA structure which avoid an energetically less favorable conformation. However, the structure of the mutant HA.Fab complex indicates that the antibody binds selectively to mutant HA in a wild type-like distorted conformation. The association of an antibody with a less favored HA conformation represents an alternative to previously described mechanisms of escape from neutralization by antibodies.

Studies of the binding properties of influenza hemagglutinin receptor-site mutants.

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

Adaptation of egg-grown and transfectant influenza viruses for growth in mammalian cells: selection of hemagglutinin mutants with elevated pH of membrane fusion.

A series of eight transfectant influenza viruses was generated by reverse genetics for studies of the palmitylated cysteine residues in the cytoplasmic tail of the hemagglutinin glycoprotein (HA). Following amplification of these viruses in MDCK cells we found that all had developed an elevated pH of membrane fusion--an unexpected result since previous mutant HA expression studies had shown that substitutions of the cysteine residues had no effect on fusion properties. Sequence analyses revealed that each of the viruses had at least one additional mutation in the ectodomain of HA which was responsible for the increase in fusion pH. Similarly, when we passaged egg-grown wild-type X-31 virus in three different lines of MDCK cells or in MDBK cells, high pH fusion mutants were selected within a few passages in every case. The locations of the substitutions in the HA structure are in or near the "fusion peptide" or at subunit interfaces throughout the length of the trimer--reminiscent of the changes selected in earlier studies on amantadine resistance. The observation that passage of certain viruses in mammalian cells can result in the selection of mutants with elevated fusion pH has potential implications both for reverse genetic experiments and, perhaps more importantly, for the choice of substrates for propagation of vaccine viruses.

Studies using double mutants of the conformational transitions in influenza hemagglutinin required for its membrane fusion activity.

Amino acid substitutions widely distributed throughout the influenza hemagglutinin (HA) influence the pH of its membrane fusion activity. We have combined a number of these substitutions in double mutants and determined the effects on the pH of fusion and on the pH at which the refolding of HA required for fusion occurs. By analyzing combinations of mutations in three regions of the metastable neutral-pH HA that are rearranged at fusion pH we obtain evidence for both additive and nonadditive effects and for an apparent order of dominance in the effects of amino acid substitutions in particular regions on the pH of fusion. We conclude that there are at least three components in the structural transition required for membrane fusion activity and consider possible pathways for the transition in relation to the known differences between neutral and fusion pH HA structures.

Effect of the N-terminal glycine on the secondary structure, orientation, and interaction of the influenza hemagglutinin fusion peptide with lipid bilayers.

The amino-terminal segment of the membrane-anchored subunit of influenza hemagglutinin (HA) plays a crucial role in membrane fusion and, hence, has been termed the fusion peptide. We have studied the secondary structure, orientation, and effects on the bilayer structure of synthetic peptides corresponding to the wild-type and several fusogenic and nonfusogenic mutants with altered N-termini of the influenza HA fusion peptide by fluorescence, circular dichroism, and Fourier transform infrared spectroscopy. All peptides contained segments of alpha-helical and beta-strand conformation. In the wild-type fusion peptide, 40% of all residues were in alpha-secondary and 30% in beta-secondary structures. By comparison, the nonfusogenic peptides exhibited larger beta/alpha secondary structure ratios. The order parameters of the helices and the amide carbonyl groups of the beta-strands of the wild-type fusion peptide were measured separately, based on the infrared dichroism of the respective absorption bands. Order parameters in the range 0.1-0.7 were found for both segments of the wild-type peptide, which indicates that they are most likely aligned at oblique angles to the membrane normal. The nonfusogenic but not the fusogenic peptides induced splitting of the infrared absorption band at 1735 cm(-1), which is assigned to stretching vibrations of the lipid ester carbonyl bond. This splitting, which reports on an alteration of the hydrogen bonds formed between the lipid ester carbonyls and water and/or hydrogen-donating groups of the fusion peptides, correlated with the beta/alpha ratio of the peptides, suggesting that unpaired beta-strands may replace water molecules and hydrogen-bond to the lipid ester carbonyl groups. The profound structural changes induced by single amino acid replacements at the extreme N-terminus of the fusion peptide further suggest that tertiary or quaternary structural interactions may be important when fusion peptides bind to lipid bilayers.

The ectodomain of HIV-1 env subunit gp41 forms a soluble, alpha-helical, rod-like oligomer in the absence of gp120 and the N-terminal fusion peptide.

The human immunodeficiency virus-1 (HIV-1) envelope glycoprotein is composed of a soluble glycopolypeptide gp120 and a transmembrane glycopolypeptide gp41. These subunits form non-covalently linked oligomers on the surface of infected cells, virions and cells transfected with the complete env gene. Two length variants of the extracellular domain of gp41 (aa 21-166 and aa 39-166), that both lack the N-terminal fusion peptide and the C-terminal membrane anchor and cytoplasmic domain, have been expressed in insect cells to yield soluble oligomeric gp41 proteins. Oligomerization was confirmed by chemical cross-linking and gel filtration. Electron microscopy and circular dichroism measurements indicate a rod-like molecule with a high alpha-helical content and a high melting temperature (78 degrees C). The binding of monoclonal antibody Fab fragments dramatically increased the solubility of both gp41 constructs. We propose that gp41 folds into its membrane fusion-active conformation, when expressed alone.

A soluble domain of the membrane-anchoring chain of influenza virus hemagglutinin (HA2) folds in Escherichia coli into the low-pH-induced conformation.

The extensive refolding of the membrane-anchoring chain of hemagglutinin (HA) of influenza virus (termed HA2) in cellular endosomes, which initiates viral entry by membrane fusion, suggests that viral HA is meta-stable. HA2 polypeptide residues 38-175 expressed in Escherichia coli are reported here to fold in vivo into a soluble trimer. The structure appears to be the same as the low-pH-induced conformation of viral HA2 by alpha-helical content, thermodynamic stability, protease dissection, electron microscopy, and antibody binding. These results provide evidence that the structure of the low-pH-induced fold of viral HA2 (TBHA2) observed crystallographically is the lowest-energy-state fold of the HA2 polypeptide. They indicate that the HA2 conformation in viral HA before low pH activation of its fusion potential is metastable and suggest that removal of the receptor-binding chain (HA1) is enough to allow HA2 to adopt the stable state. Further, they provide direct evidence that low pH is not required to form the membrane-fusion conformation but acts to make this state kinetically accessible in viral HA.

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.

Structure of influenza virus haemagglutinin complexed with a neutralizing antibody.

Haemagglutinin (HA) is the influenza surface glycoprotein that interacts with infectivity-neutralizing antibodies. As a consequence of this immune pressure, it is the variable virus component, which is important in antigenic drift, that results in recurrent epidemics of influenza. We have determined the crystallographic structure of a complex formed between the antigen-binding fragment (Fab) of a neutralizing antibody and the membrane-distal domain ('HA top') of a HA subunit prepared from HA in its membrane-fusion-active conformation. A dramatic change is seen in the structure of the Fab-combining site on complex formation. Our results indicate that neutralization of infectivity by this antibody involves the inhibition of receptor binding, and demonstrate how influenza virus can maintain its conserved receptor-binding site despite the immune selective pressure for change in this region of the molecule; they also contribute to a complete description of the endosomal pH-induced fusion-active HA structure.

Electron microscopy of antibody complexes of influenza virus haemagglutinin in the fusion pH conformation.

Activation of the membrane fusion potential of influenza haemagglutinin (HA) at endosomal pH requires changes in its structure. X-ray analysis of TBHA2, a proteolytic fragment of HA in the fusion pH conformation, indicates that at the pH of fusion the 'fusion peptide' is displaced by > 10 nm from its location in the native structure to the tip of an 11 nm triple-stranded coiled coil, and that the formation of this structure involves extensive re-folding or reorganization of HA. Here we examine the structure of TBHA2 with the electron microscope and compare it with the fusion pH structure of HA2 in virosomes, HA2 in aggregates formed at fusion pH by the soluble, bromelain-released ectodomain BHA and HA2 in liposomes with which BHA associates at fusion pH. We have oriented each HA2 preparation for comparison, using site-specific monoclonal antibodies. We conclude that the structural changes in membrane-anchored and soluble HA preparations at the pH of fusion appear to be the same; that in the absence of a target membrane, the 'fusion peptide' of HA in virosomes associates with the virosome membrane so that HA2 is membrane bound at both N- and C-termini, which implies that inversion of the re-folded HA can occur; and that the structural changes observed by X-ray analysis do not result from the proteolytic digestions used in the preparation of TBHA2.