Membrane structure and fusion-triggering conformational change of the fusion domain from influenza hemagglutinin

The N-terminal domain of the influenza hemagglutinin (HA) is the only portion of the molecule that inserts deeply into membranes of infected cells to mediate the viral and the host cell membrane fusion. This domain constitutes an autonomous folding unit in the membrane, causes hemolysis of red blood cells and catalyzes lipid exchange between juxtaposed membranes in a pH-dependent manner. Combining NMR structures determined at pHs 7.4 and 5 with EPR distance constraints, we have deduced the structures of the N-terminal domain of HA in the lipid bilayer. At both pHs, the domain is a kinked, predominantly helical amphipathic structure. At the fusogenic pH 5, however, the domain has a sharper bend, an additional 3(10)-helix and a twist, resulting in the repositioning of Glu 15 and Asp 19 relative to that at the nonfusogenic pH 7.4. Rotation of these charged residues out of the membrane plane creates a hydrophobic pocket that allows a deeper insertion of the fusion domain into the core of the lipid bilayer. Such an insertion mode could perturb lipid packing and facilitate lipid mixing between juxtaposed membranes.     

Membrane interaction and structure of the transmembrane domain of influenza hemagglutinin and its fusion peptide complex

Background  

To study the organization and interaction with the fusion domain (or fusion peptide, FP) of the transmembrane domain (TMD) of influenza virus envelope glycoprotein for its role in membrane fusion which is also essential in the cellular trafficking of biomolecules and sperm-egg fusion.     

Modification of the cytoplasmic domain of influenza virus hemagglutinin affects enlargement of the fusion pore

The fusion activity of chimeras of influenza virus hemagglutinin (HA) (from A/fpv/Rostock/34; subtype H7) with the transmembrane domain (TM) and/or cytoplasmic tail (CT) either from the nonviral, nonfusogenic T-cell surface protein CD4 or from the fusogenic Sendai virus F-protein was studied. Wild-type or chimeric HA was expressed in CV-1 cells by the transient T7-RNA-polymerase vaccinia virus expression system. Subsequently, the fusion activity of the expression products was monitored with red blood cells or ghosts as target cells. To assess the different steps of fusion, target cells were labeled with the fluorescent membrane label octadecyl rhodamine B-chloride (R18) (membrane fusion) and with the cytoplasmic fluorophores calcein (molecular weight [MW], 623; formation of small aqueous fusion pore) and tetramethylrhodamine-dextran (MW, 10,000; enlargement of fusion pore). All chimeric HA/F-proteins, as well as the chimera with the TM of CD4 and the CT of HA, were able to mediate the different steps of fusion very similarly to wild-type HA. Quite differently, chimeric proteins with the CT of CD4 were strongly impaired in mediating pore enlargement. However, membrane fusion and formation of small pores were similar to those of wild-type HA, indicating that the conformational change of the ectodomain and earlier fusion steps were not inhibited. Various properties of the CT which may affect pore enlargement are considered. We surmise that the hydrophobicity of the sequence adjacent to the transmembrane domain is important for pore dilation.     

Architecture of the influenza hemagglutinin membrane fusion site

The mechanism of influenza hemagglutinin (HA) mediated membrane fusion has been intensively studied for over 20 years after the bromelain-released ectodomain of HA at neutral pH was first crystallized. Nearly 10 years ago, the low-pH-induced “spring coiled” conformational change of HA was predicted from peptide chemistry and confirmed by crystallography. Other work has yielded a wealth of knowledge on the observed changes in HA fusion/hemifusion phenotypes as a function of site-specific mutations of HA, or added amphipathic molecules or particular IgGs. It is becoming clear that the conformational changes predicted by the crystallography are necessary to cause fusion and that interfering with these changes can block fusion or reduce it to hemifusion. What is not known is how the conformational changes cause fusion. In particular, while it is generally agreed that fusion requires an aggregate of HAs, how the aggregate may act to transduce the energy of the HA conformational changes to creating the initial fusion defect is not known. We have used a comprehensive mass action kinetic model of HA-mediated fusion to carry out a “meta-analysis” of several key data sets, using HA-expressing cells and using virions. The consensus result of these detailed kinetic studies was that the fusion site of influenza hemagglutinin (HA) is an aggregate with at least eight HAs. The high-energy conformational change of only two of these HAs within the aggregate permits the formation of the first fusion pore. This “8 and 2” result was required to best fit all the data. We review these studies and how this kinetic result can guide and constrain HA fusion models. The kinetic analysis suggests that the sequence of fusion intermediates starts with protein control and ends with lipid control, which makes sense. While curvature intermediates, e.g. the lipid stalk, are almost certainly within the fusion sequence, the “8 and 2” result does not suggest that they are the first step after HA aggregation. The stabilized hydrophobic defect model we have proposed as a precursor to the lipid stalk can form and is consistent with the “8 and 2” result.     

Architecture of the influenza hemagglutinin membrane fusion site

The mechanism of influenza hemagglutinin (HA) mediated membrane fusion has been intensively studied for over 20 years after the bromelain-released ectodomain of HA at neutral pH was first crystallized. Nearly 10 years ago, the low-pH-induced "spring coiled" conformational change of HA was predicted from peptide chemistry and confirmed by crystallography. Other work has yielded a wealth of knowledge on the observed changes in HA fusion/hemifusion phenotypes as a function of site-specific mutations of HA, or added amphipathic molecules or particular IgGs. It is becoming clear that the conformational changes predicted by the crystallography are necessary to cause fusion and that interfering with these changes can block fusion or reduce it to hemifusion. What is not known is how the conformational changes cause fusion. In particular, while it is generally agreed that fusion requires an aggregate of HAs, how the aggregate may act to transduce the energy of the HA conformational changes to creating the initial fusion defect is not known. We have used a comprehensive mass action kinetic model of HA-mediated fusion to carry out a "meta-analysis" of several key data sets, using HA-expressing cells and using virions. The consensus result of these detailed kinetic studies was that the fusion site of influenza hemagglutinin (HA) is an aggregate with at least eight HAs. The high-energy conformational change of only two of these HAs within the aggregate permits the formation of the first fusion pore. This "8 and 2" result was required to best fit all the data. We review these studies and how this kinetic result can guide and constrain HA fusion models. The kinetic analysis suggests that the sequence of fusion intermediates starts with protein control and ends with lipid control, which makes sense. While curvature intermediates, e.g. the lipid stalk, are almost certainly within the fusion sequence, the "8 and 2" result does not suggest that they are the first step after HA aggregation. The stabilized hydrophobic defect model we have proposed as a precursor to the lipid stalk can form and is consistent with the "8 and 2" result.     

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.     

Kinetically differentiating influenza hemagglutinin fusion and hemifusion machines

Membrane fusion mediated by influenza virus hemagglutinin (HA) yields different phenotypes depending on the surface density of activated HAs. A key question is whether different phenotypes arise from different fusion machines or whether different numbers of identical fusion machines yield different probabilistic outcomes. If fusion were simply a less probable event than hemifusion, requiring a larger number of identical fusion machines to occur first, then two predictions can be made. First, fusion should have a shorter average delay time than hemifusion, since there are more machines. Second, fusion should have a longer execution time of lipid mixing after it begins than hemifusion, since the full event cannot be faster than the partial event. Using a new automated video microscopy technique, we simultaneously monitored many HA-expressing cells fusing with erythrocytes and identified individual cell pairs with either full or only partial redistribution of fluorescent lipids. The full lipid mixing phenotype also showed contents mixing, i.e., fusion. Kinetic screening of the digitized fluorescence data showed that the execution of lipid mixing after the onset is faster for fusion than hemifusion. We found no correlation between the delay times before the onset of lipid mixing and the final fusion phenotype. We also found that the execution time for fusion was faster than that for hemifusion. Thus, we provide the first experimental evidence for fusion and hemifusion arising from different machines.     

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.     

pH-dependent self-association of influenza hemagglutinin fusion peptides in lipid bilayers

We have recently designed a host-guest peptide system that allows us to quantitatively measure the energetics of interaction of viral fusion peptides with lipid bilayers. Here, we show that fusion peptides of influenza hemagglutinin reversibly associate with one another at membrane surfaces above critical surface concentrations, which range from one to five peptides per 1000 lipids in the systems that we investigated. It is further demonstrated by using circular dichroism and Fourier transform infrared spectroscopy that monomeric peptides insert into the bilayers in a predominantly alpha-helical conformation, whereas self-associated fusion peptides adopt predominantly antiparallel beta-sheet structures at the membrane surface. The two forms are readily interconvertible and the equilibrium between them is determined by the pH and ionic strength of the surrounding solution. Lowering the pH favors the monomeric alpha-helical conformation, whereas increasing the ionic strength shifts the equilibrium towards the membrane-associated beta-aggregates. The binding data are interpreted in terms of a cooperative binding model that yields free energies of insertion and free energies of self-association for each of the peptides studied at pH 7.4 and pH 5. At pH 5 and 35 mM ionic strength, the insertion energy of the 20 residue influenza hemagglutinin fusion peptide is -7.2 kcal/mol and the self-association energy is -1.9 kcal/mol. We propose that self-association of fusion peptides could be a major driving force for recruiting a small number of hemagglutinin trimers into a fusion site.     

The transmembrane domain of influenza hemagglutinin exhibits a stringent length requirement to support the hemifusion to fusion transition

Glycosylphosphatidylinositol-anchored influenza hemagglutinin (GPI-HA) mediates hemifusion, whereas chimeras with foreign transmembrane (TM) domains mediate full fusion. A possible explanation for these observations is that the TM domain must be a critical length in order for HA to promote full fusion. To test this hypothesis, we analyzed biochemical properties and fusion phenotypes of HA with alterations in its 27-amino acid TM domain. Our mutants included sequential 2-amino acid (Delta2-Delta14) and an 11-amino acid deletion from the COOH-terminal end, deletions of 6 or 8 amino acids from the NH(2)-terminal and middle regions, and a deletion of 12 amino acids from the NH(2)-terminal end of the TM domain. We also made several point mutations in the TM domain. All of the mutants except Delta14 were expressed at the cell surface and displayed biochemical properties virtually identical to wild-type HA. All the mutants that were expressed at the cell surface promoted full fusion, with the notable exception of deletions of >10 amino acids. A mutant in which 11 amino acids were deleted was severely impaired in promoting full fusion. Mutants in which 12 amino acids were deleted (from either end) mediated only hemifusion. Hence, a TM domain of 17 amino acids is needed to efficiently promote full fusion. Addition of either the hydrophilic HA cytoplasmic tail sequence or a single arginine to Delta12 HA, the hemifusion mutant that terminates with 15 (hydrophobic) amino acids of the HA TM domain, restored full fusion activity. Our data support a model in which the TM domain must span the bilayer to promote full fusion.     

Conformational intermediates and fusion activity of influenza virus hemagglutinin

Three strains of influenza virus (H1, H2, and H3) exhibited similar characteristics in the ability of their hemagglutinin (HA) to induce membrane fusion, but the HAs differed in their susceptibility to inactivation. The extent of inactivation depended on the pH of preincubation and was lowest for A/Japan (H2 subtype), in agreement with previous studies (A. Puri, F. Booy, R. W. Doms, J. M. White, and R. Blumenthal, J. Virol. 64:3824-3832, 1990). While significant inactivation of X31 (H3 subtype) was observed at 37 degrees C at pH values corresponding to the maximum of fusion (about pH 5.0), no inactivation was seen at preincubation pH values 0.2 to 0.4 pH units higher. Surprisingly, low-pH preincubation under those conditions enhanced the fusion rates and extents of A/Japan as well as those of X31. For A/PR 8/34 (H1 subtype), neither a shift of the pH (to >5.0) nor a decrease of the temperature to 20 degrees C was sufficient to prevent inactivation. We provide evidence that the activated HA is a conformational intermediate distinct from the native structure and from the final structure associated with the conformational change of HA, which is implicated by the high-resolution structure of the soluble trimeric fragment TBHA2 (P. A. Bullough, F. M. Hughson, J. J. Skehel, and D. C. Wiley, Nature 371:37-43, 1994).     

How to lose a kink and gain a helix: pH independent conformational changes of the fusion domains from influenza hemagglutinin in heterogeneous lipid bilayers

We have simulated two conformations of the fusion domain of influenza hemagglutinin (HA) within explicit water, salt, and heterogeneous lipid bilayers composed of POPC:POPG (4:1). Each conformation has seven different starting points in which the initial peptide structure is the same for each conformation, but the location across the membrane normal and lipid arrangement around the peptide are varied, giving a combined total simulation time of 140 ns. For the HA5 conformation (primary structure from recent NMR spectroscopy at pH = 5), the peptide exhibits a stable and less kinked structure in the lipid bilayer compared to that from the NMR studies. The relative fusogenic behavior of the different conformations has been investigated by calculation of the relative free energy of insertion into the hydrophobic region of lipid bilayer as a function of the depth of immersion. For the HA7 conformations (primary structure from recent NMR spectroscopy at pH = 7.4), while the N-terminal helix preserves its initial structure, the flexible C-terminal chain produces a transient helical motif inside the lipid bilayer. This conformational change is pH-independent, and is closely related to the peptide insertion into the lipid bilayer.     

Membrane fusion mechanisms: the influenza hemagglutinin paradigm and its implications for intracellular fusion

The mechanism of membrane fusion induced by the influenza virus hemagglutinin (HA) has been extensively characterized. Fusion is triggered by low pH, which induces conformational changes in the protein, leading to insertion of a hydrophobic 'fusion peptide' into the viral membrane and the target membrane for fusion. Insertion perturbs the target membrane, and hour glass-shaped lipidic fusion intermediates, called stalks, fusing the outer monolayers of the two membranes, are formed. Stalk formation is followed by complete fusion of the two membranes. Structures similar to those formed by HA at the pH of fusion are found not only in many other viral fusion proteins, but are also formed by SNAREs, proteins involved in intracellular fusion. Substances that inhibit or promote HA-induced fusion because they affect stalk formation, also inhibit or promote intracellular fusion, cell–cell fusion and even intracellular fission similarly. Therefore, the mechanism of influenza HA-induced fusion may be a paradigm for many intracellular fusion events.     

Secondary structure, phospholipid membrane interactions, and fusion activity of two glutamate-rich analogs of influenza hemagglutinin fusion peptide

Two synthetic mutants of influenza HA2 fusion peptide (residues 1-25), containing Glu on the polar (residues 4,8-E5(4,8)) or the hydrophobic (residues 3,7-E5(3,7)) face of the amphipathic helix, were synthesized and labeled with NBD at the N-terminus. Introduction of Glu residues into the fusion peptide leads to increased sensitivity of various biochemical properties to pH compared to the wild type. The E5 peptides showed a decrease of alpha-helix content and increase of beta-sheet structure. Lipid binding was diminished, but not abolished even at high pH. The E5 analogs penetrate the lipid bilayer less deeply than the wild type, especially at high pH. The N-terminal half of the peptide showed significant variation of the depth of the penetration into the lipid bilayer. Both E5 peptides were fusion active. The properties of E5(3,7) were more affected by the Glu substitution and showed greater variation with pH than E5(4,8).     

Configuration of influenza hemagglutinin fusion peptide monomers and oligomers in membranes

The 20 N-terminal residues of the HA2 subunit of influenza hemagglutinin (HA), known as the fusion peptide, play a crucial role in membrane fusion. Molecular dynamics simulations with implicit solvation are employed here to study the structure and orientation of the fusion peptide in membranes. As a monomer the alpha-helical peptide adopts a shallow, slightly tilted orientation along the lipid tail-head group interface. The average angle of the peptide with respect to membrane plane is 12.4 degrees . We find that the kinked structure proposed on the basis of NMR data is not stable in our model because of the high energy cost related to the membrane insertion of polar groups. Because hemagglutinin-mediated membrane fusion is promoted by low pH, we examined the effect of protonation of the Glu and Asp residues. The configurations of the protonated peptides were slightly deeper in the membrane but at similar angles. Finally, because HA is a trimer, we modeled helical fusion peptide trimers. We find that oligomerization affects the insertion depth of the peptide and its orientation with respect to the membrane: a trimer exhibits equally favorable configurations in which some or all of the helices in the bundle insert obliquely deep into the membrane.     

Secondary structure, orientation, oligomerization, and lipid interactions of the transmembrane domain of influenza hemagglutinin

Influenza virus hemagglutinin (HA), the viral envelope glycoprotein that mediates fusion between the viral and cellular membranes, is a homotrimer of three subunits, each containing two disulfide-linked polypeptide chains, HA(1) and HA(2). Each HA(2) chain spans the viral membrane with a single putative transmembrane alpha-helix near its C-terminus. Fusion experiments with recombinant HAs suggest that this sequence is required for a late step of membrane fusion, as a glycosylphosphatidylinositol-anchored analogue of HA only mediates "hemifusion" of membranes, i.e., the merging of the proximal, but not distal, leaflets of the two juxtaposed lipid bilayers [Kemble et al. (1994) Cell 76, 383-391]. To find a structural explanation for the function of the transmembrane domain of HA(2) in membrane fusion, we have studied the secondary structure, orientation, oligomerization, and lipid interactions of a synthetic peptide representing the transmembrane segment of X:31 HA (TMX31) by circular dichroism and attenuated total reflection Fourier transform infrared spectroscopy and by gel electrophoresis. The peptide was predominantly alpha-helical in detergent micelles and in phospholipid bilayers. The helicity was increased in lipid bilayers composed of acidic lipids compared to pure phosphatidylcholine bilayers. In planar lipid bilayers, the helices were oriented close to the membrane normal. TMX31 aggregated into small heat-resistant oligomers composed of two to five subunits in SDS micelles. Amide hydrogen exchange experiments indicated that a large fraction of the helical residues were accessible to water, suggesting the possibility that TMX31 forms pores in lipid bilayers. Finally, the peptide increased the acyl chain order in lipid bilayers, which may be related to the preferential association of HA with lipid "rafts" in the cell surface and which may be an important prerequisite for complete membrane fusion.     

Configuration of influenza hemagglutinin fusion peptide monomers and oligomers in membranes

The 20 N-terminal residues of the HA2 subunit of influenza hemagglutinin (HA), known as the fusion peptide, play a crucial role in membrane fusion. Molecular dynamics simulations with implicit solvation are employed here to study the structure and orientation of the fusion peptide in membranes. As a monomer the α-helical peptide adopts a shallow, slightly tilted orientation along the lipid tail-head group interface. The average angle of the peptide with respect to membrane plane is 12.4 °. We find that the kinked structure proposed on the basis of NMR data is not stable in our model because of the high energy cost related to the membrane insertion of polar groups. Because hemagglutinin-mediated membrane fusion is promoted by low pH, we examined the effect of protonation of the Glu and Asp residues. The configurations of the protonated peptides were slightly deeper in the membrane but at similar angles. Finally, because HA is a trimer, we modeled helical fusion peptide trimers. We find that oligomerization affects the insertion depth of the peptide and its orientation with respect to the membrane: a trimer exhibits equally favorable configurations in which some or all of the helices in the bundle insert obliquely deep into the membrane.     

Heterogeneity of early intermediates in cell-liposome fusion mediated by influenza hemagglutinin

To explore early intermediates in membrane fusion mediated by influenza virus hemagglutinin (HA) and their dependence on the composition of the target membrane, we studied lipid mixing between HA-expressing cells and liposomes containing phosphatidylcholine (PC) with different hydrocarbon chains. For all tested compositions, our results indicate the existence of at least two types of intermediates, which differ in their lifetimes. The composition of the target membrane affects the stability of fusion intermediates at a stage before lipid mixing. For less fusogenic distearoyl PC-containing liposomes at 4 degrees C, some of the intermediates inactivate, and no intermediates advance to lipid mixing. Fusion intermediates that formed for the more fusogenic dioleoyl PC-containing liposomes did not inactivate and even yielded partial lipid mixing at 4 degrees C. Thus, a more fusogenic target membrane effectively blocks nonproductive release of the conformational energy of HA. Even for the same liposome composition, HA forms two types of fusion intermediates, dissimilar in their stability and propensity to fuse. This diversity of fusion intermediates emphasizes the importance of local membrane composition and local protein concentration in fusion of heterogeneous biological membranes.     

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.     

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.