Early steps of the conformational change of influenza virus hemagglutinin to a fusion active state: Stability and energetics of the hemagglutinin

A conformational change of the homotrimeric glycoprotein hemagglutinin (HA) of influenza virus mediates fusion between the viral envelope and the endosome membrane. The conformational change of the HA ectodomain is triggered by the acidic pH of the endosome lumen. An essential step of the conformational change is the formation of an extended coiled-coil motif exposing the hydrophobic fusion peptide toward the target membrane. The structures of the neutral-pH, non-fusion active conformation of the HA ectodomain and of a fragment of the ectodomain containing the coiled-coil motif are known. However, it is not known by which mechanism protonation triggers the conformational change of the stable neutral-pH conformation of the ectodomain. Here, recent studies on the stability of the HA ectodomain at neutral pH, the energetics of the conformational change toward the fusion-active state and of the unfolding of the HA ectodomain are summarised. A model for the early steps of the conformational change of the HA ectodomain is presented. The model implicates that protonation leads to a partial dissociation of the distal domains of the HA monomers that is driven by electrostatic repulsion. The opening of the ectodomain enables water to enter the ectodomain. The interaction of water with respective sequences originally shielded from contact with water drives the formation of the coiled-coil structure.     

Energetics of the loop-to-helix transition leading to the coiled-coil structure of influenza virus hemagglutinin HA2 subunits

Fusion of influenza virus with the endosomal membrane of the host cell is mediated by the homotrimer-organized glycoprotein hemagglutinin (HA). Its fusion activity is triggered by a low pH-mediated conformational change affecting the structure of the HA1 and HA2 subunits. The HA2 subunits undergo a loop-to-helix transition leading to a coiled-coil structure, a highly conserved motif for many fusion mediating viral proteins. However, experimental studies showed that the HA2 coiled-coil structure is stable at neutral and low pH, implying that there is no direct relationship between low pH and the HA2 loop-to-helix transition. To interpret this observation, we used a computational approach based on the dielectric continuum solvent model to explore the influence of water and pH on the free energy change of the transition. The computations showed that the electrostatic interaction between HA2 fragments and water is the major driving force of the HA2 loop-to-helix transition leading to the coiled-coil structure, as long as the HA1 globular domain covering the HA2 subunits in the nonfusion competent conformation is reorganized and thereby allows water molecules to interact with the whole loop segments of the HA2 subunits. Moreover, we show that the energy released by the loop-to-helix transition may account for those energies required for driving the subsequent steps of membrane fusion. Such a water-driven process may resemble a general mechanism for the formation of the highly conserved coiled-coil motif of enveloped viruses.     

Protonation and stability of the globular domain of influenza virus hemagglutinin.

A partial dissociation of the HA1 subunits of influenza virus hemagglutinin (HA) is considered to be the initial step of conformational changes of the HA ectodomain leading to a membrane fusion active conformation (L. Godley, J. Pfeifer, D. Steinhauer, B. Ely, G. Shaw, R. Kaufman, E. Suchanek, C. Pabo, J.J. Skehel, D.C. Wiley, and S. Wharton, 1992, Cell 68:635-645; G.W. Kemble, D.L.Bodian, J. Rose, I.A. Wilson, and J.M. White, 1992, J. Virol. 66:4940-4950). Here, we explore a mechanism that provides an understanding of the physical and chemical basis for such dissociation and relies on two essential observations. First, based on the x-ray structure of HA from X31 (I.A. Wilson, J.J. Skehel, and D.C. Wiley, 1981, Nature 289:366-373), and by employing techniques of molecular modeling, we show that the protonation of the HA1 subunits is enhanced at the conditions known to trigger conformational changes of the HA ectodomain. Second, we found that the dependence of the calculated relative degree of protonation of the HA1 domain on temperature and pH is similar to that observed experimentally for the conformational change of HA assessed by proteinase K sensitivity. We suggest that at the pH-temperature conditions typical for the conformational change of HA and membrane fusion, dissociation of the HA1 subunits is caused by the enhanced protonation of the HA1 subunits leading to an increase in the positive net charge of these subunits and, in turn, to a weakened attraction between them.     

The final conformation of the complete ectodomain of the HA2 subunit of influenza hemagglutinin can by itself drive low pH-dependent fusion

One of the best characterized fusion proteins, the influenza virus hemagglutinin (HA), mediates fusion between the viral envelope and the endosomal membrane during viral entry into the cell. In the initial conformation of HA, its fusogenic subunit, the transmembrane protein HA2, is locked in a metastable conformation by the receptor-binding HA1 subunit of HA. Acidification in the endosome triggers HA2 refolding toward the final lowest energy conformation. Is the fusion process driven by this final conformation or, as often suggested, by the energy released by protein restructuring? Here we explored structural properties as well as the fusogenic activity of the full sized trimeric HA2(1–185) (here called HA2*) that presents the final conformation of the HA2 ectodomain. We found HA2* to mediate fusion between lipid bilayers and between biological membranes in a low pH-dependent manner. Two mutations known to inhibit HA-mediated fusion strongly inhibited the fusogenic activity of HA2*. At surface densities similar to those of HA in the influenza virus particle, HA2* formed small fusion pores but did not expand them. Our results confirm that the HA1 subunit responsible for receptor binding as well as the transmembrane and cytosolic domains of HA2 is not required for fusion pore opening and substantiate the hypothesis that the final form of HA2 is more important for fusion than the conformational change that generates this form.     

Identification of a pH sensor in Influenza hemagglutinin using X-ray crystallography

Hemagglutnin (HA) mediates entry of influenza virus through a series of conformational changes triggered by the low pH of the endosome. The residue or combination of residues acting as pH sensors has not yet been fully elucidated. In this work, we assay pH effects on the structure of H5 HA by soaking HA crystallized at pH 6.5 in a series of buffers with lower pH, mimicking the conditions of the endosome. We find that HA1-H38, which is conserved in Group 1 HA, undergoes a striking change in side chain conformation, which we attribute to its protonation and cation-cation repulsion with conserved HA1-H18. This work suggests that x-ray crystallography can be applied for studying small-scale pH-induced conformational changes providing valuable information on the location of pH sensors in HA. Importantly, the observed change in HA1-H38 conformation is further evidence that the pH-induced conformational changes of HA are the result of a series of protonation events to conserved and non-conserved pH sensors.     

Membrane fusion activity of the influenza virus hemagglutinin. The low pH-induced conformational change

The hemagglutinin (HA) spike glycoprotein of influenza virus catalyzes a low pH-induced membrane fusion event which releases the viral genome into the host cell cytoplasm. To study the fusion mechanism in more detail, we have prepared the ectodomain of HA in water-soluble form by treating virus particles with bromelain. Under mildly acidic conditions (pH less than or equal to 5.8), the ectodomain undergoes a conformational change which we found to be biochemically and immunologically equivalent to that in native viral HA. It became sensitive to proteinase K, it exposed new antigenic epitopes in its HA1 chain, and it acquired amphiphilic properties, notably the ability to bind to liposomes. The attachment to liposomes exhibited the same pH dependence and rapid kinetics as the conformational change and was mediated by HA2. The nature of the attachment resembled that of an integral membrane protein except that the bound HA was partially removed by base. As observed for virus fusion, attachment is independent of divalent cations and lipid composition. Temperature was found to be a critical parameter only with dimyristoylphosphatidycholine vesicles where attachment was partially blocked below the major phase transition. These and other results obtained indicated that the low pH-induced conformational change in the isolated ectodomain is equivalent to that occurring in intact viral HA, and that its attachment to liposomes can serve as a model for the initial stages in the HA-induced membrane fusion reaction.     

Conformational change of influenza virus hemagglutinin is sensitive to ionic concentration

The homotrimeric spike glycoprotein hemagglutinin (HA) of influenza virus undergoes a low pH-mediated conformational change which mediates the fusion of the viral envelope with the target membrane. Previous approaches predict that the interplay of electrostatic interactions between and within HA subunits, HA 1 and HA2, are essential for the metastability of the HA ectodomain. Here, we show that suspension media of low ionic concentration promote fusion of fluorescent labelled influenza virus X31 with erythrocyte ghosts and with ganglioside containing liposomes. By measuring the low pH mediated inactivation of the fusion competence of HA and the Proteinase K sensitivity of low pH incubated HA we show that the conformational change is promoted by low ionic concentration. We surmise that electrostatic attraction within the HA ectodomain is weakened by lowering the ionic concentration facilitating the conformational change at low pH. Dedicated to Prof. K. Arnold on the occasion of his 65th birthday.     

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.     

The influenza hemagglutinin precursor as an acid-sensitive probe of the biosynthetic pathway.

The hemagglutinin of influenza virus (HA), an acid-activated membrane fusion protein, is synthesized in the endoplasmic reticulum and transported through the Golgi complex to the cell surface of infected cells as an uncleaved, fusion-incompetent precursor, HA0. The mature, proteolytically activated HA is known to undergo a rapid, irreversible, acid-induced conformational change which mediates membrane fusion and virus penetration. On the basis of antigenic modifications and the acquisition of trypsin susceptibility, we demonstrate here that HA0, while unable to cause fusion, is acid sensitive. It undergoes irreversible conformational changes quite similar to those of HA at mildly acidic pH (pH less than 6.0). The ectodomain of HA0 does not, however, acquire hydrophobic properties and the changes occur in a less concerted manner (the pH dependence is much broader and the rate of conversion slower). These differences are likely to account for the inability of acid-treated HA0 to trigger membrane fusion. It was shown, moreover, that HA0 acquired its acid-sensitive properties immediately following trimerization in the endoplasmic reticulum. Since HA0 did not convert to the acid form at any point during its intracellular transport, we concluded that the trans-Golgi compartment, known to be more acidic than the cytosol and involved in constitutive membrane transport, is not likely to have a pH less than 6.0.     

Investigation of pathways for the low-pH conformational transition in influenza hemagglutinin

Targeted molecular dynamics simulations were used to study the conformational transition of influenza hemagglutinin (HA) from the native conformation to putative fusogenic or postfusion conformations populated at low pH. Three pathways for this conformational change were considered. Complete dissociation of the globular domains of HA was observed in one pathway, whereas smaller rearrangements were observed in the other two. The fusion peptides became exposed and moved toward the target membrane, although occasional movement toward the viral membrane was also observed. The effective energy profiles along the paths show multiple barriers. The final low-pH structures, which are consistent with available experimental data, are comparable in effective energy to native HA. As a control, the uncleaved precursor HA0 was also forced along the same pathway. In this case both the final energy and the energy barrier were much higher than in the cleaved protein. This study suggests that 1) as proposed, the native conformation is the global minimum energy conformation for the uncleaved precursor but a metastable state for cleaved HA; 2) the spring-loaded conformational change is energetically plausible in full-length HA; and 3) complete globular domain dissociation is not necessary for extension of the coiled coil and fusion peptide exposure, but the model with complete dissociation has lower energy.     

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.     

The 1-127 HA2 construct of influenza virus hemagglutinin induces cell-cell hemifusion.

Conformational changes in the HA2 subunit of influenza hemagglutinin (HA) are coupled to membrane fusion. We investigated the fusogenic activity of the polypeptide FHA2 representing 127 amino-terminal residues of the ectodomain of HA2. While the conformation of FHA2 both at neutral and at low pH is nearly identical to the final low-pH conformation of HA2, FHA2 still induces lipid mixing between liposomes in a low-pH-dependent manner. Here, we found that FHA2 induces lipid mixing between bound cells, indicating that the "spring-loaded" energy is not required for FHA2-mediated membrane merger. Although, unlike HA, FHA2 did not form an expanding fusion pore, both acidic pH and membrane concentrations of FHA2, required for lipid mixing, have been close to those required for HA-mediated fusion. Similar to what is observed for HA, FHA2-induced lipid mixing was reversibly blocked by lysophosphatidylcholine and low temperature, 4 degrees C. The same genetic modification of the fusion peptide inhibits both HA- and FHA2-fusogenic activities. The kink region of FHA2, critical for FHA2-mediated lipid mixing, was exposed in the low-pH conformation of the whole HA prior to fusion. The ability of FHA2 to mediate lipid mixing very similar to HA-mediated lipid mixing is consistent with the hypothesis that hemifusion requires just a portion of the energy released in the conformational change of HA at acidic pH.     

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.     

Conformational aspects of the acid-induced fusion mechanism of influenza virus hemagglutinin. Circular dichroism and fluorescence studies

Circular dichroism and tryptophan fluorescence spectroscopy have been used to investigate the structures of the influenza virus membrane glycoprotein hemagglutinin, acid-treated hemagglutinin, and fragments of hemagglutinin derived by proteolysis. The conformational change in hemagglutinin which occurs at the pH of membrane fusion (pH 5-6) was associated with a significant change of the environment of tyrosine residues, a change in the environment of tryptophan residues, but no changes in secondary structure. Tryptic digestion of the hemagglutinin in its low pH conformation which releases one of the subunit polypeptides (HA1) caused minimal changes in tyrosine and tryptophan environments but a small secondary structural change in HA1. The secondary structure of the remainder of the molecule (HA2) was very similar to that predicted from the known x-ray crystallographic structure of the native molecule. However, fluorescence spectroscopy indicated a tertiary change in structure in the coiled coil of alpha-helices which form the fibrous central stem of the molecule. These results are consistent with a conformational change required for membrane fusion which involves a decrease of HA1/HA1, HA1/HA2 interactions and changes in tertiary structure not accompanied by changes in secondary structure.     

Exploring the early stages of the pH‐induced conformational change of influenza hemagglutinin

Hemagglutinin (HA) mediates the membrane fusion process of influenza virus through its pH‐induced conformational change. However, it remains challenging to study its structure reorganization pathways in atomic details. Here, we first applied continuous constant pH molecular dynamics approach to predict the pK_a values of titratable residues in H2 subtype HA. The calculated net‐charges in HA1 globular heads increase from 0e (pH 7.5) to +14e (pH 4.5), indicating that the charge repulsion drives the detrimerization of HA globular domains. In HA2 stem regions, critical pH sensors, such as Glu103₂, His18₁, and Glu89₁, are identified to facilitate the essential structural reorganizations in the fusing pathways, including fusion peptide release and interhelical loop transition. To probe the contribution of identified pH sensors and unveil the early steps of pH‐induced conformational change, we carried out conventional molecular dynamics simulations in explicit water with determined protonation state for each titratable residue in different environmental pH conditions. Particularly, energy barriers involving previously uncharacterized hydrogen bonds and hydrophobic interactions are identified in the fusion peptide release pathway. Nevertheless, comprehensive comparisons across HA family members indicate that different HA subtypes might employ diverse pH sensor groups along with different fusion pathways. Finally, we explored the fusion inhibition mechanism of antibody CR6261 and small molecular inhibitor TBHQ, and discovered a novel druggable pocket in H2 and H5 subtypes. Our results provide the underlying mechanism for the pH‐driven conformational changes and also novel insight for anti‐flu drug development. Proteins 2014; 82:2412–2428. © 2014 Wiley Periodicals, Inc.     

Structural basis of local, pH-dependent conformational changes in glycoprotein B from herpes simplex virus type 1

Herpesviruses enter cells by membrane fusion either at the plasma membrane or in endosomes, depending on the cell type. Glycoprotein B (gB) is a conserved component of the multiprotein herpesvirus fusion machinery and functions as a fusion protein, with two internal fusion loops, FL1 and FL2. We determined the crystal structures of the ectodomains of two FL1 mutants of herpes simplex virus type 1 (HSV-1) gB to clarify whether their fusion-null phenotypes were due to global or local effects of the mutations on the structure of the gB ectodomain. Each mutant has a single point mutation of a hydrophobic residue in FL1 that eliminates the hydrophobic side chain. We found that neither mutation affected the conformation of FL1, although one mutation slightly altered the conformation of FL2, and we conclude that the fusion-null phenotype is due to the absence of a hydrophobic side chain at the mutated position. Because the ectodomains of the wild-type and the mutant forms of gB crystallized at both low and neutral pH, we were able to determine the effect of pH on gB conformation at the atomic level. For viruses that enter cells by endocytosis, the low pH of the endosome effects major conformational changes in their fusion proteins, thereby promoting fusion of the viral envelope with the endosomal membrane. We show here that upon exposure of gB to low pH, FL2 undergoes a major relocation, probably driven by protonation of a key histidine residue. Relocation of FL2, as well as additional small conformational changes in the gB ectodomain, helps explain previously noted changes in its antigenic and biochemical properties. However, no global pH-dependent changes in gB structure were detected in either the wild-type or the mutant forms of gB. Thus, low pH causes local conformational changes in gB that are very different from the large-scale fusogenic conformational changes in other viral fusion proteins. We propose that these conformational changes, albeit modest, play an important functional role during endocytic entry of HSV.     

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.     

Structure of a flavivirus envelope glycoprotein in its low-pH-induced membrane fusion conformation

Enveloped viruses enter cells via a membrane fusion reaction driven by conformational changes of specific viral envelope proteins. We report here the structure of the ectodomain of the tick-borne encephalitis virus envelope glycoprotein, E, a prototypical class II fusion protein, in its trimeric low-pH-induced conformation. We show that, in the conformational transition, the three domains of the neutral-pH form are maintained but their relative orientation is altered. Similar to the postfusion class I proteins, the subunits rearrange such that the fusion peptide loops cluster at one end of an elongated molecule and the C-terminal segments, connecting to the viral transmembrane region, run along the sides of the trimer pointing toward the fusion peptide loops. Comparison with the low-pH-induced form of the alphavirus class II fusion protein reveals striking differences at the end of the molecule bearing the fusion peptides, suggesting an important conformational effect of the missing membrane connecting segment.     

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.     

Measuring pKa of activation and pKi of inactivation for influenza hemagglutinin from kinetics of membrane fusion of virions and of HA expressing cells

The data for the pH dependence of lipid mixing between influenza virus (A/PR/8/34 strain) and fluorescently labeled liposomes containing gangliosides has been analyzed using a comprehensive mass action kinetic model for hemaglutinin (HA)-mediated fusion. Quantitative results obtained about the architecture of HA-mediated membrane fusion site from this analysis are in agreement with the previously reported results from analyses of data for HA-expressing cells fusing with various target membranes. Of the eight or more HAs forming a fusogenic aggregate, only two have to undergo the "essential" conformational change needed to initiate fusion. The mass action kinetic model has been extended to allow the analysis of the pKa for HA activation and pKi for HA inactivation. Inactivation and activation of HA following protonation were investigated for various experimental systems involving different strains of HA (A/PR/8/34, X:31, A/Japan). We find that the pKa for the final protonation site on each monomer of the trimer molecule is 5.6 to 5.7, irrespective of the strain. We also find that the pKi for the PR/8 strain is 4.8 to 4.9. The inactivation rate constants for HA, measured from experiments done with PR/8 virions fusing with liposomes and X:31 HA-expressing cells fusing with red blood cells, were both found to be of the order of 10(-4) s(-1). This number appears to be the minimal rate for HA's essential conformational change at low HA surface density. At high HA surface densities, we find evidence for cooperativity in the conformational change, as suggested by other studies.