Autocatalytic activation of influenza hemagglutinin

Enveloped viruses contain surface proteins that mediate fusion between the viral and target cell membranes following an activating stimulus. Acidic pH induces the influenza virus fusion protein hemagglutinin (HA) via irreversible refolding of a trimeric conformational state leading to exposure of hydrophobic fusion peptides on each trimer subunit. Herein, we show that cells expressing fowl plague virus HA demonstrate discrete switching behavior with respect to the HA conformational change. Partially activated states do not exist at the scale of the cell, activation of HA leads to aggregation of cell surface trimers, and newly synthesized HA refold spontaneously in the presence of previously activated HA. These observations imply a feedback mechanism involving self-catalyzed refolding of HA and thus suggest a mechanism similar to the autocatalytic refolding and aggregation of prions.     

Reversible merger of membranes at the early stage of influenza hemagglutinin-mediated fusion

Fusion mediated by influenza hemagglutinin (HA), a prototype fusion protein, is commonly detected as lipid and content mixing between fusing cells. Decreasing the surface density of fusion-competent HA inhibited these advanced fusion phenotypes and allowed us to identify an early stage of fusion at physiological temperature. Although lipid flow between membranes was restricted, the contacting membrane monolayers were apparently transiently connected, as detected by the transformation of this fusion intermediate into complete fusion after treatments known to destabilize hemifusion diaphragms. These reversible connections disappeared within 10-20 min after application of low pH, indicating that after the energy released by HA refolding dissipated, the final low pH conformation of HA did not support membrane merger. Although the dynamic character and the lack of lipid mixing at 37 degrees C distinguish the newly identified fusion intermediate from the intermediate arrested at 4 degrees C described previously, both intermediates apparently belong to the same family of restricted hemifusion (RH) structures. Because the formation of transient RH structures at physiological temperatures was as fast as fusion pore opening and required less HA, we hypothesize that fusion starts with the formation of multiple RH sites, only a few of which then evolve to become expanding fusion pores.     

Delay of influenza hemagglutinin refolding into a fusion-competent conformation by receptor binding: a hypothesis

Two subunits of influenza hemagglutinin (HA), HA1 and HA2, represent one of the best-characterized membrane fusion machines. While a low pH conformation of HA2 mediates the actual fusion, HA1 establishes a specific connection between the viral and cell membranes via binding to the sialic acid-containing receptors. Here we propose that HA1 may also be involved in modulating the kinetics of HA refolding. We hypothesized that binding of the HA1 subunit to its receptor restricts the major refolding of the low pH-activated HA to a fusion-competent conformation and, in the absence of fusion, to an HA-inactivated state. Dissociation of the HA1-receptor connection was considered to be a slow kinetic step. To verify this hypothesis, we first analyzed a simple kinetic scheme accounting for the stages of dissociation of the HA1/receptor bonds, inactivation and fusion, and formulated experimentally testable predictions. Second, we verified these predictions by measuring the extent of fusion between HA-expressing cells and red blood cells. Three experimental approaches based on 1) the temporal inhibition of fusion by lysophosphatidylcholine, 2) rapid dissociation of the HA1-receptor connections by neuraminidase treatment, and 3) substitution of membrane-anchored receptors by a water-soluble sialyllactose all provided support for the proposed role of the release of HA1-receptor connections. Possible biological implications of this stage in HA refolding and membrane fusion are being discussed.     

Anti-peptide antibodies detect steps in a protein conformational change: low-pH activation of the influenza virus hemagglutinin

At low pH, the hemagglutinin (HA) of influenza virus undergoes an irreversible conformational change that potentiates its essential membrane fusion function. We have probed the details of this conformational change using a panel of 14 anti-HA-peptide antibodies. Whereas some antibodies reacted equally well with both the neutral and low-pH HA conformations, others reacted to a significantly greater extent with the low-pH form. The locations of the peptides recognized by the latter antibodies in the three-dimensional HA structure indicated regions of the protein that change in response to low pH. Moreover, kinetic experiments suggested steps in the conformational change. In addition to their relevance to membrane fusion, our results show that anti-peptide antibodies can be used to study some types of biologically important protein conformational changes.     

Role of metastability and acidic pH in membrane fusion by tick-borne encephalitis virus.

The envelope protein E of the flavivirus tick-borne encephalitis (TBE) virus is, like the alphavirus E1 protein, a class II viral fusion protein that differs structurally and probably mechanistically from class I viral fusion proteins. The surface of the native TBE virion is covered by an icosahedrally symmetrical network of E homodimers, which mediate low-pH-induced fusion in endosomes. At the pH of fusion, the E homodimers are irreversibly converted to a homotrimeric form, which we have found by intrinsic fluorescence measurements to be more stable than the native dimers. Thus, the TBE virus E protein is analogous to the prototypical class I fusion protein, the influenza virus hemagglutinin (HA), in that it is initially synthesized in a metastable state that is energetically poised to be converted to the fusogenic state by exposure to low pH. However, in contrast to what has been observed with influenza virus HA, this transition could not be triggered by input of heat energy alone and membrane fusion could be induced only when the virus was exposed to an acidic pH. In a previous study we showed that the dimer-to-trimer transition appears to be a two-step process involving a reversible dissociation of the dimer followed by an irreversible trimerization of the dissociated monomeric subunits. Because the dimer-monomer equilibrium in the first step apparently depends on the protonation state of E, the lack of availability of monomers for the trimerization step at neutral pH could explain why low pH is essential for fusion in spite of the metastability of the native E dimer.     

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.     

Structural changes in Influenza virus at low pH characterized by cryo-electron tomography

Influenza virus enters host cells by endocytosis. The low pH of endosomes triggers conformational changes in hemagglutinin (HA) that mediate fusion of the viral and endosomal membranes. We have used cryo-electron tomography to visualize influenza A virus at pH 4.9, a condition known to induce fusogenicity. After 30 min, when all virions are in the postfusion state, dramatic changes in morphology are apparent: elongated particles are no longer observed, larger particles representing fused virions appear, the HA spikes become conspicuously disorganized, a layer of M1 matrix protein is no longer resolved on most virions, and the ribonucleoprotein complexes (RNPs) coagulate on the interior surface of the virion. To probe for intermediate states, preparations were imaged after 5 min at pH 4.9. These virions could be classified according to their glycoprotein arrays (organized or disorganized) and whether or not they have a resolved M1 layer. Employing subtomogram averaging, we found, in addition to the neutral-pH state of HA, two intermediate conformations that appear to reflect an outwards movement of the fusion peptide and rearrangement of the HA1 subunits, respectively. These changes are reversible. The tomograms also document pH-induced changes affecting the M1 layer that appear to render the envelope more pliable and hence conducive to fusion. However, it appears desirable for productive infection that fusion should proceed before the RNPs become coagulated with matrix protein, as eventually happens at low pH.     

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.     

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.     

pH-induced conformational changes of membrane-bound influenza hemagglutinin and its effect on target lipid bilayers.

Influenza virus hemagglutinin (HA) has served as a paradigm for both pH-dependent and -independent viral membrane fusion. Although large conformational changes were observed by X-ray crystallography when soluble fragments of HA were subjected to fusion-pH conditions, it is not clear whether the same changes occur in membrane-bound HA, what the spatial relationship is between the conformationally changed HA and the target and viral membranes, and in what way HA perturbs the target membrane at low pH. We have taken a spectroscopic approach using an array of recently developed FTIR techniques to address these questions. Difference attenuated total reflection FTIR spectroscopy was employed to reveal reversible and irreversible components of the pH-induced conformational change of the membrane-bound bromelain fragment of HA, BHA. Additional proteolytic fragments of BHA were produced which permitted a tentative assignment of the observed changes to the HA1 and HA2 subunits, respectively. The membrane-bound HA1 subunit undergoes a reversible conformational change, which most likely involves the loss of a small proportion of beta-sheet at low pH. BHA was found to undergo a partially reversible tilting motion relative to the target membrane upon exposure to pH 5, indicating a previously undescribed hinge near the anchoring point to the target membrane. Time-resolved amide H/D exchange experiments revealed a more dynamic (tertiary) structure of membrane-bound BHA and its HA2, but not its HA1, subunit. Finally BHA and, to a lesser degree, HA1 perturbed the lipid bilayer of the target membrane at the interface, as assessed by spectral changes of the lipid ester carbonyl groups. These results are discussed in the context of a complementary study of HA that was bound to viral membranes through its transmembrane peptide (Gray C, Tamm LK, 1997, Protein Sci 6:1993-2006). A distinctive role for the HA1 subunit in the conformational change of HA becomes apparent from these combined studies.     

Class II fusion protein of alphaviruses drives membrane fusion through the same pathway as class I proteins

Viral fusion proteins of classes I and II differ radically in their initial structures but refold toward similar conformations upon activation. Do fusion pathways mediated by alphavirus E1 and influenza virus hemagglutinin (HA) that exemplify classes II and I differ to reflect the difference in their initial conformations, or concur to reflect the similarity in the final conformations? Here, we dissected the pathway of low pH–triggered E1-mediated cell–cell fusion by reducing the numbers of activated E1 proteins and by blocking different fusion stages with specific inhibitors. The discovered progression from transient hemifusion to small, and then expanding, fusion pores upon an increase in the number of activated fusion proteins parallels that established for HA-mediated fusion. We conclude that proteins as different as E1 and HA drive fusion through strikingly similar membrane intermediates, with the most energy-intensive stages following rather than preceding hemifusion. We propose that fusion reactions catalyzed by all proteins of both classes follow a similar pathway.     

pH-dependence of intermediate steps of membrane fusion induced by the influenza fusion peptide

Membrane fusion mediated by the influenza-virus fusion protein is activated by low pH via a cascade of reactions. Some processes among them are irreversible, such as helix hairpin formation of the ectodomain, whereas others are reversible, such as exposure of the fusion peptide. Using this property, we attempted to dissect, in temporal order, different stages of the fusion reaction involving the fusion peptide by an acidic-neutral-acidic pH cycle. The fluorescence-quenching data indicated that both insertion depth and self-assembly are pH-reversible. In addition, lipid mixing assay was demonstrated to be arrested by neutral pH. By contrast, membrane leakage was shown to be irreversible with respect to pH. Our results, along with those from other studies on the pH-dependence of membrane fusion, are used to build a model for the virus-mediated fusion event from the perspective of pH-reversibility.     

Minimal aggregate size and minimal fusion unit for the first fusion pore of influenza hemagglutinin-mediated membrane fusion

The data of Melikyan et al. (J. Gen. Physiol. 106:783, 1995) for the time required for the first measurable step of fusion, the formation of the first flickering conductivity pore between influenza hemagglutinin (HA) expressing cells and planar bilayers, has been analyzed using a new mass action kinetic model. The analysis incorporates a rigorous distinction between the minimum number of HA trimers aggregated at the nascent fusion site (which is denoted the minimal aggregate size) and the number of those trimers that must to undergo a slow essential conformational change before the first fusion pore could form (which is denoted the minimal fusion unit). At least eight (and likely more) HA trimers aggregated at the nascent fusion site. Remarkably, of these eight (or more) HAs, only two or three must undergo the essential conformational change slowly before the first fusion pore can form. Whether the conformational change of these first two or three HAs are sufficient for the first fusion pore to form or whether the remaining HAs within the aggregate must rapidly transform in a cooperative manner cannot be determined kinetically. Remarkably, the fitted halftime for the essential HA conformational change is roughly 10(4) s, which is two orders of magnitude slower than the observed halftime for fusion. This is because the HAs refold with distributed kinetics and because the conductance assay monitored the very first aggregate to succeed in forming a first fusion pore from an ensemble of hundreds or thousands (depending upon the cell line) of fusogenic HA aggregates within the area of apposition between the cell and the planar bilayer. Furthermore, the average rate constant for this essential conformational change was at least 10(7) times slower than expected for a simple coiled coil conformational change, suggesting that there is either a high free energy barrier to fusion and/or very many nonfusogenic conformations in the refolding landscape. Current models for HA-mediated fusion are examined in light of these new constraints on the early structure and evolution of the nascent fusion site. None completely comply with the data.     

Kinetics of pH-dependent fusion between influenza virus and liposomes

The pH-dependent fusion between influenza virus and liposomes (large unilamellar vesicles) has been investigated as a model for the fusion step in the infectious entry of the virus into cells. Fusion was monitored continuously, with a fluorescence assay based on resonance energy transfer (RET) [Struck, D. K., Hoekstra, D., & Pagano, R. E. (1981) Biochemistry 20, 4093-4099], which allows an accurate quantitation of the fusion process. Evidence is presented indicating that the dilution of the RET probes from the liposomal bilayer into the viral membrane is not due to transfer of individual lipid molecules. The initial rate and final extent of the fusion reaction increase dramatically with decreasing pH, fusion being virtually complete within 1 min at pH 4.5-5.0. From experiments in which the ratio of virus to liposomes was varied, it is concluded that virus-liposome fusion products continue to fuse with liposomes, but not with virus. Fusion is most efficient with liposomes consisting of negatively charged phospholipids, while phosphatidylcholine and sphingomyelin are inhibitory. The reaction is completely blocked by an antiserum against the virus and inhibited by pretreatment of the virus with trypsin. The effect of proteolytic pretreatment at pH 7.4 is enhanced after preincubation of the virus at pH 5.0, consistent with the occurrence of a low pH induced, irreversible, conformational change in the viral fusion protein, the hemagglutinin (HA), exposing trypsin cleavage sites. When, after initiation of the fusion reaction at pH 5.0, the pH is readjusted to neutral, the process is arrested instantaneously, indicating that the low pH induced conformational change in the HA protein, in itself, is not sufficient to trigger fusion activity.     

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.     

The mature avian leukosis virus subgroup A envelope glycoprotein is metastable, and refolding induced by the synergistic effects of receptor binding and low pH is coupled to infection

The spring-loaded model stipulates that influenza virus infection is coupled to the transition of the virus hemagglutinin (HA) from a metastable conformation to a highly stable conformation at low pH. The properties of retrovirus envelope glycoproteins indicate that infection is coupled to an analogous conformational change. As a test of this hypothesis, the requirements for avian leukosis virus A (ALV-A) infection were examined. These studies indicate that, like HA, the conformation of the mature ALV-A envelope glycoprotein is metastable and that infection is linked to refolding at low pH. However, unlike HA, low-pH activation is only observed after priming by receptor. Therefore, ALV-A infection is dependent on the synergistic effects of receptor binding and low pH, suggesting that receptor binding superimposes an additional constraint on activation of ALV-A fusion that proceeds by a mechanism comparable to that of influenza virus.     

Reversible and irreversible unfolding of multi-domain proteins

In contrast to single-domain proteins unfolding of larger multi-domain proteins is often irreversible. In a comparative case study on three different multi-domain proteins (phosphoglycerate kinase: PGK and two homologous alpha-amylases: TAKA and BLA) we investigated properties of unfolded states and their ability to fold back into the native state. For this purpose guanidine hydrochloride, alkaline pH, and thermal unfolded states were characterized. Structural alterations upon unfolding and refolding transitions were monitored using fluorescence and CD spectroscopy. Static and dynamic light scattering was employed to follow aggregation processes. Furthermore, proper refolding was also investigated by enzyme activity measurements. While for PGK at least partial reversible unfolding transitions were observed in most cases, we found reversible unfolding for TAKA in the case of alkaline pH and GndHCl induced unfolding. BLA exhibits reversible unfolding only under conditions with high concentrations of protecting osmolytes (glycerol), indicating that aggregation of the unfolded state is the main obstacle to achieve proper refolding for this protein. Structural properties, such as number and size of domains, secondary structure contents and compositions within domains, and domain topology were analyzed and considered in the interpretation of differences in refolding behavior of the investigated proteins.     

Kinetics of pH-dependent fusion between 3T3 fibroblasts expressing influenza hemagglutinin and red blood cells. Measurement by dequenching of fluorescence

Fusion between membranes of 3T3 fibroblasts expressing hemagglutinin (HA) from the Japan strain of influenza virus and human red blood cells (RBC) was measured using an assay for lipid mixing based on the relief of self-quenching (dequenching) of fluorescence of the lipid probe octadecylrhodamine (R18). The probe was incorporated into the membrane of intact RBC at self-quenching concentrations, and the RBCs were bound to the 3T3 cells. Fusion, which allowed movement of R18 into 3T3 cell membranes, was monitored by spectrofluorometry as an increase in fluorescence. Upon lowering the pH below 5.4, the fluorescence increased after a delay of about 30 s at 37 degrees C, and leveled off within 2 min. In control experiments where R18 RBCs bound to 3T3 cells expressing the uncleaved precursor hemagglutinin (HA0) were incubated at 37 degrees C and low pH, no fluorescence increase was observed. This indicated that the R18 dequenching occurred as a result of HA-induced fusion of plasma membranes. Fusion showed a very steep pH dependence with a threshold at pH 5.4 and a maximum at pH 5.0, similar to HA-induced fusion seen previously using cell biological techniques. The fusion rate increased and the delay for the onset of fusion decreased as the temperature was raised above 20 degrees C. Low pH activation of the fusion process at 37 degrees C could be partially arrested by raising the pH after 2-10 s, but not after 15 s, indicating that the irreversible pH-activated conformational change of HA necessary for fusion was complete within about 15 s. Analysis of the data indicates that the pH-induced membrane fusion activity of HA is a highly cooperative event.     

Influence of calcium on lipid mixing mediated by influenza hemagglutinin

We studied the influence of calcium on lipid mixing mediated by influenza hemagglutinin (HA). Lipid mixing between HA-expressing cells and liposomes containing disialoganglioside, influenza virus receptor, was studied at 37 degrees C and neutral pH after a low-pH pulse at 4 degrees C. With DSPC/cholesterol liposomes, calcium present after raising the temperature significantly promoted lipid mixing only when it was triggered by a short low-pH application. In case of DOPC/cholesterol liposomes, calcium promotion was observed regardless of the duration of the low-pH pulse. Calcium present during a short, but not long, low-pH application to HA-expressing cells with bound DSPC/cholesterol liposomes at 4 degrees C inhibited subsequent lipid mixing. We hypothesize that calcium influences lipid mixing because it binds to a vestigial esterase domain of hemagglutinin or causes expulsion of the fusion peptide from an electronegative cavity. We suggest that calcium promotes the transition from early and reversible conformation(s) of low pH-activated HA towards an irreversible conformation that underlies both HA-mediated lipid mixing and HA inactivation.     

Temperature-dependent kinetics of the activities of influenza virus

The temperature dependence of membrane interactions between PR8 influenza virus and virus receptor (GD1a)-containing liposomes was studied. For quantitation, the octadecylrhodamine B chloride (R18) membrane marker was incorporated into liposomes at quenched concentrations. Upon interaction with target membranes, the marker gets diluted, and dequenching can be measured in a fluorescence spectrophotometer. Rate constants were calculated from the dequenching curves under low pH conditions, which allow for fusion, and at neutral pH, where no specific fusion occurs. Activation energies were determined from Arrhenius plots. The results were compared with the temperature dependence of other viral activities like infectivity, hemolysis, and fusion with erythrocytes. For the slow reaction at pH 7.4, where only non-specific lipid transfer takes place, the activation energy was about 24 kcal/mole between 15 degrees C and 45 degrees C. For the fast, hemagglutinin (HA)-specific fusion reaction (pH 5.3), a very low activation energy (approximately 7 kcal/mole) was found between 25 degrees C and 37 degrees C, whereas below 25 degrees C it was much higher (approximately 34 kcal/mole). The temperature range with low activation energy coincides with the one for optimal infectivity, hemolysis, and fusion with erythrocytes. Furthermore, it is the same range in which the conformational change of HA takes place, which in the absence of a partner membrane leads to an irreversible inactivation of the fusion protein.