PH-Dependent Mechanisms of Influenza Infection Mediated by Hemagglutinin.
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ABSTRACT:
SUBMITTER: Caffrey M
PROVIDER: S-EPMC8718792 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:During the fusion of the influenza virus to the host cell, bending of the HA2 chain of hemagglutinin into a hairpin-shaped structure in a pH-dependent manner facilitates the fusion of the viral envelope and the endosomal membrane. To characterize the structural and dynamical responses of the hinge region of HA2 to pH changes and examine the role of a conserved histidine in this region (the hinge histidine), we have performed an extensive set of molecular dynamics (MD) simulations of 26-residue peptides encompassing the hinge regions of several hemagglutinin subtypes under both neutral and low pH conditions, modeled by the change of the protonation state of the hinge histidine. More than 70 sets of MD simulations (collectively amounting to 25.1 μs) were performed in both implicit and explicit solvents to study the effect of histidine protonation on structural dynamics of the hinge region. In both explicit and implicit solvent simulations, hinge bending was consistently observed upon the protonation of the histidine in all the simulations starting with an initial straight helical conformation, whereas the systems with a neutral histidine retained their primarily straight conformation throughout the simulations. Conversely, the MD simulations starting from an initially bent conformation resulted in the formation of a straight helical structure upon the neutralization of the hinge histidine, whereas the bent structure was maintained when the hinge histidine remained protonated. Finally, mutation of the hinge histidine to alanine abolishes the bending response of the peptide altogether. A molecular mechanism based on the interaction of the hinge histidine with neighboring acidic residues is proposed to be responsible for its role in controlling the conformation of the hinge. We propose that this might present a common mechanism for pH-controlled structural changes in helical structures when histidines act as the pH sensor.
| S-EPMC3752100 | biostudies-literature
Project description:Bat influenza virus H17N10 represents a distinct lineage of influenza A viruses with gene segments coding for proteins that are homologs of the surface antigens, hemagglutinin (HA) and neuraminidase (NA). Our recent study of the N10 NA homolog revealed an NA-like structure, but with a highly divergent putative active site exhibiting little or no NA activity, and provided strong motivation for performing equivalent structural and functional analyses of the H17 HA protein. The overall structure of the H17 HA homolog from A/little yellow-shouldered bat/Guatemala/060/2010 at 3.18 Å resolution is very similar to other influenza HAs, with a putative receptor-binding site containing some conserved aromatic residues that form the base of the sialic acid binding site. However, the rest of the H17 receptor-binding site differs substantially from the other HA subtypes, including substitution of other conserved residues associated with receptor binding. Significantly, electrostatic potential analyses reveal that this putative receptor-binding site is highly acidic, making it unfavorable to bind any negatively charged sialylated receptors, consistent with the recombinant H17 protein exhibiting no detectable binding to sialylated glycans. Furthermore, the fusion mechanism is also distinct; trypsin digestion with recombinant H17 protein, when exposed to pH 4.0, did not degrade the HA1 and HA2, in contrast to other HAs. These distinct structural features and functional differences suggest that the H17 HA behaves very differently compared with other influenza HAs.
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Project description:Human monoclonal antibodies have been identified which neutralize broad spectra of influenza A or B viruses. Here, we dissect the mechanisms by which such antibodies interfere with infectivity. We distinguish four mechanisms that link the conserved hemagglutinin (HA) epitopes of broadly neutralizing antibodies to critical processes in the viral life cycle. HA-stem binding antibodies can act intracellularly by blocking fusion between the viral and endosomal membranes and extracellularly by preventing the proteolytic activation of HA. HA-head binding antibodies prevent viral attachment and release. These insights into newly identified ways by which the human immune system can interfere with influenza virus infection may aid the development of novel universal vaccines and antivirals.
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Project description:Background. Detection of neutralizing antibodies (nAbs) to influenza A virus hemagglutinin (HA) antigens by conventional serological assays is currently the main immune correlate of protection for influenza vaccines However, current prepandemic avian influenza vaccines are poorly immunogenic in inducing nAbs despite considerable protection conferred. Recent studies show that Ab-dependent cell-mediated cytotoxicity (ADCC) to HA antigens are readily detectable in the sera of healthy individuals and patients with influenza infection. Methods. Virus neutralization and ADCC activities of serum samples from individuals who received either seasonal or a stock-piled H5N1 avian influenza vaccine were evaluated by hemagglutination inhibition assay, microneutralization assay, and an improved ADCC natural killer (NK) cell activation assay. Results. Immunization with inactivated seasonal influenza vaccine led to strong expansion of both nAbs and ADCC-mediating antibodies (adccAbs) to H3 antigen of the vaccine virus in 24 postvaccination human sera. In sharp contrast, 18 individuals vaccinated with the adjuvanted H5N1 avian influenza vaccine mounted H5-specific antibodies with strong ADCC activities despite moderate virus neutralization capacity. Strength of HA-specific ADCC activities is largely associated with the titers of HA-binding antibodies and not with the fine antigenic specificity of anti-HA nAbs. Conclusions. Detection of both nAbs and adccAbs may better reflect protective capacity of HA-specific antibodies induced by avian influenza vaccines.
| S-EPMC4943536 | biostudies-literature
Project description:The hemagglutinin (HA) glycoproteins of influenza viruses play a key role in binding host cell receptors and in mediating virus-host cell membrane fusion during virus infection. Upon virus entry, HA is triggered by low pH and undergoes large structural rearrangements from a prefusion state to a postfusion state. While structures of prefusion state and postfusion state of HA have been reported, the intermediate structures remain elusive. Here, we report two distinct low pH intermediate conformations of the influenza virus HA using cryo-electron microscopy (cryo-EM). Our results show that a decrease in pH from 7.8 to 5.2 triggers the release of fusion peptides from the binding pockets and then causes a dramatic conformational change in the central helices, in which the membrane-proximal ends of the central helices unwind to an extended form. Accompanying the conformational changes of the central helices, the stem region of the HA undergoes an anticlockwise rotation of 9.5 degrees and a shift of 15 Å. The HA head, after being stabilized by an antibody, remains unchanged compared to the neutral pH state. Thus, the conformational change of the HA stem region observed in our research is likely to be independent of the HA head. These results provide new insights into the structural transition of HA during virus entry.
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Project description:The subunits of the influenza hemagglutinin (HA) trimer are synthesized as single-chain precursors (HA0s) that are proteolytically cleaved into the disulfide-linked polypeptides HA1 and HA2. Cleavage is required for activation of membrane fusion at low pH, which occurs at the beginning of infection following transfer of cell-surface-bound viruses into endosomes. Activation results in extensive changes in the conformation of cleaved HA. To establish the overall contribution of cleavage to the mechanism of HA-mediated membrane fusion, we used cryogenic electron microscopy (cryo-EM) to directly image HA0 at neutral and low pH. We found extensive pH-induced structural changes, some of which were similar to those described for intermediates in the refolding of cleaved HA at low pH. They involve a partial extension of the long central coiled coil formed by melting of the preexisting secondary structure, threading it between the membrane-distal domains, and subsequent refolding as extended helices. The fusion peptide, covalently linked at its N terminus, adopts an amphipathic helical conformation over part of its length and is repositioned and packed against a complementary surface groove of conserved residues. Furthermore, and in contrast to cleaved HA, the changes in HA0 structure at low pH are reversible on reincubation at neutral pH. We discuss the implications of covalently restricted HA0 refolding for the cleaved HA conformational changes that mediate membrane fusion and for the action of antiviral drug candidates and cross-reactive anti-HA antibodies that can block influenza infectivity.
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Project description: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.
| S-EPMC7111647 | biostudies-literature
Project description: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.
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Project description:Enveloped viruses enter cells via a process of membrane fusion between the viral envelope and a cellular membrane. For influenza virus, mutational data have shown that the membrane-inserted portions of the hemagglutinin protein play a critical role in achieving fusion. In contrast to the relatively well-understood ectodomain, a predictive mechanistic understanding of the intramembrane mechanisms by which influenza hemagglutinin drives fusion has been elusive. We used molecular dynamics simulations of fusion between a full-length hemagglutinin proteoliposome and a lipid bilayer to analyze these mechanisms. In our simulations, hemagglutinin first acts within the membrane to increase lipid tail protrusion and promote stalk formation and then acts to engage the distal leaflets of each membrane and promote stalk widening, curvature, and eventual fusion. These two sequential mechanisms, one occurring before stalk formation and one after, are consistent with our experimental measurements of single-virus fusion kinetics to liposomes of different sizes. The resulting model also helps explain and integrate previous mutational and biophysical data, particularly the mutational sensitivity of the fusion peptide N terminus and the length sensitivity of the transmembrane domain. We hypothesize that entry by other enveloped viruses may also use sequential processes of acyl tail exposure, followed by membrane curvature and distal leaflet engagement.
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Project description:Broadly neutralizing antibodies against highly variable pathogens have stimulated the design of vaccines and therapeutics. We report the use of diverse camelid single-domain antibodies to influenza virus hemagglutinin to generate multidomain antibodies with impressive breadth and potency. Multidomain antibody MD3606 protects mice against influenza A and B infection when administered intravenously or expressed locally from a recombinant adeno-associated virus vector. Crystal and single-particle electron microscopy structures of these antibodies with hemagglutinins from influenza A and B viruses reveal binding to highly conserved epitopes. Collectively, our findings demonstrate that multidomain antibodies targeting multiple epitopes exhibit enhanced virus cross-reactivity and potency. In combination with adeno-associated virus-mediated gene delivery, they may provide an effective strategy to prevent infection with influenza virus and other highly variable pathogens.
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