Influenza hemagglutinin membrane anchor.
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ABSTRACT:
SUBMITTER: Benton DJ
PROVIDER: S-EPMC6176637 | biostudies-literature | 2018 Oct
REPOSITORIES: biostudies-literature
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Project description:Membrane fusion is the critical step for infectious cell penetration by enveloped viruses. We have previously used single-virion measurements of fusion kinetics to study the molecular mechanism of influenza-virus envelope fusion. Published data on fusion inhibition by antibodies to the 'stem' of influenza virus hemagglutinin (HA) now allow us to incorporate into simulations the provision that some HAs are inactive. We find that more than half of the HAs are unproductive even for virions with no bound antibodies, but that the overall mechanism is extremely robust. Determining the fraction of competent HAs allows us to determine their rates of target-membrane engagement. Comparison of simulations with data from H3N2 and H1N1 viruses reveals three independent functional variables of HA-mediated membrane fusion closely linked to neutralization susceptibility. Evidence for compensatory changes in the evolved mechanism sets the stage for studies aiming to define the molecular constraints on HA evolvability.
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Project description:The influenza viral membrane protein hemagglutinin (HA) is required at high concentrations on virion and host-cell membranes for infectivity. Because the role of actin in membrane organization is not completely understood, we quantified the relationship between HA and host-cell actin at the nanoscale. Results obtained using superresolution fluorescence photoactivation localization microscopy (FPALM) in nonpolarized cells show that HA clusters colocalize with actin-rich membrane regions (ARMRs). Individual molecular trajectories in live cells indicate restricted HA mobility in ARMRs, and actin disruption caused specific changes to HA clustering. Surprisingly, the actin-binding protein cofilin was excluded from some regions within several hundred nanometers of HA clusters, suggesting that HA clusters or adjacent proteins within the same clusters influence local actin structure. Thus, with the use of imaging, we demonstrate a dynamic relationship between glycoprotein membrane organization and the actin cytoskeleton at the nanoscale.
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Project description:Hemagglutinin (HA) is a protein located on the surface of the influenza virus that mediates viral fusion to the host cellular membrane. During the fusion process the HA fusion peptide (HAfp), formed by the first 23 N-terminal residues of HA and structurally characterized by two alpha helices (Helix A and Helix B) tightly packed in a hairpin-like arrangement, is the only part of the virus in direct contact with the host membrane. After encountering the host cell, HAfp is believed to insert into the membrane, thereby initiating the fusion of the viral and host membranes. Detailed characterization of the interactions between the HAfp and cellular membrane is therefore of high relevance to the mechanism of viral entry into the host cell. Employing HMMM membrane representation with enhanced lipid mobility, we have performed a large set of independent simulations of unbiased membrane binding of HAfp. We have been able to capture spontaneous binding and insertion of HAfp consistently in nearly all the simulations. A reproducible membrane-bound configuration emerges from these simulations, despite employing a diverse set of initial configurations. Extension of several of the simulations into full membrane systems confirms the stability of the membrane-bound form obtained from HMMM binding simulations. The resulting model allows for the characterization of important interactions between the peptide and the membrane and the details of the binding process of the peptide for the first time. Upon membrane binding, Helix A inserts much deeper into the membrane than Helix B, suggesting that the former is responsible for hydrophobic anchoring of the peptide into the membrane. Helix B, in contrast, is found to establish major amphipathic interactions at the interfacial region thereby contributing to binding strength of HAfp.
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Project description:The highly conserved cytoplasmic tail of influenza virus glycoprotein hemagglutinin (HA) contains three cysteines, posttranslationally modified by covalently bound fatty acids. While viral HA acylation is crucial in virus replication, its physico-chemical role is unknown. We used virus-like particles (VLP) to study the effect of acylation on morphology, protein incorporation, lipid composition, and membrane fusion. Deacylation interrupted HA-M1 interactions since deacylated mutant HA failed to incorporate an M1 layer within spheroidal VLP, and filamentous particles incorporated increased numbers of neuraminidase (NA). While HA acylation did not influence VLP shape, lipid composition, or HA lateral spacing, acylation significantly affected envelope curvature. Compared to wild-type HA, deacylated HA is correlated with released particles with flat envelope curvature in the absence of the matrix (M1) protein layer. The spontaneous curvature of palmitate was calculated by molecular dynamic simulations and was found to be comparable to the curvature values derived from VLP size distributions. Cell-cell fusion assays show a strain-independent failure of fusion pore enlargement among H2 (A/Japan/305/57), H3 (A/Aichi/2/68), and H3 (A/Udorn/72) viruses. In contradistinction, acylation made no difference in the low-pH-dependent fusion of isolated VLPs to liposomes: fusion pores formed and expanded, as demonstrated by the presence of complete fusion products observed using cryo-electron tomography (cryo-ET). We propose that the primary mechanism of action of acylation is to control membrane curvature and to modify HA's interaction with M1 protein, while the stunting of fusion by deacylated HA acting in isolation may be balanced by other viral proteins which help lower the energetic barrier to pore expansion.IMPORTANCE Influenza A virus is an airborne pathogen causing seasonal epidemics and occasional pandemics. Hemagglutinin (HA), a glycoprotein abundant on the virion surface, is important in both influenza A virus assembly and entry. HA is modified by acylation whose removal abrogates viral replication. Here, we used cryo-electron tomography to obtain three-dimensional images to elucidate a role for HA acylation in VLP assembly. Our data indicate that HA acylation contributes to the capability of HA to bend membranes and to HA's interaction with the M1 scaffold protein during virus assembly. Furthermore, our data on VLP and, by hypothesis, virus suggests that HA acylation, while not critical to fusion pore formation, contributes to pore expansion in a target-dependent fashion.
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Project description:Our previous studies showed that an angled boomerang-shaped structure of the influenza hemagglutinin (HA) fusion domain is critical for virus entry into host cells by membrane fusion. Because the acute angle of ?105° of the wild-type fusion domain promotes efficient non-leaky membrane fusion, we asked whether different angles would still support fusion and thus facilitate virus entry. Here, we show that the G13A fusion domain mutant produces a new leaky fusion phenotype. The mutant fusion domain structure was solved by NMR spectroscopy in a lipid environment at fusion pH. The mutant adopted a boomerang structure similar to that of wild type but with a shallower kink angle of ?150°. G13A perturbed the structure of model membranes to a lesser degree than wild type but to a greater degree than non-fusogenic fusion domain mutants. The strength of G13A binding to lipid bilayers was also intermediate between that of wild type and non-fusogenic mutants. These membrane interactions provide a clear link between structure and function of influenza fusion domains: an acute angle is required to promote clean non-leaky fusion suitable for virus entry presumably by interaction of the fusion domain with the transmembrane domain deep in the lipid bilayer. A shallower angle perturbs the bilayer of the target membrane so that it becomes leaky and unable to form a clean fusion pore. Mutants with no fixed boomerang angle interacted with bilayers weakly and did not promote any fusion or membrane perturbation.
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Project description:Hemagglutinin (HA) plays an important role in the first step of influenza virus (IFV) infection because it initiates the binding of the virus to the sialylgalactose linkages of the receptors on the host cells. We herein demonstrate that a HA-binding peptide immobilized on a solid support is available to bind to HA and IFV. We previously obtained a HA-binding pentapeptide (Ala-Arg-Leu-Pro-Arg), which was identified by phage-display selection against HAs from random peptide libraries. This peptide binds to the receptor-binding site of HA by mimicking sialic acid. A peptide-conjugated lipid (pep-PE) was chemically synthesized from the peptide and a saturated phospholipid. A lipid bilayer composed of pep-PE and an unsaturated phospholipid (DOPC) was immobilized on a mica plate; and the interaction between HA and the pep-PE/DOPC membrane was investigated using atomic force microscopy. The binding of IFV to the pep-PE/DOPC membrane was detected by an enzyme-linked immunosorbent assay and real-time reverse transcription PCR. Our results indicate that peptide-conjugated lipids are a useful molecular device for the detection of HA and IFV.
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Project description:The influenza surface glycoprotein hemagglutinin (HA) is a potential target for antiviral drugs because of its key roles in the initial stages of infection: receptor binding and the fusion of virus and cell membranes. The structure of HA in complex with a known inhibitor of membrane fusion and virus infectivity, tert-butyl hydroquinone (TBHQ), shows that the inhibitor binds in a hydrophobic pocket formed at an interface between HA monomers. Occupation of this site by TBHQ stabilizes the neutral pH structure through intersubunit and intrasubunit interactions that presumably inhibit the conformational rearrangements required for membrane fusion. The nature of the binding site suggests routes for the chemical modification of TBHQ that could lead to the development of more potent inhibitors of membrane fusion and potential anti-influenza drugs.
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Project description:Membrane fusion is involved in many fundamental cellular processes and entry of enveloped viruses into host cells. Influenza type A virus HA has long served as a paradigm for mechanistic studies of protein-mediated membrane fusion via large-scale structural rearrangements induced by acidic pH. Here we report the newly determined crystal structure of influenza B virus HA2 in the postfusion state. Together with a large number of previously determined prefusion structures of influenza A and B virus HA and a postfusion structure of influenza A/H3N2 HA2, we identified conserved features that are shared between influenza A and B virus HA in the conformational transition and documented substantial differences that likely influence the detailed mechanisms of this process. Further studies are needed to dissect the effects of these and other structural differences in HA conformational changes and influenza pathogenicity and transmission, which may ultimately expedite the discovery of novel anti-influenza fusion inhibitors.
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Project description:Influenza A virus (IAV) mutates rapidly, preventing a single seasonal vaccine from targeting more than one strain, and year-to-year vaccine effectiveness is low. One challenge in designing effective vaccines is that genetic mutations frequently cause amino acid variations in IAV envelope protein hemagglutinin (HA) that create new N-glycosylation sequons; resulting N-glycans cause antigenic shielding, allowing viral escape from adaptive immune responses. Vaccine candidate strain selection currently involves correlating antigenicity with HA protein sequence among circulating strains, but quantitative comparison of site-specific glycosylation information is likely to improve the ability to design vaccines with broader effectiveness against evolved strains. However, there is poor understanding of the influence of glycosylation on immunodominance, antigenicity, and immunogenicity of HA, and there are no well-tested methods for comparing glycosylation similarity among virus samples. Here, we present a method for statistically rigorous quantification of similarity between two related virus strains that considers the presence and abundance of glycopeptide glycoforms. We demonstrate the strength of our approach by determining that there was a quantifiable difference in glycosylation at the protein level between wild-type IAV HA from A/Switzerland/9715293/2013 (SWZ13) and a mutant strain of SWZ13, even though no N-glycosylation sequons were changed. We determined site-specifically that WT and mutant HA have varying similarity at the glycosylation sites of the head domain, reflecting competing pressures to evade host immune response while retaining viral fitness. To our knowledge, our results are the first to quantify changes in glycosylation state that occur in related proteins of considerable glycan heterogeneity. Our results provide a method for understanding how changes in glycosylation state are correlated with variations in protein sequence, which is necessary for improving IAV vaccine strain selection. Understanding glycosylation will be especially important as we find new expression vectors for vaccine production, as glycosylation state depends greatly on the host species.
2020-07-03 | PXD018920 | Pride
Project description:Influenza virus penetrates cells by fusion of viral and endosomal membranes catalyzed by the viral hemagglutinin (HA). Structures of the initial and final states of the HA trimer define the fusion endpoints, but do not specify intermediates. We have characterized these transitions by analyzing low-pH-induced fusion kinetics of individual virions and validated the analysis by computer simulation. We detect initial engagement with the target membrane of fusion peptides from independently triggered HAs within the larger virus-target contact patch; fusion then requires engagement of three or four neighboring HA trimers. Effects of mutations in HA indicate that withdrawal of the fusion peptide from a pocket in the pre-fusion trimer is rate-limiting for both events, but the requirement for cooperative action of several HAs to bring the fusing membranes together leads to a long-lived intermediate state for single, extended HA trimers. This intermediate is thus a fundamental aspect of the fusion mechanism. DOI:http://dx.doi.org/10.7554/eLife.00333.001.
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