Influenza hemagglutinin membrane anchor

Benton, D.J.; Nans, Andrea; Calder, Lesley J.; Turner, Jack; Neu, Ursula; Lin, Yi Pu; Ketelaars, Esther; Kallewaard, Nicole L.; Corti, Davide; Lanzavecchia, Antonio; Gamblin, S.J.; Rosenthal, Peter B.; Skehel, J.J.

doi:10.1073/pnas.1810927115

Cited by 117 publications

(145 citation statements)

References 59 publications

(66 reference statements)

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“…This is followed by formation of 6HB and membrane fusion. Flexibility at the juxtamembrane region and the tilted orientation against the viral membrane required for membrane fusion were recently reported by a study examining influenza HA in detergent micelles analyzed by cryoEM (45). We anticipate that the mechanism suggested by Western blot analysis here will be confirmed by other approaches such as high-resolution cryoEM and/or in silico simulation.…”

Section: Discussionsupporting

confidence: 70%

Biochemical Analysis of Coronavirus Spike Glycoprotein Conformational Intermediates during Membrane Fusion

Kawase

Kataoka

Shirato

et al. 2019

J Virol

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A fusion protein expressed on the surface of enveloped viruses mediates fusion of the viral and cellular membranes to facilitate virus infection. Pre-and postfusion structures of viral fusion proteins have been characterized, but conformational changes between them remain poorly understood. Here, we examined the intermediate conformation of the murine coronavirus fusion protein, called the spike protein, which must be cleaved by a cellular protease following receptor binding. Western blot analysis of protease digestion products revealed that two subunits (67 and 69 kDa) are produced from a single spike protein (180 kDa). These two subunits were considered to be by-products derived from conformational changes and were useful for probing the intermediate conformation of the spike protein. Interaction with a heptad repeat (HR) peptide revealed that these subunits adopt packed and unpacked conformations, respectively, and two-dimensional electrophoresis revealed a trimeric assembly. Based on biochemical observations, we propose an asymmetric trimer model for the intermediate structure of the spike protein. Receptor binding induces the membrane-binding potential of the trimer, in which at least one HR motif forms a packed-hairpin structure, while membrane fusion subunits are covered by the receptor-binding subunit, thereby preventing the spike protein from forming the typical homotrimeric prehairpin structure predicted by the current model of class I viral fusion protein. Subsequent proteolysis induces simultaneous packing of the remaining unpacked HRs upon assembly of three HRs at the central axis to generate a six-helix bundle. Our model proposes a key mechanism for membrane fusion of enveloped viruses. IMPORTANCE Recent studies using single-particle cryo-electron microscopy (cryoEM) revealed the mechanism underlying activation of viral fusion protein at the priming stage. However, characterizing the subsequent triggering stage underpinning transition from pre-to postfusion structures is difficult because single-particle cryoEM excludes unstable structures that appear as heterogeneous shapes. Therefore, population-based biochemical analysis is needed to capture features of unstable proteins. Here, we analyzed protease digestion products of a coronavirus fusion protein during activation; their sizes appear to be affected directly by the conformational state. We propose a model for the viral fusion protein in the intermediate state, which involves a compact structure and conformational changes that overcome steric hindrance within the three fusion protein subunits.

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Section: Discussionsupporting

confidence: 70%

Biochemical Analysis of Coronavirus Spike Glycoprotein Conformational Intermediates during Membrane Fusion

Kawase

Kataoka

Shirato

et al. 2019

J Virol

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“…Interestingly, a tilted orientation of TMDs could be determined in micelles with a crossing point at conserved residue R696. Influenza hemagglutinin was shown to have similar TMD dynamics with tilted and straight orientations, suggesting that these could be common type I viral fusion proteins TMD topologies (Benton et al, 2018). The arrangement of helices shown here for HIV Env is different than the three-helix bundle topology observed in NMR structures of the TM helices alone (Chen and Chou, 2017;Chiliveri et al, 2018;Dev et al, 2016;Hollingsworth et al, 2018;Kwon et al, 2018a).…”

Section: Discussionmentioning

confidence: 60%

HIV-1 Envelope and MPER antibody structures in lipid assemblies

Rantalainen

Berndsen

Antanasijevic

et al. 2019

Preprint

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SummaryStructural and functional studies of HIV Env as a transmembrane protein have long been complicated by challenges associated with inherent flexibility of the molecule and the membrane-embedded hydrophobic regions. Thus, most structural studies have utilized soluble forms where the regions C-terminal to the ectodomain are deleted. Here, we present approaches for incorporating full-length, wild-type HIV-1 Env, as well as C-terminally truncated and stabilized versions, into lipid assemblies, providing a modular platform for Env structural studies by single particle electron microscopy. We reconstituted a full-length Env clone into a nanodisc with MSP1D1 scaffold, complexed it with an MPER targeting antibody 10E8, and structurally defined the full quaternary epitope of 10E8 consisting of lipid, MPER and ectodomain contacts. By aligning this and other Env-MPER antibody complex reconstructions with the lipid bilayer, we observe evidence of Env tilting as part of the neutralization mechanism for MPER-targeting antibodies. We also adapted the platform toward vaccine design purposes by introducing stabilizing mutations that allow purification of unliganded Env with peptidisc scaffold.

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“…None of the classes gave density that could be fit with a single MPER-TM model, however. The corresponding linkage in influenza virus hemagglutinin is also flexible, but in that case, classification yielded a reasonable map for at least two different orientations of the TM segments relative to the ectodomain [34]. We could nonetheless introduce the model of a bicelle-embedded MPER-TM structure (PDB ID: 6E8W), determined by NMR [28], by aligning its known axial register with respect to phospholipid headgroups with the headgroup density in Env structures derived from cryo-electron tomography and subtomogram averaging [13] ( Fig.…”

Section: Model Buildingmentioning

confidence: 99%

Cryo-EM structure of full-length HIV-1 Env bound with the Fab of antibody PG16

Pan¹,

Peng²,

Chen³

2019

Preprint

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The HIV-1 envelope protein (Env) is the target of neutralizing antibodies and the template for vaccine immunogen design. The dynamic conformational equilibrium of trimeric Env influences its antigenicity and potential immunogenicity. Antibodies that bind at the trimer apex stabilize a "closed" conformation characteristic of the most difficult to neutralize isolates. A goal of vaccine development is therefore to mimic the closed conformation in a designed immunogen. A disulfide-stabilized, trimeric Env ectodomain --the "SOSIP" construct --has many of the relevant properties; it is also particularly suitable for structure determination. Some singlemolecule studies have, however, suggested that the SOSIP trimer is not a good representation of Env on the surface of a virion or an infected cell. We isolated Env (fully cleaved to gp120 and gp41) from the surface of expressing cells using tagged, apex-binding Fab PG16 and determined the structure of the PG16-Env complex by cryo-EM to an overall resolution of 4.6 Å.Placing the only purification tag on the Fab ensured that the isolated Env was continuously stabilized in its closed, native conformation. The Env structure in this complex corresponds closely to the SOSIP structures determined by both x-ray crystallography and cryo-EM.Although the membrane-interacting elements are not resolved in our reconstruction, we can make inferences about the connection between ectodomain and membrane-proximal external region (MPER) by reference to the published cryo-tomography structure of an Env "spike" and the NMR structure of the MPER-transmembrane segment. We discuss these results in view of the conflicting interpretations in the literature.

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Influenza hemagglutinin membrane anchor

Cited by 117 publications

References 59 publications

Biochemical Analysis of Coronavirus Spike Glycoprotein Conformational Intermediates during Membrane Fusion

Biochemical Analysis of Coronavirus Spike Glycoprotein Conformational Intermediates during Membrane Fusion

HIV-1 Envelope and MPER antibody structures in lipid assemblies

Cryo-EM structure of full-length HIV-1 Env bound with the Fab of antibody PG16

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