|Entry||Database: EMDB / ID: 8843|
|Title||Influenza A virus-like particles containing HA-MAY, NA, M1, and M2 proteins (MAY denotes three amino acids that replace three cysteine residues in the cytoplasmic tail of hemagglutinin)|
|Map data||Influenza A virus-like particles containing HA-MAY, NA, M1, and M2 proteins (MAY denotes three amino acids that replace three cysteine residues in the cytoplasmic tail of hemagglutinin)|
|Sample||Influenza A virus:|
|Source||Influenza A virus|
|Method||electron tomography / cryo EM|
|Authors||Chlanda P / Zimmerberg J|
|Citation||Journal: J. Virol. / Year: 2017|
Title: Palmitoylation Contributes to Membrane Curvature in Influenza A Virus Assembly and Hemagglutinin-Mediated Membrane Fusion.
Authors: Petr Chlanda / Elena Mekhedov / Hang Waters / Alexander Sodt / Cindi Schwartz / Vinod Nair / Paul S Blank / Joshua Zimmerberg
Abstract: The highly conserved cytoplasmic tail of influenza virus glycoprotein hemagglutinin (HA) contains three cysteines, posttranslationally modified by covalently bound fatty acids. While viral HA ...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. 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.
|Date||Deposition: Jul 24, 2017 / Header (metadata) release: Aug 9, 2017 / Map release: Aug 9, 2017 / Last update: Oct 25, 2017|
|Structure viewer||EM map: |
Downloads & links
|File||emd_8843.map.gz (map file in CCP4 format, 568807 KB)|
|Voxel size||X=Y=Z: 7.367 Å|
CCP4 map header:
-Entire Influenza A virus
|Entire||Name: Influenza A virus / Number of components: 1|
-Component #1: virus, Influenza A virus
|Virus||Name: Influenza A virus / Class: VIRUS-LIKE PARTICLE / Empty: Yes / Enveloped: Yes / Isolate: OTHER|
|Species||Species: Influenza A virus|
|Specimen||Specimen state: particle / Method: cryo EM|
|Sample solution||pH: 7|
|Vitrification||Cryogen name: ETHANE|
-Electron microscopy imaging
Model: Titan Krios / Image courtesy: FEI Company
|Imaging||Microscope: FEI TITAN KRIOS|
|Electron gun||Electron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Electron dose: 1.3 e/Å2 / Illumination mode: FLOOD BEAM|
|Lens||Imaging mode: BRIGHT FIELD|
|Specimen Holder||Model: OTHER|
|Camera||Detector: FEI FALCON II (4k x 4k)|
|Processing||Method: electron tomography / Number of sections: 61|
|3D reconstruction||Algorithm: BACK PROJECTION / Software: IMOD|
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