EMDB-8846: Influenza A virus-like particles containing HA-MAY, NA, M1, and M...

Basic information

• Entry: Database: EMDB / ID: EMD-8846
• Title: Influenza A virus-like particles containing HA-MAY, NA, M1, and M2 proteins incubated with liposomes at pH 5 (MAY denotes three amino acids that replace three cysteines in the cytoplasmic tail of hemagglutinin)
• Map data: Influenza A virus-like particles containing HA-MAY, NA, M1, and M2 proteins incubated with liposomes at pH 5 (MAY denotes three amino acids that replace three cysteines in the cytoplasmic tail of hemagglutinin)

Sample

Influenza A virus-like particles containing HA-MAY, NA, M1, and M2 proteins incubated with liposomes at pH 5 != Influenza A virus

Influenza A virus-like particles containing HA-MAY, NA, M1, and M2 proteins incubated with liposomes at pH 5

• Virus: Influenza A virus

• Biological species: 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 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.

History

Deposition	Jul 24, 2017	-
Header (metadata) release	Aug 9, 2017	-
Map release	Aug 9, 2017	-
Update	Oct 25, 2017	-
Current status	Oct 25, 2017	Processing site: RCSB / Status: Released

Map

• File: Download / File: emd_8846.map.gz / Format: CCP4 / Size: 1.2 GB / Type: IMAGE STORED AS SIGNED BYTE
• Annotation: Influenza A virus-like particles containing HA-MAY, NA, M1, and M2 proteins incubated with liposomes at pH 5 (MAY denotes three amino acids that replace three cysteines in the cytoplasmic tail of hemagglutinin)
• Voxel size: X=Y=Z: 7.367 Å

Density

Minimum - Maximum	-128. - 127.
Average (Standard dev.)	21.919567000000001 (±13.273326000000001)

• Symmetry: Space group: 1

Details

EMDB XML:

Map geometry	Axis order X Y Z Origin 0 0 0 Dimensions 2048 2048 315 Spacing 2048 2048 315
Cell	A: 15087.616 Å / B: 15087.616 Å / C: 2320.605 Å α=β=γ: 90.0 °

CCP4 map header:

mode	envelope stored as signed bytes (from -128 lowest to 127 highest)
Å/pix. X/Y/Z	7.367	7.367	7.367
M x/y/z	2048	2048	315
origin x/y/z	0.000	0.000	0.000
length x/y/z	15087.616	15087.616	2320.605
α/β/γ	90.000	90.000	90.000
MAP C/R/S	1	2	3
start NC/NR/NS	0	0	0
NC/NR/NS	2048	2048	315
D min/max/mean	-128.000	127.000	21.920

Supplemental data

Sample components

Entire : Influenza A virus-like particles containing HA-MAY, NA, M1, and M...

• Entire: Name: Influenza A virus-like particles containing HA-MAY, NA, M1, and M2 proteins incubated with liposomes at pH 5

Components

• Virus: Influenza A virus

Supramolecule #1: Influenza A virus

• Supramolecule: Name: Influenza A virus / type: virus / ID: 1 / Parent: 0 / NCBI-ID: 11320 / Sci species name: Influenza A virus / Virus type: VIRUS-LIKE PARTICLE / Virus isolate: OTHER / Virus enveloped: Yes / Virus empty: Yes

Experimental details

Structure determination

• Method: cryo EM
• Processing: electron tomography
• Aggregation state: particle

Sample preparation

• Buffer: pH: 5
• Vitrification: Cryogen name: ETHANE
• Sectioning: Other: NO SECTIONING
• Fiducial marker: Manufacturer: EMS / Diameter: 10 nm

Electron microscopy

• Microscope: FEI TITAN KRIOS
• Image recording: Film or detector model: FEI FALCON II (4k x 4k) / Average electron dose: 1.3 e/Ų
• Electron beam: Acceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
• Electron optics: Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus min: 5.0 µm
• Experimental equipment: Model: Titan Krios / Image courtesy: FEI Company

Image processing

• Final reconstruction: Algorithm: BACK PROJECTION / Software - Name: IMOD / Number images used: 61