EMDB-15234: Structure of self-assembling engineered protein nanocage (EPN) fu...

Basic information

Entry

Database: EMDB / ID: EMD-15234

• Title: Structure of self-assembling engineered protein nanocage (EPN) fused with hepatitis A pX protein

Sample

• Complex: EPN-pX
  • Protein or peptide: EPN-pX

• Function / homology: KDPG/KHG aldolase / KDPG and KHG aldolase / Viral coat protein subunit / Aldolase-type TIM barrel / lyase activity / symbiont entry into host cell / virion attachment to host cell / 2-dehydro-3-deoxyphosphogluconate aldolase/4-hydroxy-2-oxoglutarate aldolase / Genome polyprotein
• Biological species: Homo sapiens (human) / Human immunodeficiency virus type 1 BH10
• Method: single particle reconstruction / cryo EM / Resolution: 6.7 Å
• Authors: Duyvesteyn HME / Stuart DI

Funding support

United States, United Kingdom, 6 items

Organization	Grant number	Country
National Institutes of Health/National Cancer Institute (NIH/NCI)	R01-AI103083	United States
National Institutes of Health/National Cancer Institute (NIH/NCI)	R01-AI131685	United States
National Institutes of Health/National Cancer Institute (NIH/NCI)	R01-AI088255	United States
National Institutes of Health/National Cancer Institute (NIH/NCI)	R01-AI150095	United States
Wellcome Trust	ALR00750	United Kingdom
Medical Research Council (MRC, United Kingdom)	MR/N00065X/1	United Kingdom

• Citation: Journal: PLoS Pathog / Year: 2022; Title: Nonlytic cellular release of hepatitis A virus requires dual capsid recruitment of the ESCRT-associated Bro1 domain proteins HD-PTP and ALIX.; Authors: Takayoshi Shirasaki / Hui Feng / Helen M E Duyvesteyn / William G Fusco / Kevin L McKnight / Ling Xie / Mark Boyce / Sathish Kumar / Rina Barouch-Bentov / Olga González-López / Ryan McNamara / Li Wang / Adriana Hertel-Wulff / Xian Chen / Shirit Einav / Joseph A Duncan / Maryna Kapustina / Elizabeth E Fry / David I Stuart / Stanley M Lemon / ; Abstract: Although picornaviruses are conventionally considered 'nonenveloped', members of multiple picornaviral genera are released nonlytically from infected cells in extracellular vesicles. The mechanisms underlying this process are poorly understood. Here, we describe interactions of the hepatitis A virus (HAV) capsid with components of host endosomal sorting complexes required for transport (ESCRT) that play an essential role in release. We show release of quasi-enveloped virus (eHAV) in exosome-like vesicles requires a conserved export signal located within the 8 kDa C-terminal VP1 pX extension that functions in a manner analogous to late domains of canonical enveloped viruses. Fusing pX to a self-assembling engineered protein nanocage (EPN-pX) resulted in its ESCRT-dependent release in extracellular vesicles. Mutational analysis identified a 24 amino acid peptide sequence located within the center of pX that was both necessary and sufficient for nanocage release. Deleting a YxxL motif within this sequence ablated eHAV release, resulting in virus accumulating intracellularly. The pX export signal is conserved in non-human hepatoviruses from a wide range of mammalian species, and functional in pX sequences from bat hepatoviruses when fused to the nanocage protein, suggesting these viruses are released as quasi-enveloped virions. Quantitative proteomics identified multiple ESCRT-related proteins associating with EPN-pX, including ALG2-interacting protein X (ALIX), and its paralog, tyrosine-protein phosphatase non-receptor type 23 (HD-PTP), a second Bro1 domain protein linked to sorting of ubiquitylated cargo into multivesicular endosomes. RNAi-mediated depletion of either Bro1 domain protein impeded eHAV release. Super-resolution fluorescence microscopy demonstrated colocalization of viral capsids with endogenous ALIX and HD-PTP. Co-immunoprecipitation assays using biotin-tagged peptides and recombinant proteins revealed pX interacts directly through the export signal with N-terminal Bro1 domains of both HD-PTP and ALIX. Our study identifies an exceptionally potent viral export signal mediating extracellular release of virus-sized protein assemblies and shows release requires non-redundant activities of both HD-PTP and ALIX.

History

Deposition	Jun 23, 2022	-
Header (metadata) release	Aug 10, 2022	-
Map release	Aug 10, 2022	-
Update	Aug 31, 2022	-
Current status	Aug 31, 2022	Processing site: PDBe / Status: Released

Map

• File: Download / File: emd_15234.map.gz / Format: CCP4 / Size: 244.1 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
• Voxel size: X=Y=Z: 1.54 Å

Density

Contour Level	By AUTHOR: 0.0402
Minimum - Maximum	-0.07111076 - 0.2850455
Average (Standard dev.)	0.00017298825 (±0.011541795)

• Symmetry: Space group: 1

Details

EMDB XML:

Map geometry	Axis order X Y Z Origin 0 0 0 Dimensions 400 400 400 Spacing 400 400 400
Cell	A=B=C: 616.0 Å α=β=γ: 90.0 °

Supplemental data

Half map: #1

• File: emd_15234_half_map_1.map

Half map: #2

• File: emd_15234_half_map_2.map

Sample components

Entire : EPN-pX

• Entire: Name: EPN-pX

Components

• Complex: EPN-pX
  • Protein or peptide: EPN-pX

Supramolecule #1: EPN-pX

• Supramolecule: Name: EPN-pX / type: complex / Chimera: Yes / ID: 1 / Parent: 0 / Macromolecule list: all; Details: Associated model just contains EPN-01 component since associated tag and pX could not be confidentally resolved.
• Source (natural): Organism: Homo sapiens (human)
• Recombinant expression: Organism: Homo sapiens (human) / Recombinant cell: HEK293T

Macromolecule #1: EPN-pX

• Macromolecule: Name: EPN-pX / type: protein_or_peptide / ID: 1 ; Details: Model just contains EPN-01 component since associated tag and pX could not be confidentally resolved.,Model just contains EPN-01 component since associated tag and pX could not be confidentally resolved.; Number of copies: 1 / Enantiomer: LEVO
• Source (natural): Organism: Human immunodeficiency virus type 1 BH10
• Molecular weight: Theoretical: 32.934234 KDa
• Recombinant expression: Organism: Homo sapiens (human)

Sequence

String:

MGARASGSKS GSGSDSGSKI EELFKKHKIV AVLRANSVEE AKKKALAVFL GGVHLIEITF TVPDADTVIK ELSFLKEMGA IIGAGTVTS VEQCRKAVES GAEFIVSPHL DEEISQFCKE KGVFYMPGVM TPTELVKAMK LGHTILKLFP GEVVGPQFVK A MKGPFPNV KFVPTGGVNL DNVCEWFKAG VLAVGVGSAL VKGTPVEVAE KAKAFVEKIR GCTEQKLISE EDLMMSRIAA GD LESSVDD PRSEEDKRFE SHIECRKPYK ELRLEVGKQR LKYAQEELSN EVLPPPRKMK GLFSQ

Experimental details

Structure determination

• Method: cryo EM
• Processing: single particle reconstruction
• Aggregation state: particle

Sample preparation

• Buffer: pH: 7 / Details: PBS
• Grid: Model: Quantifoil R2/2 / Material: COPPER / Mesh: 200 / Pretreatment - Type: GLOW DISCHARGE
• Vitrification: Cryogen name: ETHANE / Chamber humidity: 90 % / Chamber temperature: 293 K / Instrument: GATAN CRYOPLUNGE 3 / Details: Blot time 4-5 s..

Electron microscopy

• Microscope: TFS GLACIOS
• Image recording: Film or detector model: FEI FALCON III (4k x 4k) / Detector mode: INTEGRATING / Number grids imaged: 1 / Number real images: 1803 / Average electron dose: 46.0 e/Ų
• Electron beam: Acceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
• Electron optics: C2 aperture diameter: 50.0 µm / Illumination mode: OTHER / Imaging mode: BRIGHT FIELD / Nominal defocus max: 3.0 µm / Nominal defocus min: 0.7000000000000001 µm / Nominal magnification: 92000
• Sample stage: Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN

Image processing

• Particle selection: Number selected: 1830 / Details: Manual selection.
• CTF correction: Software - Name: CTFFIND

Startup model

Type of model: PDB ENTRY
PDB model - PDB ID:

5kp9

• Final reconstruction: Applied symmetry - Point group: I (icosahedral) / Resolution.type: BY AUTHOR / Resolution: 6.7 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION / Number images used: 1830
• Initial angle assignment: Type: MAXIMUM LIKELIHOOD / Software - Name: RELION
• Final angle assignment: Type: MAXIMUM LIKELIHOOD / Software - Name: RELION
• Final 3D classification: Software - Name: RELION

Atomic model buiding 1

Initial model

(PDB ID:

5kp9

,

7b3y

)

• Details: Rigid body fit monomer in chimera, generated biological unit then checked in coot and residues to be altered done so in coot followed by one cycle of rigid body refinement of entire icosahedral assembly in phenix, defining each monomeric unit as a separate body and considering ncs constraints to check for clashes.
• Refinement: Space: REAL / Protocol: RIGID BODY FIT / Target criteria: Correlation coefficient

Output model

PDB-8a8n:
Structure of self-assembling engineered protein nanocage (EPN) fused with hepatitis A pX protein