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- PDB-8a8n: Structure of self-assembling engineered protein nanocage (EPN) fu... -
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Open data
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Basic information
Entry | Database: PDB / ID: 8a8n | |||||||||||||||||||||
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Title | Structure of self-assembling engineered protein nanocage (EPN) fused with hepatitis A pX protein | |||||||||||||||||||||
![]() | EPN-pX | |||||||||||||||||||||
![]() | STRUCTURAL PROTEIN / hepatitis A / pX / VP1-pX / nanocage / icosahedral / protein nanoparticle / virus assembly / enveloped viruses | |||||||||||||||||||||
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 | ![]() | |||||||||||||||||||||
Method | ELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 6.7 Å | |||||||||||||||||||||
![]() | Duyvesteyn, H.M.E. / Stuart, D.I. | |||||||||||||||||||||
Funding support | ![]() ![]()
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![]() | ![]() 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 ...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 ...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 |
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Structure visualization
Structure viewer | Molecule: ![]() ![]() |
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Downloads & links
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Download
PDBx/mmCIF format | ![]() | 48.9 KB | Display | ![]() |
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PDB format | ![]() | 34.3 KB | Display | ![]() |
PDBx/mmJSON format | ![]() | Tree view | ![]() | |
Others | ![]() |
-Validation report
Arichive directory | ![]() ![]() | HTTPS FTP |
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-Related structure data
Related structure data | ![]() 15234MC M: map data used to model this data C: citing same article ( |
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Similar structure data | Similarity search - Function & homology ![]() |
Experimental dataset #1 | Data reference: ![]() |
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Links
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Assembly
Deposited unit | ![]()
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Components
#1: Protein | Mass: 32934.234 Da / Num. of mol.: 1 Source method: isolated from a genetically manipulated source 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. Source: (gene. exp.) ![]() Cell line (production host): HEK293T / Production host: ![]() |
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-Experimental details
-Experiment
Experiment | Method: ELECTRON MICROSCOPY |
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EM experiment | Aggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction |
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Sample preparation
Component | Name: EPN-pX / Type: COMPLEX Details: Associated model just contains EPN-01 component since associated tag and pX could not be confidentally resolved. Entity ID: all / Source: RECOMBINANT |
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Molecular weight | Experimental value: NO |
Source (natural) | Organism: ![]() |
Source (recombinant) | Organism: ![]() |
Buffer solution | pH: 7 / Details: PBS |
Specimen | Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES |
Specimen support | Grid material: COPPER / Grid mesh size: 200 divisions/in. / Grid type: Quantifoil R2/2 |
Vitrification | Instrument: GATAN CRYOPLUNGE 3 / Cryogen name: ETHANE / Humidity: 90 % / Chamber temperature: 293 K / Details: Blot time 4-5 s. |
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Electron microscopy imaging
Microscopy | Model: TFS GLACIOS |
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Electron gun | Electron source: ![]() |
Electron lens | Mode: BRIGHT FIELD / Nominal magnification: 92000 X / Nominal defocus max: 3000 nm / Nominal defocus min: 700 nm / C2 aperture diameter: 50 µm |
Specimen holder | Cryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER |
Image recording | Electron dose: 46 e/Å2 / Detector mode: INTEGRATING / Film or detector model: FEI FALCON III (4k x 4k) / Num. of grids imaged: 1 / Num. of real images: 1803 |
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Processing
EM software |
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CTF correction | Type: PHASE FLIPPING AND AMPLITUDE CORRECTION | |||||||||||||||||||||||||||||||||
Particle selection | Num. of particles selected: 1830 / Details: Manual selection. | |||||||||||||||||||||||||||||||||
Symmetry | Point symmetry: I (icosahedral) | |||||||||||||||||||||||||||||||||
3D reconstruction | Resolution: 6.7 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 1830 / Symmetry type: POINT | |||||||||||||||||||||||||||||||||
Atomic model building | Protocol: RIGID BODY FIT / Space: REAL / Target criteria: Correlation coefficient 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 ...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. | |||||||||||||||||||||||||||||||||
Atomic model building |
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