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- PDB-8a8n: Structure of self-assembling engineered protein nanocage (EPN) fu... -

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Basic information

Entry
Database: PDB / ID: 8a8n
TitleStructure of self-assembling engineered protein nanocage (EPN) fused with hepatitis A pX protein
ComponentsEPN-pX
KeywordsSTRUCTURAL PROTEIN / hepatitis A / pX / VP1-pX / nanocage / icosahedral / protein nanoparticle / virus assembly / enveloped viruses
Function / homology
Function and homology information


D-galactonate catabolic process / 2-dehydro-3-deoxy-6-phosphogalactonate aldolase activity / virion component / host cell / symbiont entry into host cell / virion attachment to host cell
Similarity search - Function
KDPG/KHG aldolase / KDPG and KHG aldolase / Viral coat protein subunit / Aldolase-type TIM barrel
Similarity search - Domain/homology
2-dehydro-3-deoxyphosphogluconate aldolase/4-hydroxy-2-oxoglutarate aldolase / Genome polyprotein
Similarity search - Component
Biological speciesHuman immunodeficiency virus type 1 BH10
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 6.7 Å
AuthorsDuyvesteyn, H.M.E. / Stuart, D.I.
Funding support United States, United Kingdom, 6items
OrganizationGrant numberCountry
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 TrustALR00750 United Kingdom
Medical Research Council (MRC, United Kingdom)MR/N00065X/1 United Kingdom
CitationJournal: 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 ...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
DepositionJun 23, 2022Deposition site: PDBE / Processing site: PDBE
Revision 1.0Aug 10, 2022Provider: repository / Type: Initial release
Revision 1.1Aug 24, 2022Group: Database references / Category: citation / citation_author
Item: _citation.journal_volume / _citation.pdbx_database_id_PubMed ..._citation.journal_volume / _citation.pdbx_database_id_PubMed / _citation.title / _citation_author.identifier_ORCID / _citation_author.name
Revision 1.2Aug 31, 2022Group: Database references / Category: citation / citation_author
Item: _citation.page_first / _citation.page_last / _citation_author.identifier_ORCID
Revision 1.3Nov 6, 2024Group: Data collection / Refinement description / Structure summary
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / em_3d_fitting_list / em_admin / pdbx_entry_details / pdbx_initial_refinement_model / pdbx_modification_feature
Item: _em_3d_fitting_list.accession_code / _em_3d_fitting_list.initial_refinement_model_id ..._em_3d_fitting_list.accession_code / _em_3d_fitting_list.initial_refinement_model_id / _em_3d_fitting_list.source_name / _em_3d_fitting_list.type / _em_admin.last_update

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: EPN-pX


Theoretical massNumber of molelcules
Total (without water)32,9341
Polymers32,9341
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein EPN-pX


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.) Human immunodeficiency virus type 1 BH10
Cell line (production host): HEK293T / Production host: Homo sapiens (human) / References: UniProt: Q9WXS1, UniProt: V9Z3B5
Has protein modificationY

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: 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
Molecular weightExperimental value: NO
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Homo sapiens (human) / Cell: HEK293T
Buffer solutionpH: 7 / Details: PBS
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid mesh size: 200 divisions/in. / Grid type: Quantifoil R2/2
VitrificationInstrument: 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

MicroscopyModel: TFS GLACIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 200 kV / Illumination mode: OTHER
Electron lensMode: BRIGHT FIELD / Nominal magnification: 92000 X / Nominal defocus max: 3000 nm / Nominal defocus min: 700 nm / C2 aperture diameter: 50 µm
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingElectron 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
IDNameCategory
1RELIONparticle selection
2EPUimage acquisition
4CTFFINDCTF correction
7Cootmodel fitting
8UCSF Chimeramodel fitting
10RELIONinitial Euler assignment
11RELIONfinal Euler assignment
12RELIONclassification
13RELION3D reconstruction
14PHENIXmodel refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 1830 / Details: Manual selection.
SymmetryPoint symmetry: I (icosahedral)
3D reconstructionResolution: 6.7 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 1830 / Symmetry type: POINT
Atomic model buildingProtocol: 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
IDPDB-ID 3D fitting-IDAccession codeInitial refinement model-IDSource nameType
15KP9

5kp9

15KP91PDBexperimental model
27B3Y

7b3y

17B3Y2PDBexperimental model

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